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MtExpress, a Comprehensive and Curated RNAseq-based Gene Expression Atlas for the Model Legume <i>Medicago truncatula</i>
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bioRxiv (Cold Spring Harbor Laboratory)
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Abstract Although RNA sequencing has been becoming the main transcriptomic approach in the model legume
Medicago truncatula, there is currently no genome-wide gene expression atlas covering the whole set of
RNAseq data published for this species. Nowadays, such tool is highly valuable to provide a global view of gene
expression in a wide range of conditions and tissues/organs. Here, we present MtExpress, a gene expression atlas that compiles an exhaustive set of published M. Here, we present MtExpress, a gene expression atlas that compiles an exhaustive set of published M. truncatula RNAseq data (https://medicago.toulouse.inrae.fr/MtExpress). MtExpress makes use of recent
releases of M. truncatula genome sequence and annotation, as well as up-to-date tools to perform mapping,
quality control, statistical analysis and normalization of RNAseq data. MtExpress combines semi-automated
pipelines with manual re-labelling and organization of samples, to produce an attractive and user-friendly
interface, fully integrated with other available Medicago genomic resources. Importantly, MtExpress is highly
flexible, in terms of both queries, e.g. allowing searches with gene names and orthologous gene IDs from
Arabidopsis and other legume species, and outputs, to customize visualization and redirect gene study to
relevant Medicago webservers. Code de champ modifié Thanks to its semi-automated pipeline, MtExpress will be frequently updated to follow the rapid pace of M. truncatula RNAseq data publications, as well as the constant improvement of genome annotation. MtExpress
also hosts legacy GeneChip expression data originally stored in the Medicago Gene Expression Atlas (MtGEA),
as a very valuable and complementary resource. MtExpress, a Comprehensive and Curated RNAseq-based Gene Expression Atlas for the Model Legume
Medicago truncatula MtExpress, a Comprehensive and Curated RNAseq-based Gene Expression Atlas for the Model Legume
Medicago truncatula Running head: MtExpress database Corresponding author: S. Carrere, LIPME, Université de Toulouse, INRAE, CNRS, Castanet-Tolosan, France ,
b
ti
@i
f Code de champ modifié Subject areas: regulation of gene expression; genomics Number of colour figures: 3 1 MtExpress, a Comprehensive and Curated RNAseq-based Gene Expression Atlas for the Model Legume
Medicago truncatula Running head: MtExpress database
Sebastien Carrere*1, Jerome Verdier2 and Pascal Gamas1
1 LIPME, Université de Toulouse, INRAE, CNRS, Castanet-Tolosan, France
2 Univ Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, F-49000 Angers, France
*Corresponding author E-mail: Sebastien.Carrere@inrae.fr Running head: MtExpress database Sebastien Carrere*1, Jerome Verdier2 and Pascal Gamas1 2 RNAseq, SRA, TMM, Medicago, legumes, atlas, GeneChip Keywords RNAseq, SRA, TMM, Medicago, legumes, atlas, GeneChip 3 Introduction Retrieval, quality checking and organization of RNAseq data To build MtExpress, RNAseq data (i.e. sequenced reads, fastq) generated from Medicago were
downloaded from the Sequence Read Archive (SRA) and the European Nucleotide Archive (ENA) (Fig. 1), along
with associated metadata. In order to ensure that MtExpress data are reliable and well documented, only
sequencing data associated with peer-reviewed publications were conserved, representing 431 different samples
(1374 if considering biological replicates) (supplemental data set 1). The nf-core/rnaseq bioinformatic pipeline
version 3.0 (Patel et al. 2020) was used to automatically analyze the RNAseq libraries. First, FastQC/MultiQC were
used to perform quality checking and reporting of sequencing data. Sequencing reads were, then, mapped to the
Medicago genome version 5.1.7 (Pecrix et al. 2018) (52,723 genes) and reference genomes of Medicago
symbionts or pathogens used in the selected SRA projects (listed in supplemental table 1), using STAR mapper
(Dobin et al. 2013). Finally, SALMON (Patro et al. 2017) was used to quantify read counts of transcripts. To enable
cross-comparisons between different experiments, we normalized the complete dataset by the trimmed mean
of M-values method (TMM) (Robinson and Oshlack 2010), using the EdgeR package. All samples were then
manually labelled using a coherent nomenclature and organized into different categories based on organs or
physiological conditions (18 categories in this first release; e.g. leaf, seed, abiotic factors, biotic stress, root
nodule symbiosis…; supplemental data set 1), with keywords attributed to each experiment, thereby enabling
rapid search/selection. Introduction Legumes represent a plant family of major importance for food, feed and the agro-ecosystem, thanks
to their nutritional qualities and their ability to grow efficiently in the absence of nitrogen fertilizers due to their
endosymbioses with soil microorganisms, notably with nitrogen-fixing soil bacteria (rhizobia). Indeed, legumes
are becoming a keystone in tomorrow’s agriculture to ensure food security, sustainable practices and mitigation
of greenhouse effects. Medicago truncatula was proposed as a model legume about thirty years ago, and since
then it has been widely used to study a wide range of biological issues relevant for plant biology and legume crop
improvement. Its model legume status has led to the development of important resources for genetics, reverse
genetics, structural and functional genomics (Kang et al. 2016). This notably included a gene expression atlas
(MtGEA; https://mtgea.noble.org/v3) (Benedito et al. 2008) based upon GeneChip technology developed by
Affymetrix. This atlas integrated 270 different samples (not considering biological replicates) in its last release
and has been extensively used by the Medicago community. Medicago GeneChip data are also displayed with
other visualization tools available from the Medicago ePLant BAR website (Waese et al. 2017)
(http://bar.utoronto.ca/eplant_medicago/). Unfortunately, the MtGEA website does not provide expression
profiles anymore, due to technical issues. Moreover, this resource was built upon first genome versions and only
represents 45% of the protein-coding and long non-coding RNA genes of Mt5.1.7 genome release/annotation. In the past years, RNA sequencing has become increasingly popular to study gene expression at the
plant, organ and tissue levels. The advantages of this approach are its sensitivity and its flexibility to exploit new
genome releases and annotations by the possibility of (re)mapping sequencing reads, as well as to discover
transcript isoforms or novel transcripts such as long non-coding RNAs. Two databases (db) give access to some
M. truncatula RNAseq data, the first one as part of a generic db developed by EBI on many species
(https://www.ebi.ac.uk/gxa/plant/experiments), and the second one as part of a specific study dedicated to
nutrient-
and
nodulation-
responsive
peptides
(http://mtsspdb.noble.org/atlas-
internal/3880/transcript/profile/0) (de Bang et al. 2017). However, these db only show a small selection of
publicly available RNAseq data. Here we present MtExpress, a gene expression atlas based upon M. truncatula
RNAseq data supported by peer-reviewed publications (https://medicago.toulouse.inrae.fr/MtExpress). We
ensured that MtExpress was a comprehensive, reliable, user-friendly, interconnected and upgradeable database,
by combination of automated analyses and manual curation. Code de champ modifié A flexible and user-friendly interface Thanks to synonymy links previously computed in the LeGOO knowledge base (Carrère et al. 2019),
MtExpress can be queried by any gene locus identifier from the different M. truncatula genome releases (from
Mt3.0 to Mt5.0), and identifiers of Mt16kOLI1 microarrays (Kuster et al. 2004) or Affymetrix (Benedito et al. 2008) and Nimblegen (Verdier et al. 2013) gene chips. Moreover, we extended this query flexibility to gene
acronyms, based on a list manually extracted from publications, available on the Mt5.0 genome browser
https://medicago.toulouse.inrae.fr/MtrunA17r5.0-ANR/. Furthermore, gene IDs from Arabidopsis thaliana
(A.thaliana Col-0 [Araport11]) (Cheng et al. 2017) and four major legumes, namely soybean (Glycine max Williams
82 [Phytozome.12]) (Schmutz et al. 2010)), Lotus japonicus (L. japonicus Miyakojima MG-20 [3.0] (Li et al. 2020)),
common bean (Phaseolus vulgaris BAT93 [EnsemblPlants.38] (Schmutz et al. 2014)) and pea (Pisum sativum cv. 5 ’Caméor’ v1a (Kreplak et al. 2019), can be used to query the expression pattern of putative orthologues in M. truncatula (Carrère et al. 2019). The query output (Fig. 2) was set up to display by default the expression profile of the queried gene
using a ‘reference dataset’, containing 28 samples which provide an overview of gene expression in various plant
organs as well as symbiotic interactions. Then, the user can switch to one of the 18 pre-arranged categories, each
with a more extensive or specialized set of samples: e.g. ‘abiotic factors’ and ‘biotic stress’ categories with all
RNAseq samples related to different stresses, or ‘seed’ and ‘nodule’ categories, dedicated to samples specific to
these organs. If needed, the complete dataset containing the 431 samples can also be selected for visualization. Finally, custom-made selections can also be defined by the user by selecting/unselecting specific experiments to
visualize and export the desired graphical outputs. All of the information associated to each experiment is
provided, including the MultiQC (Ewels et al. 2016) report, and the corresponding publication, metadata and raw
counts. Gene expression levels (with a choice between log2 TMM or raw TMM values) are displayed as box plots,
with various informative elements (notably project name from SRA and descriptive statistics from biological
replicates such as mean, median, min, max, Q1, Q3), each project being distinguished by a different color. Information about the whole dataset is also available via the ‘metadata’ button, including global statistics, used
reference genomes and tools. A flexible and user-friendly interface Additional information on queried genes is easily accessible via the ‘synonymous’
and ‘annotation’ buttons, as well as links to the Mt5.0 genome browser and the LeGOO knowledge database. Conversely, a link to MtExpress is provided for all annotated genes in the Mt5.0 genome browser. As case studies, we examined the expression profile of various gene markers described to be specifically
expressed in certain organs, tissues, or conditions. We found that their expression patterns were as expected
from the literature, thereby supporting our automated processes. Three of them are shown in Fig. 3, using the
reference sample set: MtPT4 (PHOSPHATE TRANSPORTER 4) (Harrison et al. 2002) (arbuscular mycorrhyzal
symbiosis: arbuscule-containing cells), MtNF-YA1 (NUCLEAR FACTOR YA1) (Combier et al. 2006) (root nodule
symbiosis: response to bacterial Nod factors, rhizobium infection, nodule inception and apical nodule zones),
MtDME (DEMETER) (Satgé et al. 2016) (endosperm and nodule differentiation zone). 6 6 Retrieval and presentation of MtGEA GeneChip data As an additional tool to MtExpress, we downloaded publicly available Medicago Affymetrix GeneChip
data (supplemental data set 2), previously stored in the Medicago Gene Expression Atlas (MtGEA) site hosted by
the Noble Research Institute at https://mtgea.noble.org/v3. We implemented these data into the MtExpress
webserver to provide an alternative solution to the inoperative MtGEA webserver. Moreover, we added features
of MtExpress, such as the flexible input queries and pre-defined or custom-made categories. This alternative
resource is also available at https://medicago.toulouse.inrae.fr/MtExpress. Code de champ modifié Material and Methods Material and Methods Discussion MtExpress benefits from several important assets: (i) the use of recent releases of genome
sequences/annotations to provide an up-to-date tool; (ii) semi-automated analysis pipelines allowing frequent
updates (at least twice a year, depending on the number of new RNAseq data publications and possible new
annotation release); (iii) manual re-labelling and organization of samples to conserve an unified structure
through the updates; (iv) an attractive and flexible interface; (v) a transcriptomic platform fully integrated with
other Medicago tools such as the Medicago Mt5.0 genome browser (Pecrix et al. 2018) and the LeGOO
knowledge base (Carrère et al. 2019). The combination of manual curation with automated quality control and
statistical analyses is key to ensure the reliability of MtExpress. Furthermore, the immediate access to
accompanying metadata and publications represent an obvious gain of time and added value for users to better
evaluate the data quality. We therefore believe that this user-friendly, exhaustive and integrated resource will be of great use to
the Medicago and, widely, to the legume community to quickly obtain the expression profiles of genes of interest
in a variety of organs, tissues and conditions. This is, for instance, essential to define gene regulation patterns
and determine gene expression specificity of developmental stage(s) or plant responses to biotic or abiotic
environments. Considering the current pace of M. truncatula RNAseq data production and publication, we
anticipate that the number and diversity of sequenced RNA samples will continue to grow rapidly. MtExpress will
be updated accordingly, with creation of additional categories if needed. In the near future, we also plan to
implement analytical tools such as gene co-expression analysis and pair-wise comparisons between experiments. 7 7 Material and Methods Collecting Data RNAseq dataset. We collected all data available as of April 1st 2021 in the Sequence Read Archive (SRA), using
the Run Selector with the query "Medicago truncatula [ORG]" and the filter "AssayType = RNA-seq". We kept all
studies associated with a peer-reviewed publication, representing 51 projects altogether (supplemental data set
1). All metadata coming from SRA, ENA and PubMed were associated to each selected project and stored for
downstream
treatment
under
a
GIT
repository
[https://lipm-gitlab.toulouse.inra.fr/EXPRESSION-
ATLAS/ExpressionAtlasData]. Fastq files were directly downloaded from EBI SRA FTP mirror (ftp.sra.ebi.ac.uk )
and listed in csv files with curated sample names, replicates and library strandedness to be properly processed. Affymetrix GeneChip MtGEA dataset. We exported experiment replicates for all probesets (including Mtr, Msa,
Sme and control probesets) from https://mtgea.noble.org/v3/experiments.php downloading webpage
(Benedito et al. 2008). We excluded eight duplicated libraries as well as seven libraries for which biological
replicate data were not available, and added one missing set of published data (supplemental data set 2). All
samples were manually ordered, classified into 14 categories (supplemental data set 2) and aliases were added
to homogenize labelling. Metadata such as publication, organ, experimental factors were manually extracted
from the MtGEA website. Expression Measure of RNAseq dataset nf-core/rnaseq version 3.0 (Patel et al. 2020) was used with default parameters to run expression measure
separately on each SRA project (Fig. 1). We chose STAR (Dobin et al. 2013) as a read mapper with 20 multiple
alignments allowed for one read, and SALMON as a read quantifier (Patro et al. 2017) (see supplemental data set
3 for software versions). Of note, SALMON can deal with multi-mapped reads, that can occur with M. truncatula
gene duplications, using an expectation maximisation (EM) algorithm to attribute read counts to the most likely
transcripts (Deschamps-Francoeur, 2020). We used the version 5.1.7 of M. truncatula A17 genome annotation
(Pecrix et al. 2018) to map RNAseq data from different accessions of M. truncatula, as well as reference genomes
of Medicago symbionts or pathogens used in the selected SRA Studies (Sinorhizobium meliloti 2011,
Sinorhizobium medicae WSM419, Fusarium oxysporum f. sp. medicaginis, Rhizophagus irregularis, Ralstonia
pseudosolanacearum GMI1000, Verticillium albo-atrum V31-2, Gigaspora rosea and Rhizoctonia solani AG-3) 8 (supplemental table 1). Using quality control reports and statistical analyses provided by MultiQC (Ewels et al. 2016), we defined strandedness for each library and verified the consistency of biological replicates. RNAseq dataset. Collecting Data The complete dataset (51 SRA projects representing 431 samples) was normalized together in
order to provide consistent values across gene expression comparisons in sub-categories, using TMM
normalization from EdgeR package (Robinson and Oshlack 2010). Affymetrix GeneChip MtGEA dataset. The expression data downloaded from the MtGEA site website were
already normalized using a robust multichip average method (RMA normalization). For RNAseq and GeneChip datasets, we added functional annotations provided by an automatic pipeline (Pecrix
et al. 2018) as well as synonymy links to identifiers of different generations of M. truncatula microarrays,
GeneChips and genome sequences/annotations (Carrère et al. 2019). We also used a manually curated list of
3,760
gene
names/acronyms
and
products
(Gene
acronyms
and
IDs,
release
20210409;
https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/). Normalized expression values (TMM or RMA) from
biological replicates were summarized using descriptive statistics (mean, median, min, max, Q1, Q3) using ad-
hoc Scala/Spark scripts to ensure future scalability. All these information were merged with dataset metadata to
build a consistent database. Availability Project name: MtExpress Project name: MtExpress
Project home page: https://medicago.toulouse.inrae.fr/MtExpress
Source Code and Data: https://lipm-gitlab.toulouse.inra.fr/EXPRESSION-ATLAS
Operating system(s): Platform independent
Programming languages: Scala, R, Spark, Javascript, Perl
Other requirements: Drill 1.16, Apache web server
License: Apache License 2.0 Project name: MtExpress
Project home page: https://medicago.toulouse.inrae.fr/MtExpress
Source Code and Data: https://lipm-gitlab.toulouse.inra.fr/EXPRESSION-ATLAS
Operating system(s): Platform independent
Programming languages: Scala, R, Spark, Javascript, Perl
Other requirements: Drill 1.16, Apache web server
License: Apache License 2.0 Code de champ modifié Code de champ modifié Programming languages: Scala, R, Spark, Javascript, Perl Other requirements: Drill 1.16, Apache web server License: Apache License 2.0 Database The database consists of a repository of JSON and PARQUET files. Each dataset is documented through a JSON
definition file along with run definition files including information related to reference genome and tools versions
used for mapping. Database is accessed using Drill software (https://drill.apache.org/) version 1.16.0. Database
structure and scheme are available at https://lipm-gitlab.toulouse.inra.fr/EXPRESSION-ATLAS Code de champ modifié API All normalized data are accessible through a REST API developed using the Scalatra framework and exposed with
Jetty servlet container (supplemental data set 3). The API is fully documented using Swagger 2.0. Documentation
can be found at: https://lipm-browsers.toulouse.inra.fr/expression-atlas-api All normalized data are accessible through a REST API developed using the Scalatra framework and exposed with
Jetty servlet container (supplemental data set 3). The API is fully documented using Swagger 2.0. Documentation
can be found at: https://lipm-browsers.toulouse.inra.fr/expression-atlas-api 9 User Interface The user interface has been developed using the Vue.js library (v. 2.6.12) with a set of components, and plugins
(see supplemental data set 3), notably Chart.js for boxplot display. It aims at providing simple functionalities such
as gene searching through different accession identifiers (based on synonymous computed across different
generations of genome assemblies/annotations and arrays), gene names or functions. Expression patterns are
displayed using box-plots to show the level of variability between biological replicates. Users can easily
select/unselect SRA projects to be plotted, navigate through different subsets of projects or locus and display
raw or log2 normalized expression values, access to gene annotations, synonymous and links to relevant external
resources (genome browser, knowledge base, original data repository). For each SRA project, links to MultiQC
(Ewels et al. 2016) report, raw count and metadata files are provided. Availability Data Availability The data underlying this article are publicly available in SRA (https://trace.ncbi.nlm.nih.gov/Traces/sra/) and
MtGEA (https://mtgea.noble.org/v3/) databases. Funding This work was supported by the Agence Nationale de la Recherche [EPISYM ANR-15-CE20-0002 and DESWITCH
ANR-19-CE20-0027], Laboratoire d’Excellence TULIP ANR-10-LABX-41]; and Plant2Pro® Carnot Institute
[agreement #18-CARN-024-01 – 2018]. 10 Disclosures Conflicts of interest: No conflicts of interest declared Conflicts of interest: No conflicts of interest declared Acknowledgments We thank Jérôme Gouzy and Marie-Françoise Jardinaud (LIPME, Castanet-Tolosan) for valuable discussions on
the MtExpress set up and Nathalie Vialaneix (MIAT, Castanet-Tolosan) for her advice on statistical analyses. We
also thank Erika Sallet, Ludovic Cottret, Ludovic Legrand and Léo Géré (LIPME) for technical advice. Finally, we
are
grateful
to
the
Genotoul
bioinformatics
platform
Toulouse
Occitanie
(Bioinfo
Genotoul,
https://doi.org/10.15454/1.5572369328961167E12) for providing computing resources. References Benedito, V.A., Torres-Jerez, I., Murray, J.D., Andriankaja, A., Allen, S., Kakar, K., et al. (2008) A gene expression
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quantification of transcript expression. Nat Methods 14: 417-419. quantification of transcript expression. Nat Methods 14: 417-419. Pecrix, Y., Staton, S.E., Sallet, E., Lelandais-Briere, C., Moreau, S., Carrere, S., et al. (2018) Whole-genome Pecrix, Y., Staton, S.E., Sallet, E., Lelandais-Briere, C., Moreau, S., Carrere, S., et al. (2018) Whole-genome
landscape of Medicago truncatula symbiotic genes. Nat Plants 4: 1017-1025. landscape of Medicago truncatula symbiotic genes. Nat Plants 4: 1017-1025. Robinson, M.D. and Oshlack, A. (2010) A scaling normalization method for differential expression analysis of
RNA-seq data. Genome Biol 11: R25. Satgé, C., Moreau, S., Sallet, E., Lefort, G., Auriac, M.C., Remblière, C., et al. (2016) Reprogramming of DNA Satgé, C., Moreau, S., Sallet, E., Lefort, G., Auriac, M.C., Remblière, C., et al. (2016) Reprogramming of DNA
methylation is critical for nodule development in Medicago truncatula. Nat Plants 2: 16166. methylation is critical for nodule development in Medicago truncatula. Nat Plants 2: 16166. Schmutz, J., Cannon, S.B., Schlueter, J., Ma, J., Mitros, T., Nelson, W., et al. (2010) Genome sequence of the
palaeopolyploid soybean. Nature 463: 178-183. palaeopolyploid soybean. Nature 463: 178-183. palaeopolyploid soybean. Nature 463: 178-183. palaeopolyploid soybean. Nature 463: 178-183. Schmutz, J., McClean, P.E., Mamidi, S., Wu, G.A., Cannon, S.B., Grimwood, J., et al. (2014) A reference genome
for common bean and genome-wide analysis of dual domestications. Nat Genet 46: 707-713. 12 Verdier, J., Lalanne, D., Pelletier, S., Torres-Jerez, I., Righetti, K., Bandyopadhyay, K., et al. References (2013) A regulatory
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Levels of Data for Hypothesis Generation in Plant Biology. Plant Cell 29: 1806-1821. Supplemental data set 2. GeneChip data
Supplemental data set 3. Software, API, User interface Supplemental data set 2. GeneChip data Figures and Tables Figure 1. RNAseq analysis pipeline used to build MtExpress. The first step consists in collecting and curating
metadata (sample and project descriptions, experiment design) and raw data (fastq files) from public
databases. Then these data are processed for quality controls and expression measure using the nf-core/rnaseq
pipeline. Once all datasets are processed, count tables are merged and values are normalized with the TMM
method from EdgeR package. Normalized values, gene annotations and datasets metadata are consolidated
into a database made of parquet files accessible through an Application Programming Interface (API) to feed
the web interface. Figure 2. Interface of MtExpress, a user-friendly gene expression atlas. (A) drop-down menu for selecting dataset
or sample category. (B) query boxes, using either Mt5.0 locus IDs, keywords, gene IDs from other genome
releases or gene acronyms. (C) example of a graphical output using the reference dataset, with color-specific box
plots per SRA project showing log2(TMM) values; buttons to switch between Raw and log2 TMM values and to
display metadata, synonymous IDs and annotation information are indicated next to links to the Mt5.0 genome
portal and the Legoo knowledge base. (D) list of plotted SRA projects with associated metadata and checkboxes
to select/ unselect projects/samples. Figure 3. Expression profile of three genes with specific expression patterns. (A) MtPT4 (PHOSPHATE
TRANSPORTER 4), expressed in arbuscule containing cells of mycorrhizal roots. (B) MtNF-YA1 (NUCLEAR FACTOR
YA1), expressed in bacterial Nod factor-treated epidermal root cells, in Rhizobium-inoculated root cells, and in
nodule apical zones (zone 1, distal zone 2 and proximal zone 2). (C) MtDME (DEMETER), expressed in seed
endosperm and in the nodule differentiation zones (proximal zone and interzone). Supplemental table 1. Reference genomes used for mapping Supplemental table 1. Reference genomes used for mapping 13 14 14
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Understanding allele shift using SSR markers in pedigree, modified bulk, and SSD breeding methods in rice
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International Rice research notes/International rice research notes
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cc-by
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Understanding allele shift using SSR markers in
pedigree, modified bulk, and SSD breeding
methods in rice A. Kanbar, Department of Field Crops, Faculty of Agriculture, University of Damascus, Syria;
N. Pirany, Department of Animal Science, Faculty of Agriculture, University of Tabriz, Iran;
A. Al-Ouda, Department of Field Crops, Faculty of Agriculture, University of Damascus, Syria;
and H.E. Shashidhar, Department of Genetics and Plant Breeding, University of Agricultural
Sciences, Bangalore, India Email: adnan_mha@yahoo.com Various methods of selection can be applied to advance segregating materials in
autogamous crops. Under certain sets of conditions, one is preferred. But, in
general, a selection system of advancing generations should be such that additive
genetic variance is generated (Murty 1979). At the same time, mean performance
needs to be maintained or increased in the selected progenies and the favorite
genes from the donor parents should be increased. Comparative studies on
different breeding methods using DNA markers are not well established. g
g
In this study, three breeding procedures—pedigree, SSD, and modified
bulk—were used to advance the materials derived from a cross between
divergent rice parents Moroberekan and IR20 in an aerobic environment. The
female parent of this cross is Moroberekan, an African japonica type, which was
chosen because of its deep and thick roots and resistance to drought. It is
relatively low yielding. The male parent is IR20, an indica type with short
stature, a shallow root system, and high yield potential; however, it is susceptible
to drought. The experiment was carried out at the Hebbal campus of the
University of Agricultural Sciences, Bangalore, India, during the 2005 dry season. Sixty F6 lines (20 for each breeding method) were randomly chosen from the F5
generation and grown in the field for the different studies. Thirty-five-day-old
seedlings were used for DNA extraction. DNA extraction was done following a
modified C-TAB method (Cao and Oard 1997). Thirty-three SSR microsatellite
markers were used to compare the three breeding methods at the DNA marker
level. Based on earlier investigations, four chromosomal regions on
chromosomes 1, 3, 7, and 9, which were associated with QTLs for drought
resistance, grain yield, and yield component traits, were identified: RM84,
RM283, RM129, RM5, RM34, RM246, RM443, RM128, RM302, RM212, and
RM265 located on chromosome 1; RM231, RM545, RM546, RM7, RM218, RM251,
RM563, and RM282 on chromosome 3; RM11, RM182, RM455, RM234, RM248,
and RM420 on chromosome 7; and RM460, RM434, RM257, RM242, RM278,
RM201, RM215, and RM205 on chromosome 9. Plant breeding Plant breeding Understanding allele shift using SSR markers in
pedigree, modified bulk, and SSD breeding
methods in rice Bands showing size similarity
with Moroberekan were given a score of 1, whereas bands showing size
similarity with IR20 got a score of 3; the heterozygote had a score of 2. 1 IRRN IRRN Analysis of molecular variance (AMOVA) was used for partitioning
diversity within and among method populations using SSR marker data. This
produces estimates of variance components and F statistic analogs (designated as
phi-statistics, ΦST). The significance of the variance components and phi-statistics
is tested using a permutation approach, eliminating the normality assumption
that is inappropriate for molecular data. AMOVA was performed on Arlequin
3.0 version (Excoffier et al 1992). Genetic distance between the three breeding
methods was estimated based on Nei’s formula (Nei 1972). Cluster analysis to
show the relatedness of the three breeding methods was done and a dendrogram
was generated using the UPGMA algorithm of POPGENE version 1.31. was generated using the UPGMA algorithm of POPGENE version 1.31. ompetition
An AMOVA of the three breeding methods indicated that a large
percentage of total genetic variation (97%) was within methods, but that a small
amount (3%) was noted among methods (Table 1). The allele frequencies,
by locus, in each breeding population are shown in Figure 1. There was a
significant difference in allelic frequencies of the parents (Moroberekan and IR20
types) in the selection-bulk and pedigree methods, whereas the frequencies of
two types of alleles were numerically different in the SSD population. This
indicated a shifting of alleles in selection-bulk and pedigree populations toward
the Moroberekan parent. In the SSD population, there was equal distribution of
Moroberekan and IR20 allelic types. This shift in allelic frequency in the pedigree
and bulk populations could be due to subsequent selection pressure across
generations. Environmental changes during seasons might have contributed to
the change in allele frequencies
within and between method
lines; the better adapted
genotypes had less c
with other lines that did not
grow well under aerobic
upland conditions. The
literature has reported a large
genetic variation within
populations or species or
groups and an appreciable
amount among them in
different plants and animals
(Reeb et al 2000, Kiambi et al
2005, Vidya et al 2005). Moroberekan alleles
IR20 alleles
Allele frequencies by locus
0.420
SSD
Pedigree
Bulk
0.440
0.460
0.480
0.500
0.520
0.540
Breeding methods
Moroberekan alleles
IR20 alleles
Allele frequencies by locus
0.420
SSD
Pedigree
Bulk
0.440
0.460
0.480
0.500
0.520
0.540
Breeding methods
Fig. Understanding allele shift using SSR markers in
pedigree, modified bulk, and SSD breeding
methods in rice 1. Graphical comparison of allelic frequencies of SSR loci among
pedigree, modified bulk, and SSD lines in the F6 generation. g
g
g
An AMOVA of the three breeding methods indicated that a large
percentage of total genetic variation (97%) was within methods, but that a small
amount (3%) was noted among methods (Table 1). The allele frequencies,
by locus, in each breeding population are shown in Figure 1. There was a
significant difference in allelic frequencies of the parents (Moroberekan and IR20
types) in the selection-bulk and pedigree methods, whereas the frequencies of
two types of alleles were numerically different in the SSD population. This
indicated a shifting of alleles in selection-bulk and pedigree populations toward
the Moroberekan parent. In the SSD population, there was equal distribution of
Moroberekan and IR20 allelic types. This shift in allelic frequency in the pedigree
and bulk populations could be due to subsequent selection pressure across
generations. Environmental changes during seasons might have contributed to Moroberekan alleles
IR20 alleles
Allele frequencies by locus
0.420
SSD
Pedigree
Bulk
0.440
0.460
0.480
0.500
0.520
0.540
Breeding methods
Moroberekan alleles
IR20 alleles
Allele frequencies by locus
0.420
SSD
Pedigree
Bulk
0.440
0.460
0.480
0.500
0.520
0.540
Breeding methods
Fig. 1. Graphical comparison of allelic frequencies of SSR loci among
pedigree, modified bulk, and SSD lines in the F6 generation. Moroberekan alleles
IR20 alleles
Allele frequencies by locus
0.420
SSD
Pedigree
Bulk
0.440
0.460
0.480
0.500
0.520
0.540
Breeding methods
Moroberekan alleles
IR20 alleles
Allele frequencies by locus
0.420
SSD
Pedigree
Bulk
0.440
0.460
0.480
0.500
0.520
0.540
Breeding methods
Fig. 1. Graphical comparison of allelic frequencies of SSR loci among Fig. 1. Graphical comparison of allelic frequencies of SSR loci among
pedigree, modified bulk, and SSD lines in the F6 generation. IRRN 2 Table 1. Hierarchical AMOVA based on 33 microsatellite DNA markers,
percentage of variation explained by different levels of method (subpopulation)
structure, variance components, and F statistics. SOV
DF
SS
MS
Variance
components
Percentage
variation
Φ-statistics
P value
Among
methods
2
29.32
14.66
0.19
2.73 %
ΦST = 0.0273
< 0.0099
Within
methods
117
807.716
6.90
6.90
97.27 % Table 1. Hierarchical AMOVA based on 33 microsatellite DNA markers,
percentage of variation explained by different levels of method (subpopulatio
structure, variance components, and F statistics. Pairwise RST values differed between selection-bulk/pedigree (0.034),
selection-bulk/SSD (0.016), and pedigree/SSD (0.031) (Table 2). Understanding allele shift using SSR markers in
pedigree, modified bulk, and SSD breeding
methods in rice The RST between
selection-bulk/pedigree and pedigree/SSD was significant at 1% (P = 0.010),
whereas RST between SSD and bulk was significant at 4% (P = 0.040). The
dendrogram cluster calculated from Nei’s unbiased genetic distances between
method populations by the UPGMA method revealed two distinct clusters
(Fig. 2). The unbiased genetic distance between the two clusters was 1.71. Cluster
one comprised the bulk and SSD populations. Cluster two comprised only the
pedigree population. In plant
breeding, it is known that the
modified bulk and SSD
methods are derived from
the bulk method. Thus, the
chances of having
undesirable alleles in the
modified bulk population
are greater than those in the
pedigree population. Modified
bulk
SSD
1
2
0.62
1.08
1.71
Pedigree
Modified
bulk
SSD
1
2
0.62
1.08
1.71
Pedigree
Fig. 2. Dendrogram of the relationships between three breeding method
populations using the UPGMA algorithm of POPGENE. Pairwise RST values differed between selection-bulk/pedigree (0.034),
selection-bulk/SSD (0.016), and pedigree/SSD (0.031) (Table 2). The RST between
selection-bulk/pedigree and pedigree/SSD was significant at 1% (P = 0.010),
whereas RST between SSD and bulk was significant at 4% (P = 0.040). The
dendrogram cluster calculated from Nei’s unbiased genetic distances between
method populations by the UPGMA method revealed two distinct clusters
(Fig. 2). The unbiased genetic distance between the two clusters was 1.71. Cluster
one comprised the bulk and SSD populations. Cluster two comprised only the Modified
bulk
SSD
1
2
0.62
1.08
1.71
Pedigree
Modified
bulk
SSD
1
2
0.62
1.08
1.71
Pedigree
Fig. 2. Dendrogram of the relationships between three breeding method
populations using the UPGMA algorithm of POPGENE. Fig. 2. Dendrogram of the relationships between three breeding method
populations using the UPGMA algorithm of POPGENE. Table 2. RST (above the diagonal line) and probability (below the diagonal line)
values between method groups based on microsatellite DNA. Method
Modified bulk
Pedigree
SSD
Modified bulk
0.034
0.016
Pedigree
0.01
0.031
SSD
0.04
0.01
References
Cao D, Oard JH. 1997. Pedigree and RAPD-based DNA analysis of commercial US rice
cultivars. Crop Sci. 37:1630-1635. Excoffier L, Smouse PE, Quattro JM. 1992. Analysis of molecular variance inferred from
metric distances among DNA haplotypes: application to human mitochondrial
DNA restriction data. Genetics 131:479-491. Table 2. RST (above the diagonal line) and probability (below the diagonal line)
values between method groups based on microsatellite DNA. Understanding allele shift using SSR markers in
pedigree, modified bulk, and SSD breeding
methods in rice Method
Modified bulk
Pedigree
SSD
Modified bulk
0.034
0.016
Pedigree
0.01
0.031
SSD
0.04
0.01 erences
D, Oard JH. 1997. Pedigree and RAPD-based DNA analysis of commercial US rice
cultivars. Crop Sci. 37:1630-1635. Excoffier L, Smouse PE, Quattro JM. 1992. Analysis of molecular variance inferred from
metric distances among DNA haplotypes: application to human mitochondrial
DNA restriction data. Genetics 131:479-491. IRRN 3 Kiambi DK, Newbury HJ, Ford-Loyd BV, Dawson I. 2005. Contrasting genetic diversity
among Oryza longistaminata (A. Chevet Roehr) populations from different
geographic origins using AFLP. Afr. J. Biotechnol. 4:308-317. Murty BR. 1979. Selection of parental material, breeding methods and evaluation
procedures in developing improved crop varieties. Indian J. Genet. 39:305-314. p
p
g
p
p
Nei M. 1972. Genetic distance between populations. Am. Nat. 106:283-292. p p
Reeb CA, Arcangeli L, Block BA. 2000. Structure and migration corridors in Pacific
populations of the swordfish Xiphius gladius (Sic.), as inferred through analyses
of mitochondrial DNA. Mar. Biol. 136:1123-1131. Reeb CA, Arcangeli L, Block BA. 2000. Structure and migration corridors in Pacific
populations of the swordfish Xiphius gladius (Sic.), as inferred through analyses
of mitochondrial DNA. Mar. Biol. 136:1123-1131. Vidya TNC, Fernando P, Melnick DJ, Sukumar R. 2005. Population differentiation
within and among Asian elephant (Elephas maximus) populations in southern
India. Heredity 94:71-80. Vidya TNC, Fernando P, Melnick DJ, Sukumar R. 2005. Population differentiation
within and among Asian elephant (Elephas maximus) populations in southern
India. Heredity 94:71-80. IRRN 4
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Knowledge, attitudes and awareness of the human papillomavirus among health professionals in New Zealand
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bioRxiv preprint 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals
2
New Zealand
3
4
5
6
7
Susan M. Sherman1, Karen Bartholomew2, Hayley J. Denison3, Hersha Patel4, Esther L. Moss4,5, Jer
8
Douwes3 and Collette Bromhead6
9
10
11
12
1School of Psychology, Keele University, Keele, Staffs, ST5 5BG, UK. 13
2Waitemata District Health Board (DHB) and Auckland DHB, Auckland, New Zealand
14
3Centre for Public Health Research, Massey University, Wellington, New Zealand
15
4Department of Gynaecology, University Hospitals Leicester, Leicester LE5 4PW, UK
16
5Leicester Cancer Research Centre, University of Leicester, LE2 7LX, UK
17
6Massey University, School of Health Sciences, Wellington, New Zealand
18
19
20
21
22
23
24
25
*Corresponding author
26
Email: C.Bromhead@massey.ac.nz (CB)
27
28 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand san M. Sherman1, Karen Bartholomew2, Hayley J. Denison3, Hersha Patel4, Esther L. Moss4,5, Jeroen
Douwes3 and Collette Bromhead6 1 1 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 29
Abstract
30
Background
31
Human papillomavirus (HPV) is a common sexually transmitted infection that is implicated in 99.7%
32
of cervical cancers and several other cancers that affect both men and women. Despite the role that
33
HPV plays in an estimated 5% of all cancers and the evolving role of HPV vaccination and testing in
34
protecting the public against these cancers, preliminary research in New Zealand health care
35
professionals suggest knowledge about HPV may not be sufficient. 36
Methods
37
A total of 230 practice nurses, smear takers and other clinical and laboratory staff who attended a
38
range of training events completed a cross-sectional survey between April 2016 and July 2017. The
39
survey explored four broad areas: demographics and level of experience, HPV knowledge (general
40
HPV knowledge, HPV triage and test of cure (TOC) knowledge and HPV vaccine knowledge), attitudes
41
towards the HPV vaccine and self-perceived adequacy of HPV knowledge. 42
Results
43
The mean score on the general HPV knowledge questions was 13.2 out of 15, with only 25.2% of
44
respondents scoring 100%. In response to an additional question, 14.0% thought (or were unsure)
45
that HPV causes HIV/AIDS. The mean score on the HPV Triage and TOC knowledge questions was 7.4
46
out of 10, with only 9.1% scoring 100%. The mean score on the HPV vaccine knowledge questions
47
was 6.0 out of 7 and 44.3% scored 100%. Only 62.6% of respondents agreed or strongly agreed that
48
they were adequately informed about HPV, although 71.8% agreed or strongly agreed that they
49
could confidently answer HPV-related questions asked by patients. Multivariate analyses revealed
50
that knowledge in each domain predicted confidence in responding to patient questions. 51
Furthermore, the number of years since training predicted both HPV knowledge and Triage and TOC
52
knowledge. .
CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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;
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doi:
bioRxiv preprint 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand 53
Discussion
54
Although overall level of knowledge was adequate, there were significant gaps in knowledge,
55
particularly about the role of HPV testing in the New Zealand National Cervical Screening
56
Programme. More education is required to ensure that misinformation and stigma do not
57
inadvertently result from interactions between health practitioners and the public. 2 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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bioRxiv preprint 59
Introduction
60
Human papillomavirus (HPV) is responsible for 99.7% of cases of cervical cancer along with some
61
head and neck, penile and anal cancers. There are approximately 150 new diagnoses and 50 deaths
62
from cervical cancer in New Zealand (NZ) every year1, while head and neck cancers attributable to
63
HPV are increasing in both men and women with 94 new cases and 43 deaths estimated for 20122. In
64
addition, there are longstanding ethnic inequalities in cervical cancer incidence and mortality, and
65
cervical screening coverage remains low (and cancer incidence and mortality high) for indigenous
66
Māori women as well as Pacific women3. 67
The NZ National Cervical Screening Programme (NCSP), which was established in 19904,
68
recommends 3-yearly routine screening with liquid-based cytology (LBC) for 20–69 year-old women,
69
with HPV triage testing for low grade (ASC-US/LSIL) cytology in women 30+ years. The programme
70
also recommends testing of cure following treatment for a high-grade lesion4, with a modified
71
version of the Bethesda System used for cytology classification. However, from late 2018 the NCSP
72
will introduce HPV testing as the primary screening test for women aged 25-68 years on a 5 yearly
73
basis5. 74
75
To reduce infection with high-risk types of HPV and its related cancers, the NZ National HPV
76
Immunisation Programme was introduced in September 2008, offering free HPV vaccination
77
(Gardasil®, Merck) for females born in 1990 or later. 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand School-based immunisation for 12–13 year-old
78
girls commenced in most regions in 20091 and the three-dose coverage achieved by the program in
79
cohorts born in 1991–2002 reached approximately 48–66% nationwide1. In January 2017, the free
80
programme was extended to boys and young men, the upper age for free vaccination was increased
81
to 26 years, a two-dose schedule was implemented for individuals aged 14 and under, and the
82
vaccine used was changed to nonavalent Gardasil 9 (Merck)1. 67
The NZ National Cervical Screening Programme (NCSP), which was established in 19904, 83 3 3 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
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bioRxiv preprint 84
Previous research has shown that the public is not well informed about HPV. For example, in a
85
survey of 200 NZ university health science students (mean age 19.8 years), 50.8% were both
86
unaware of the sexual transmission of HPV and unwilling to accept a free HPV vaccine, highlighting
87
the need for education in this age group6. 88
89
Furthermore, it has been shown that there is considerable stigma associated with a diagnosis of
90
HPV7, 8. For example, in a qualitative study, McCaffrey et al., found that HPV positive women7
91
reported levels of stigma and anxiety suggesting that “testing positive for HPV was associated with
92
adverse social and psychological consequences that were beyond those experienced by an abnormal
93
smear alone” (p173). Daley et al., found that younger age and less education were associated with
94
more negative emotions (e.g., anger, shock and worry) and stigma beliefs (e.g., feeling ashamed,
95
guilty and unclean) in HPV positive women8. In addition, a survey of Hong Kong Chinese health care
96
providers exploring levels of knowledge about HPV and attitudes revealed that more knowledge
97
about HPV predicted less stigmatising attitudes from health care providers9. 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand Since NZ women will,
98
from late 2018, be tested for HPV every 5 years, it is crucial that the health care professionals who
99
administer the screening programme, and who will be best placed to counsel women who receive a
100
positive HPV test, are themselves well informed about HPV. This will also likely reduce inequalities in
101
screening uptake. In particular, previous research concerning the diagnosis of depression identified
102
that effective patient-clinician communication and an established relationship in which the patient
103
trusted the general practitioner (GP) were especially important for Māori patients10. 104
Previous research exploring the knowledge of GPs and practice nurses (PNs) in Christchurch, New
105
Zealand about HPV used 5 questions as part of a larger survey exploring attitudes towards HPV 88
89
Furthermore, it has been shown that there is considerable stigma associated with a diagnosis of
90
HPV7, 8. For example, in a qualitative study, McCaffrey et al., found that HPV positive women7
91
reported levels of stigma and anxiety suggesting that “testing positive for HPV was associated with
92
adverse social and psychological consequences that were beyond those experienced by an abnormal
93
smear alone” (p173). Daley et al., found that younger age and less education were associated with
94
more negative emotions (e.g., anger, shock and worry) and stigma beliefs (e.g., feeling ashamed,
95
guilty and unclean) in HPV positive women8. In addition, a survey of Hong Kong Chinese health care
96
providers exploring levels of knowledge about HPV and attitudes revealed that more knowledge
97
about HPV predicted less stigmatising attitudes from health care providers9. Since NZ women will,
98
from late 2018, be tested for HPV every 5 years, it is crucial that the health care professionals who
99
administer the screening programme, and who will be best placed to counsel women who receive a
100
positive HPV test, are themselves well informed about HPV. This will also likely reduce inequalities in
101
screening uptake. In particular, previous research concerning the diagnosis of depression identified
102
that effective patient-clinician communication and an established relationship in which the patient
103
trusted the general practitioner (GP) were especially important for Māori patients10. 4
104
Previous research exploring the knowledge of GPs and practice nurses (PNs) in Christchurch, New
105
Zealand about HPV used 5 questions as part of a larger survey exploring attitudes towards HPV
106
vaccination11. 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand Whilst performance across the 5 questions was reasonable, there was uncertainty as
107
indicated by the number of ‘not sure’ responses, as well as some variability across questions. For
108
example, while more than 90% of GPs and PNs knew that HPV vaccination would not eliminate the 4
104
Previous research exploring the knowledge of GPs and practice nurses (PNs) in Christchurch, New
105
Zealand about HPV used 5 questions as part of a larger survey exploring attitudes towards HPV
106
vaccination11. Whilst performance across the 5 questions was reasonable, there was uncertainty as
107
indicated by the number of ‘not sure’ responses, as well as some variability across questions. For
108
example, while more than 90% of GPs and PNs knew that HPV vaccination would not eliminate the 4 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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doi:
bioRxiv preprint 109
need for cervical screening, only 33% of GPs and 7% of PNs knew that anogenital warts caused by
110
HPV 6 and 11 are not a precursor to cervical cancer. Only half of GPs and 42% of PNs knew that most
111
HPV infections will clear without medical treatment and a quarter of GPs and nearly a third of PNs
112
did not know, or were unsure, whether persistent HPV was a necessary cause of cervical cancer. 113
A recent study in the UK conducted a more detailed online survey of PNs in Leicestershire12. While
114
overall awareness of the basic facts about HPV was adequate, there were some worrying gaps in
115
knowledge, for example nearly 10% of PNs did not know that HPV causes cervical cancer and 63%
116
believed that HPV requires treatment. Some of the PNs reported not feeling adequately informed
117
about HPV and that training provision could be improved. 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand There was no correlation found between
118
self-perceived adequacy of knowledge and HPV knowledge scores. 113
A recent study in the UK conducted a more detailed online survey of PNs in Leicestershire12. While
114
overall awareness of the basic facts about HPV was adequate, there were some worrying gaps in
115
knowledge, for example nearly 10% of PNs did not know that HPV causes cervical cancer and 63%
116
believed that HPV requires treatment. Some of the PNs reported not feeling adequately informed
117
about HPV and that training provision could be improved. There was no correlation found between
118
self-perceived adequacy of knowledge and HPV knowledge scores. 119
To our knowledge there are no studies exploring what primary care staff such as GPs, PNs and smear
120
takers in NZ know about HPV since 2009. In light of the recent changes to the immunisation
121
programme and the forthcoming changes to the NSCP, it is important to benchmark what nurses and
122
smear takers understand about HPV, whether they feel well informed and assess any training needs
123
they might identify. 119
To our knowledge there are no studies exploring what primary care staff such as GPs, PNs and smear
120
takers in NZ know about HPV since 2009. In light of the recent changes to the immunisation
121
programme and the forthcoming changes to the NSCP, it is important to benchmark what nurses and
122
smear takers understand about HPV, whether they feel well informed and assess any training needs
123
they might identify. 124
Methods
125
An anonymous cross-sectional survey was conducted between April 2016 and July 2017. GPs,
126
practice nurses, smear takers and other clinical and laboratory staff who attended a variety of
127
training events in Auckland District Health Board (DHB) and Waitemata DHB catchment areas were
128
invited to complete the paper-based survey. 124
Methods
125
An anonymous cross-sectional survey was conducted between April 2016 and July 2017. GPs,
126
practice nurses, smear takers and other clinical and laboratory staff who attended a variety of
127
training events in Auckland District Health Board (DHB) and Waitemata DHB catchment areas were
128
invited to complete the paper-based survey. 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand Then, variables
146
with a p value <0.05 were entered into a multivariate model to estimate the association between
147
these predictors and knowledge score, while controlling for potential confounders. 148
Factors affecting self-perceived adequacy of HPV knowledge were also explored, using binary logisti
149
regression. Feeling adequately informed and feeling confident in answering patient questions were
150
converted from 5-point Likert scales to binary variables of yes (strongly agree, agree) and no or
151
undecided (strongly disagree, disagree or undecided) as the dependent variables. Again, predictor
152
variables which were associated with the dependent variables in univariate analyses were entered
153
into a multivariate model to estimate the association between knowledge score and self-perceived
154
adequacy while controlling for potential confounders. 1
Knowledge, attitudes and awareness of the human papillomavirus among health professionals in
2
New Zealand 129
The survey was taken from Patel et al.,12 who had incorporated most of the items from Waller et
130
al.,13 and was adapted by adding back in a question about HPV and HIV/AIDS from Waller et al., and
131
by changing some wording to make the terminology or protocols New Zealand-specific. The overall 129
The survey was taken from Patel et al.,12 who had incorporated most of the items from Waller et
130
al.,13 and was adapted by adding back in a question about HPV and HIV/AIDS from Waller et al., and
131
by changing some wording to make the terminology or protocols New Zealand-specific. The overall 5 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
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bioRxiv preprint 132
face validity of the adapted instrument was confirmed by peer review by members of the
133
immunisation team in Auckland and cervical screening expert doctors/nurse practitioners. 134
The final survey explored four broad categories: demographics and level of experience; HPV
135
knowledge (general HPV knowledge, HPV triage and test of cure (TOC) knowledge and HPV vaccine
136
knowledge), which were assessed using a true, false, don’t know format; and attitudes towards the
137
HPV vaccine and self-perceived adequacy of HPV knowledge, which were assessed using 5-point
138
Likert scales (the survey is publicly available here: osf.io/ub7g2, DOI 10.17605/OSF.IO/UB7G2). 139
Statistical analyses
140
Demographic factors included age, profession and years since HPV training. For analyses, profession
141
was collapsed into four categories (nurse; general practitioner (GP); colposcopy, which included
142
colposcopists and colposcopy nurses; and laboratory staff and other), and years since HPV training
143
was collapsed into 3 categories (never; ≤ 1 year; > 1 year). 144
Factors affecting HPV knowledge were explored using ordinal regression analysis. First, univariate
145
regression analyses were conducted to identify variables associated with knowledge. 132
face validity of the adapted instrument was confirmed by peer review by members of the
133
immunisation team in Auckland and cervical screening expert doctors/nurse practitioners. CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 132
face validity of the adapted instrument was confirmed by peer review by members of the
133
immunisation team in Auckland and cervical screening expert doctors/nurse practitioners. 134
The final survey explored four broad categories: demographics and level of experience; HPV
135
knowledge (general HPV knowledge, HPV triage and test of cure (TOC) knowledge and HPV vaccine
136
knowledge), which were assessed using a true, false, don’t know format; and attitudes towards the
137
HPV vaccine and self-perceived adequacy of HPV knowledge, which were assessed using 5-point
138
Likert scales (the survey is publicly available here: osf.io/ub7g2, DOI 10.17605/OSF.IO/UB7G2). 140
Demographic factors included age, profession and years since HPV training. For analyses, profession
141
was collapsed into four categories (nurse; general practitioner (GP); colposcopy, which included
142
colposcopists and colposcopy nurses; and laboratory staff and other), and years since HPV training
143
was collapsed into 3 categories (never; ≤ 1 year; > 1 year). 140
Demographic factors included age, profession and years since HPV training. For analyses, profession
141
was collapsed into four categories (nurse; general practitioner (GP); colposcopy, which included
142
colposcopists and colposcopy nurses; and laboratory staff and other), and years since HPV training
143
was collapsed into 3 categories (never; ≤ 1 year; > 1 year). 144
Factors affecting HPV knowledge were explored using ordinal regression analysis. First, univariate
145
regression analyses were conducted to identify variables associated with knowledge. Then, variables
146
with a p value <0.05 were entered into a multivariate model to estimate the association between
147
these predictors and knowledge score, while controlling for potential confounders. 144
Factors affecting HPV knowledge were explored using ordinal regression analysis. First, univariate
145
regression analyses were conducted to identify variables associated with knowledge. Then, variables
146
with a p value <0.05 were entered into a multivariate model to estimate the association between
147
these predictors and knowledge score, while controlling for potential confounders. 148
Factors affecting self-perceived adequacy of HPV knowledge were also explored, using binary logistic
149
regression. Feeling adequately informed and feeling confident in answering patient questions were
150
converted from 5-point Likert scales to binary variables of yes (strongly agree, agree) and no or
151
undecided (strongly disagree, disagree or undecided) as the dependent variables. Again, predictor
152
variables which were associated with the dependent variables in univariate analyses were entered
153
into a multivariate model to estimate the association between knowledge score and self-perceived
154
adequacy while controlling for potential confounders. 6 . 155
Results ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 165
General HPV knowledge 166
Out of a maximum knowledge score of 15 (see individual questions in Table 2 and excluding the
167
question about HIV/AIDS), the mean score achieved by participants was 13.2 (standard deviation
168
(SD) 2.0) and the median score was 14 (range 0-15, interquartile range (IQR) 13-15), with 25.2%
169
(N=58) achieving 100%. One individual did not answer any questions correctly. 166
Out of a maximum knowledge score of 15 (see individual questions in Table 2 and excluding the
167
question about HIV/AIDS), the mean score achieved by participants was 13.2 (standard deviation
168
(SD) 2.0) and the median score was 14 (range 0-15, interquartile range (IQR) 13-15), with 25.2%
169
(N=58) achieving 100%. One individual did not answer any questions correctly. 0
Table 2. 155
Results 156
A total of 234 individuals completed the survey. The data for four individuals were removed, as there
157
were large sections that had been left unanswered. The majority (212) were female and one person
158
did not identify a gender. Of the 230 participants, 107 had never taken a smear. For the 123 who
159
had, the years of experience ranged from 0.1 to 42 years (mean 8.7 years, median 6.5 years). Details
160
about age categories, profession and date of most recent training, if any, are presented in Table 1. 156
A total of 234 individuals completed the survey. The data for four individuals were removed, as there
157
were large sections that had been left unanswered. The majority (212) were female and one person
158
did not identify a gender. Of the 230 participants, 107 had never taken a smear. For the 123 who
159
had, the years of experience ranged from 0.1 to 42 years (mean 8.7 years, median 6.5 years). Details
160
about age categories, profession and date of most recent training, if any, are presented in Table 1. 161
Table 1. Participant characteristics. Age
Age bracket N (%)
20-24
8 (3.5)
25-35
45 (19.6)
36-45
51 (22.2)
46-55
63 (27.4)
56-65
52 (22.6)
66-75
Blank
10 (4.3)
1 (0.4)
Profession
Profession N (%)
Registered nurse
150 (65.2)
General practitioner
12 (5.2)
Colposcopy nurse
2 (0.9)
Laboratory staff
25 (10.9)
Not disclosed
11 (4.8)
Colposcopist
2 (0.9)
Other
26 (11.3)
Enrolled nurse
2 (0.9)
HPV
training
Date of last
training
N (%)
Never
99 (43.0)
last 6 months
16 (7.0)
7-12 months
30 (13.0)
13-24 months
35 (15.2)
>2yrs
30 (13.0)
Blank
20 (8.7)
162 162 164 7 7 . CC-BY 4.0 International license
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this version posted May 8, 2018. 155
Results HPV and vaccine knowledge questions
Correct
Response
N (%)
Incorrect
Response
N (%)
Answer “don’t
know” or blank
N (%)
General HPV knowledge questions
HPV can cause cervical cancer
228 (99.1)
1 (0.4)
1 (0.4)
Having many sexual partners increases
the risk of getting HPV
223 (97.0)
5 (2.2)
2 (0.9)
HPV can be passed on during sexual
intercourse
218 (94.8)
2 (0.9)
10 (4.3)
A person could have HPV for many years
without knowing it
217 (94.3)
0 (0)
13 (5.7)
HPV always has visible signs or symptoms
216 (93.9)
5 (2.2)
9 (3.9)
HPV is very rare
215 (93.5)
7 (3.0)
8 (3.5)
There are many types of HPV
214 (93.0)
4 (1.7)
12 (5.2)
Men cannot get HPV
213 (92.6)
8 (3.5)
9 (3.9)
Using condoms reduces the risk of getting
HPV
204 (88.7)
17 (7.4)
9 (3.9)
HPV can be passed on by genital skin-to-
skin contact
203 (88.3)
9 (3.9)
18 (7.8)
HPV can cause genital warts
203 (88.3)
13 (5.7)
14 (6.1)
HPV can be cured with antibiotics
201 (87.4)
14 (6.1)
15 (6.5)
HPV can cause HIV/AIDS1
198 (86.1)
13 (5.7)
19 (8.3)
Most sexually active people will get HPV
at some point in their lives
171 (74.3)
29 (12.6)
30 (13.0)
Having sex at an early age increases the
risk of getting HPV
169 (73.5)
44 (19.1)
17 (7.4)
HPV usually doesn’t need any treatment
147 (63.9)
67 (29.1)
16 (7.0)
HPV Triage and TOC knowledge
questions
If a woman tests positive for HPV she will
definitely get cervical cancer
220 (95.7)
5 (2.2)
5 (2.2)
An HPV test can be done at the same time
as a Smear test
205 (89.1)
7 (3.0)
18 (7.8)
HPV testing is used to indicate if the HPV
vaccine is needed
199 (86.5)
10 (4.3)
21 (9.1)
An HPV test can tell how long you have
190 (82.6)
8 (3.5)
32 (13.9) 8 . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 155
Results CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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bioRxiv preprint 178
of getting HPV (26.5%); Most sexually active people will get HPV at some point in their lives (25.7%). 179
In addition, more than 10% of individuals incorrectly thought (or were not sure) that HPV cannot be
180
passed on by genital skin-to-skin contact, that HPV does not cause genital warts, that using condoms
181
does not reduce the risk of getting HPV and that HPV can be cured with antibiotics. 178
of getting HPV (26.5%); Most sexually active people will get HPV at some point in their lives (25.7%). 179
In addition, more than 10% of individuals incorrectly thought (or were not sure) that HPV cannot be
180
passed on by genital skin-to-skin contact, that HPV does not cause genital warts, that using condoms
181
does not reduce the risk of getting HPV and that HPV can be cured with antibiotics. 182
Following Waller et al.,13 the item about HIV/AIDS was analysed separately from the rest of the
183
questions. In total, 86.1% of respondents correctly identified that HPV does not cause HIV/AIDS. 185
Out of a maximum knowledge score of 10 (see individual questions in Table 2), the mean score
186
achieved by the participants was 7.4 (SD 2.1) and the median score was 8 (range 0-10, IQR 6-9), with
187
9.1% (N=21) achieving 100%. Three individuals had no correct answers. 185
Out of a maximum knowledge score of 10 (see individual questions in Table 2), the mean score
186
achieved by the participants was 7.4 (SD 2.1) and the median score was 8 (range 0-10, IQR 6-9), with
187
9.1% (N=21) achieving 100%. Three individuals had no correct answers. 155
Results It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint had an HPV infection
When you have an HPV test, you get the
results the same day
189 (82.2)
8 (3.5)
33 (14.3)
If an HPV test shows that a woman does
not have HPV her risk of cervical cancer is
low
174 (75.7)
35 (15.2)
21 (9.1)
If cytology and high-risk HPV test are
negative at 12 and 24 post treatment,
they will need require a repeat smear in 3
Years2
171 (74.3)
25 (10.9)
34 (14.8)
If high-risk HPV test is negative at 12 and
24 post treatment they will still require
annual follow up for life2
140 (60.9)
52 (22.6)
38 (16.5)
All cervical samples taken 6 to 12 months
post-treatment can be tested for high-risk
HPV2
102 (44.3)
31 (13.5)
97 (42.2)
All cervical samples showing mild cellular
(ASC-US/LSIL) are tested for high-risk
HPV2
101 (43.9)
85 (37.0)
44 (19.1)
HPV vaccine knowledge questions
The HPV vaccines offer protection against
all sexually transmitted infections
218 (94.8)
3 (1.3)
9 (3.9)
Girls who have had the HPV vaccine do
not need to have smear tests when they
are older
218 (94.8)
2 (0.9)
10 (4.3)
Someone who has had HPV vaccine
cannot develop cervical cancer
205 (89.1)
8 (3.5)
17 (7.4)
The recommended number of HPV
vaccine doses is three23
202 (87.8)
7 (3.0)
21 (9.1)
The HPV vaccines are most effective if
given to people who have never had sex
189 (82.2)
25 (10.9)
16 (7.0)
The HPV vaccines offer protection against
most cervical cancers
185 (80.4)
29 (12.6)
16 (7.0)
The HPV vaccine offers protection against
genital warts
156 (67.8)
41 (17.8)
33 (14.3)
1Question from Waller et al13 and in addition to Patel et al12 The following questions were most often answered incorrectly: HPV usually doesn’t need any 9
177
treatment (36.1% answered incorrectly or weren’t sure); Having sex at an early age increases the risk 9 . CC-BY 4.0 International license
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Results CC-BY 4.0 International license
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bioRxiv preprint 201
HPV vaccine knowledge 202
Out of a maximum knowledge score of 7 (see individual questions in Table 2), the mean score
203
achieved by the participants was 6.0 (SD 1.2) and the median score was 6 (range 0-7, IQR 5-7), with
204
44.3% (N=102) achieving 100%. One individual had no answers correct. 205
The following questions were answered incorrectly most often: The HPV vaccine offers protection
206
against genital warts (32.2% answered incorrectly or weren’t sure); The HPV vaccines offer
207
protection against most cervical cancers (19.6%); The HPV vaccines are most effective if given to
208
people who have never had sex (17.8%). In addition, more than 10% of participants incorrectly
209
thought (or weren’t sure) that the recommended number of HPV vaccine doses was not three and
210
that someone who has had the HPV vaccine cannot develop cervical cancer. 205
The following questions were answered incorrectly most often: The HPV vaccine offers protection
206
against genital warts (32.2% answered incorrectly or weren’t sure); The HPV vaccines offer
207
protection against most cervical cancers (19.6%); The HPV vaccines are most effective if given to
208
people who have never had sex (17.8%). In addition, more than 10% of participants incorrectly
209
thought (or weren’t sure) that the recommended number of HPV vaccine doses was not three and
210
that someone who has had the HPV vaccine cannot develop cervical cancer. 211
Factors influencing level of HPV knowledge 212
Table 3 shows the effect of predictors on the three types of knowledge, both unadjusted (‘crude’)
213
and adjusted for the other covariates (‘full model’). Having ever taken a smear was significantly
214
positively associated with all three types of knowledge when entered into the model as the only
215
predictor. 155
Results 188
The following questions were answered incorrectly most often: All cervical samples showing mild
189
cellular (ASC-US/LSIL) are tested for high-risk HPV (56.1% answered incorrectly or were not sure); All
190
cervical samples taken 6 to 12 months post-treatment can be tested for high-risk HPV (55.7%); If
191
high-risk HPV test is negative at 12 and 24 post treatment they will still require annual follow up for
192
life (39.1%); If cytology and high-risk HPV test are negative at 12 and 24 post treatment, they will
193
require a repeat smear in 3 Years (25.7%). In addition, more than 10% of individuals incorrectly
194
thought (or weren’t sure) that an HPV test can tell how long a person has had an HPV infection; an
195
HPV test cannot be done at the same time as a Smear test; HPV testing is used to indicate if the HPV
196
vaccine is needed; when an HPV test has been done that the results are available the same day; If an
197
HPV test shows that a women does not have HPV her risk of cervical cancer is not low. 188
The following questions were answered incorrectly most often: All cervical samples showing mild
189
cellular (ASC-US/LSIL) are tested for high-risk HPV (56.1% answered incorrectly or were not sure); All
190
cervical samples taken 6 to 12 months post-treatment can be tested for high-risk HPV (55.7%); If
191
high-risk HPV test is negative at 12 and 24 post treatment they will still require annual follow up for
192
life (39.1%); If cytology and high-risk HPV test are negative at 12 and 24 post treatment, they will
193
require a repeat smear in 3 Years (25.7%). In addition, more than 10% of individuals incorrectly
194
thought (or weren’t sure) that an HPV test can tell how long a person has had an HPV infection; an
195
HPV test cannot be done at the same time as a Smear test; HPV testing is used to indicate if the HPV
196
vaccine is needed; when an HPV test has been done that the results are available the same day; If an
197
HPV test shows that a women does not have HPV her risk of cervical cancer is not low. 200 10 . 155
Results However, when adjusting for the other predictors, the association with having ever taken
216
a smear was attenuated for all knowledge types and only remained significantly associated with
217
Triage and TOC knowledge score (where those who had ever taken a smear were 4 times (OR 4.1,
218
95% CI 2.1 – 8.1) more likely to have a higher knowledge score than those who had not taken a
219
smear, p < 0.01). 212
Table 3 shows the effect of predictors on the three types of knowledge, both unadjusted (‘crude’)
213
and adjusted for the other covariates (‘full model’). Having ever taken a smear was significantly
214
positively associated with all three types of knowledge when entered into the model as the only
215
predictor. However, when adjusting for the other predictors, the association with having ever taken
216
a smear was attenuated for all knowledge types and only remained significantly associated with
217
Triage and TOC knowledge score (where those who had ever taken a smear were 4 times (OR 4.1,
218
95% CI 2.1 – 8.1) more likely to have a higher knowledge score than those who had not taken a
219
smear, p < 0.01). 1
220
Years since HPV training was also associated with knowledge level in univariate analysis, where
221
those who had had training (either ≤ 1 year ago or > 1 year ago) were more likely to have a higher
222
knowledge score than those who had never had HPV training, across all types of knowledge. The
223
association was more pronounced for those who had had more recent training (≤ 1 year ago) than
224
for those who had training longer ago (> 1 year ago), as expected. The association was attenuated 1
220
Years since HPV training was also associated with knowledge level in univariate analysis, where
221
those who had had training (either ≤ 1 year ago or > 1 year ago) were more likely to have a higher
222
knowledge score than those who had never had HPV training, across all types of knowledge. The
223
association was more pronounced for those who had had more recent training (≤ 1 year ago) than
224
for those who had training longer ago (> 1 year ago), as expected. The association was attenuated 11 . 155
Results CC-BY 4.0 International license
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when taking into other predictors on knowledge. However, having had HPV training ≤ 1 year ago
226
compared to never remained significantly independently predictive of HPV knowledge score and
227
Triage and TOC knowledge score; having had training > 1 year ago compared to never also remained
228
significantly independently predictive of Triage and TOC knowledge score. Years since training was
229
not predictive of HPV vaccine knowledge score after adjustment for the other predictors. 230
Current role was not associated with HPV knowledge score in univariate or multivariate analyses. 231
However, current role was associated with the Triage and TOC knowledge score, where, even after
232
adjustment for the other predictors, those who worked in colposcopy were 7 times (95% CI 1.0 –
233
47.8) more likely to have a higher knowledge score than nurses (who were the reference category)
234
(p = 0.05). However, the number of colposcopy workers was very small (n=4), so this result should be
235
interpreted with caution. Those that were classed as laboratory staff or other were less likely to have
236
higher Triage and TOC knowledge scores in the univariate analyses, but this association disappeared
237
after adjustment for the other predictors. The laboratory staff and other group were also more likely
238
to have lower HPV vaccine knowledge scores than nurses in both univariate (OR 0.3, 95% CI 0.2 –
239
0.5, p < 0.01) and multivariate (OR 0.4, 95% CI 0.2 – 0.7, p < 0.01) models. 240
The effect of age on knowledge score was explored in univariate analysis as a potential predictor,
241
but was not associated with scores for any of the three knowledge types (data not shown), so was
242
not included in the multivariate analysis. 225
when taking into other predictors on knowledge. 155
Results However, having had HPV training ≤ 1 year ago
226
compared to never remained significantly independently predictive of HPV knowledge score and
227
Triage and TOC knowledge score; having had training > 1 year ago compared to never also remained
228
significantly independently predictive of Triage and TOC knowledge score. Years since training was
229
not predictive of HPV vaccine knowledge score after adjustment for the other predictors. 225
when taking into other predictors on knowledge. However, having had HPV training ≤ 1 year ago
226
compared to never remained significantly independently predictive of HPV knowledge score and
227
Triage and TOC knowledge score; having had training > 1 year ago compared to never also remained
228
significantly independently predictive of Triage and TOC knowledge score. Years since training was
229
not predictive of HPV vaccine knowledge score after adjustment for the other predictors. 230
Current role was not associated with HPV knowledge score in univariate or multivariate analyses. 231
However, current role was associated with the Triage and TOC knowledge score, where, even after
232
adjustment for the other predictors, those who worked in colposcopy were 7 times (95% CI 1.0 –
233
47.8) more likely to have a higher knowledge score than nurses (who were the reference category)
234
(p = 0.05). However, the number of colposcopy workers was very small (n=4), so this result should be
235
interpreted with caution. Those that were classed as laboratory staff or other were less likely to have
236
higher Triage and TOC knowledge scores in the univariate analyses, but this association disappeared
237
after adjustment for the other predictors. The laboratory staff and other group were also more likely
238
to have lower HPV vaccine knowledge scores than nurses in both univariate (OR 0.3, 95% CI 0.2 –
239
0.5, p < 0.01) and multivariate (OR 0.4, 95% CI 0.2 – 0.7, p < 0.01) models. 230
Current role was not associated with HPV knowledge score in univariate or multivariate analyses. 231
However, current role was associated with the Triage and TOC knowledge score, where, even after
232
adjustment for the other predictors, those who worked in colposcopy were 7 times (95% CI 1.0 –
233
47.8) more likely to have a higher knowledge score than nurses (who were the reference category)
234
(p = 0.05). 155
Results However, the number of colposcopy workers was very small (n=4), so this result should be
235
interpreted with caution. Those that were classed as laboratory staff or other were less likely to have
236
higher Triage and TOC knowledge scores in the univariate analyses, but this association disappeared
237
after adjustment for the other predictors. The laboratory staff and other group were also more likely
238
to have lower HPV vaccine knowledge scores than nurses in both univariate (OR 0.3, 95% CI 0.2 –
239
0.5, p < 0.01) and multivariate (OR 0.4, 95% CI 0.2 – 0.7, p < 0.01) models. 240
The effect of age on knowledge score was explored in univariate analysis as a potential predictor,
241
but was not associated with scores for any of the three knowledge types (data not shown), so was
242
not included in the multivariate analysis. 240
The effect of age on knowledge score was explored in univariate analysis as a potential predictor,
241
but was not associated with scores for any of the three knowledge types (data not shown), so was
242
not included in the multivariate analysis. 12 12 . CC-BY 4.0 International license
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bioRxiv preprint Table 3. Ordinal regression of predictors of knowledge Table 3. Ordinal regression of predictors of knowledge Attitudes and adequacy questions
Response N (%)
Strongly
agree
Agree
Undecided
Disagree
Strongly
disagree
Blank
Would you recommend
the HPV vaccine? 179 (77.8)
41 (17.8)
8 (3.5)
0
0
2 (0.9)
Do you think that the
vaccine should be offered
to men/boys as well? 168 (73.0)
47 (20.4)
12 (5.2)
1 (0.4)
0
2 (0.9)
Do you feel adequately
informed about HPV? 30 (13.0)
114 (49.6)
52 (22.6)
27 (11.7)
3 (1.3)
4 (1.7)
Are you able to
confidently answer HPV
related questions asked
by patients? 37 (16.1)
128 (55.7)
41 (17.8)
15 (6.5)
4 (1.7)
5 (2.2)
260
. CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was no
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint Nurse
Ref
Ref
GP
0.52
0.18 - 1.50
0.26
0.58
0.18 - 1.88
0.36
Colposcopy
1.31
0.19 - 9.08
0.79
1.04
0.15 - 7.41
0.97
Laboratory staff and
other
0.29
0.16 - 0.53
<0.01
0.37
0.19 - 0.73
<0.01
251 254
Of all respondents, 95.6% (N=220) agreed or strongly agreed that they would recommend the HPV
255
vaccine (Table 4), with a further 3.5% (N=8) undecided (there were 2 (0.9%) blank responses). 256
In total, 93.4% (N=215) respondents agreed or strongly agreed that men/boys should be offered the
257
vaccine (Table 4), with 5.2% (N=12) undecided and 0.4% (N=1) in disagreement (there were 2 (0.9%)
258
blank responses). 259
Table 4. Attitudes and adequacy questions
Response N (%)
Strongly
agree
Agree
Undecided
Disagree
Strongly
disagree
Blank
Would you recommend
the HPV vaccine? Table 3. Ordinal regression of predictors of knowledge 250 HPV knowledge score
Crude
Full model
OR
95% CI
p
OR
95% CI
p
Ever taken smear
No
Ref
Ref
Yes
2.48
1.54 - 3.99
<0.01
1.88
0.97 - 3.61
0.06
Years since HPV
training
Never
Ref
Ref
≤1 year ago
2.65
1.40 - 5.02
<0.01
2.19
1.05 - 4.56
0.04
>1 year ago
2.14
1.21 - 3.77
<0.01
1.60
0.79 - 3.23
0.19
Current role
Nurse
Ref
Ref
GP
2.03
0.69 - 5.97
0.20
2.17
0.67 - 7.04
0.20
Colposcopy
1.12
0.19 - 6.63
0.90
0.87
0.15 - 5.27
0.88
Laboratory staff and
other
0.64
0.37 - 1.13
0.13
1.18
0.61 - 2.29
0.62
Triage and TOC knowledge score
Crude
Full model
OR
95% CI
p
OR
95% CI
p
Ever taken smear
No
Ref
Ref
Yes
5.77
3.48 - 9.56
<0.01
4.11
2.10 - 8.05
<0.01
Years since HPV
training
Never
Ref
Ref
≤1 year ago
5.03
2.61 - 9.68
<0.01
2.41
1.16 - 5.01
0.02
>1 year ago
5.67
3.12 - 10.32
<0.01
2.30
1.14 - 4.66
0.02
Current role
Nurse
Ref
Ref
GP
0.99
0.35 - 2.81
0.99
0.94
0.30 - 2.95
0.92
Colposcopy
8.59
1.30 - 56.77
0.03
7.02
1.03 - 47.83
0.05
Laboratory staff and
other
0.41
0.23 - 0.72
<0.01
0.97
0.51 - 1.86
0.93
HPV vaccine knowledge score
Crude
Full model
OR
95% CI
p
OR
95% CI
p
Ever taken smear
No
Ref
Ref
Yes
2.31
1.42 - 3.76
<0.01
1.56
0.79 - 3.07
0.20
Years since HPV
training
Never
Ref
Ref
≤1 year ago
2.68
1.36 - 5.26
<0.01
1.96
0.90 - 4.25
0.09
>1 year ago
2.04
1.14 - 3.67
0.02
1.13
0.55 - 2.35
0.74
Current role 13 13 Nurse
Ref
Ref
GP
0.52
0.18 - 1.50
0.26
0.58
0.18 - 1.88
0.36
Colposcopy
1.31
0.19 - 9.08
0.79
1.04
0.15 - 7.41
0.97
Laboratory staff and
other
0.29
0.16 - 0.53
<0.01
0.37
0.19 - 0.73
<0.01
251
252
253
Attitudes towards HPV vaccine
254
Of all respondents, 95.6% (N=220) agreed or strongly agreed that they would recommend the HPV
255
vaccine (Table 4), with a further 3.5% (N=8) undecided (there were 2 (0.9%) blank responses). 256
In total, 93.4% (N=215) respondents agreed or strongly agreed that men/boys should be offered the
257
vaccine (Table 4), with 5.2% (N=12) undecided and 0.4% (N=1) in disagreement (there were 2 (0.9%)
258
blank responses). 259
Table 4. Table 3. Ordinal regression of predictors of knowledge 179 (77.8)
41 (17.8)
8 (3.5)
0
0
2 (0.9)
Do you think that the
vaccine should be offered
to men/boys as well? 168 (73.0)
47 (20.4)
12 (5.2)
1 (0.4)
0
2 (0.9)
Do you feel adequately
informed about HPV? 30 (13.0)
114 (49.6)
52 (22.6)
27 (11.7)
3 (1.3)
4 (1.7)
Are you able to
confidently answer HPV
related questions asked
by patients? 37 (16.1)
128 (55.7)
41 (17.8)
15 (6.5)
4 (1.7)
5 (2.2)
260 260 263 14 14 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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doi:
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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bioRxiv preprint 264
Self-perceived adequacy of HPV knowledge 265
Only 62.6% (N=144) respondents agreed or strongly agreed that they were adequately informed
266
about HPV (see Table 4), 22.6% (N=52) were undecided, while 13.0% (N=30) disagreed or strongly
267
disagreed (there were 4 (1.7%) blank responses). 268
Despite this, 71.8% (N=165) respondents agreed or strongly agreed that they could confidently
269
answer HPV related questions asked by patients (see Table 4). A further 17.8% (N=41) were
270
undecided and 8.2% (N=19) disagreed or strongly disagreed (there were 5 (2.2%) blank responses). 271
Independent t-tests confirmed that the knowledge scores for general HPV knowledge, triage and
272
test of cure knowledge and HPV vaccine knowledge were all significantly higher for those
273
participants who felt they were adequately informed than in those who did not feel they were or
274
who were unsure (p<0.01). The same was found for the question about feeling confident in
275
answering patient questions. 276
Feeling adequately informed and feeling confident in answering patient questions were both related
277
to having ever taken a smear, years since training, and to a much lesser extent, current role (data
278
not shown). Therefore, the relationship between self-perceived adequacy and knowledge was
279
explored further in multivariate analysis using binary logistic regression (Table 5). 276
Feeling adequately informed and feeling confident in answering patient questions were both related
277
to having ever taken a smear, years since training, and to a much lesser extent, current role (data
278
not shown). Therefore, the relationship between self-perceived adequacy and knowledge was
279
explored further in multivariate analysis using binary logistic regression (Table 5). 280
Feeling adequately informed was independently predicted by HPV vaccine knowledge after
281
adjustment for the other predictors, while the association with HPV knowledge and Triage and TOC
282
knowledge disappeared after adjustment. All three knowledge domains independently predicted
283
confidence in answering patient questions, even after adjustment for the other predictors. 280
Feeling adequately informed was independently predicted by HPV vaccine knowledge after
281
adjustment for the other predictors, while the association with HPV knowledge and Triage and TOC
282
knowledge disappeared after adjustment. All three knowledge domains independently predicted
283
confidence in answering patient questions, even after adjustment for the other predictors. 15 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 264
Self-perceived adequacy of HPV knowledge CC-BY 4.0 International license
under a 287
Table 5. Logistic regression of the effect of knowledge on feeling adequately informed / confident
288
in answering patient questions
Feeling adequately informed
Crude
Adjusted for ever taken a smear,
years since training and current role
OR
95% CI
p
OR
95% CI
p
HPV knowledge
1.33
1.12 – 1.58
<0.01
1.15
0.95 – 1.39
0.15
Triage and TOC knowledge
1.31
1.13 – 1.51
<0.01
1.05
0.88 – 1.25
0.61
HPV vaccine knowledge
1.59
1.25 – 2.03
<0.01
1.53
1.14 – 2.04
<0.01
Feeling confident in answering patient questions
Crude
Adjusted for ever taken a smear,
years since training and current role
OR
95% CI
p
OR
95% CI
p
HPV knowledge
1.48
1.23 – 1.78
<0.01
1.27
1.03 – 1.56
0.03
Triage and TOC knowledge
1.68
1.41 – 2.00
<0.01
1.29
1.05 – 1.59
0.02
HPV vaccine knowledge
2.03
1.55 – 2.65
<0.01
2.03
1.41 – 2.93
<0.01
289
290
Improving training
291
Suggestions for how training might be improved were provided by 36 respondents (15.7%). They
292
wanted regular updates, more training sessions and several people felt that online training and
293
other online resources such as research, frequently asked questions and updates would be useful. A
294
request for specific advice that should be provided to parents and simple information sheets for
295
both primary care and patients was suggested. There were also requests to widen the provision of
296
training beyond practice nurses to all healthcare providers, specifically including GPs, independent
297
vaccinators and Public Health Nurses delivering the School Based Immunisation programme. 298
Discussion
299
Although mean knowledge levels for HPV and the HPV vaccine were reasonable (with each subset of
300
questions yielding a mean percentage correct score of between 88% and 85%, respectively), only
301
25.2% and 44.3% of respondents scored 100% in each category, respectively. Knowledge about 264
Self-perceived adequacy of HPV knowledge It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 287
Table 5. Logistic regression of the effect of knowledge on feeling adequately informed / confident
288
in answering patient questions
Feeling adequately informed
Crude
Adjusted for ever taken a smear,
years since training and current role
OR
95% CI
p
OR
95% CI
p
HPV knowledge
1.33
1.12 – 1.58
<0.01
1.15
0.95 – 1.39
0.15
Triage and TOC knowledge
1.31
1.13 – 1.51
<0.01
1.05
0.88 – 1.25
0.61
HPV vaccine knowledge
1.59
1.25 – 2.03
<0.01
1.53
1.14 – 2.04
<0.01
Feeling confident in answering patient questions
Crude
Adjusted for ever taken a smear,
years since training and current role
OR
95% CI
p
OR
95% CI
p
HPV knowledge
1.48
1.23 – 1.78
<0.01
1.27
1.03 – 1.56
0.03
Triage and TOC knowledge
1.68
1.41 – 2.00
<0.01
1.29
1.05 – 1.59
0.02
HPV vaccine knowledge
2.03
1.55 – 2.65
<0.01
2.03
1.41 – 2.93
<0.01
289
290
Improving training
291
Suggestions for how training might be improved were provided by 36 respondents (15.7%). They
292
wanted regular updates, more training sessions and several people felt that online training and
293
other online resources such as research, frequently asked questions and updates would be useful. A
294
request for specific advice that should be provided to parents and simple information sheets for
295
both primary care and patients was suggested. There were also requests to widen the provision of
296
training beyond practice nurses to all healthcare providers, specifically including GPs, independent
297
vaccinators and Public Health Nurses delivering the School Based Immunisation programme. 298
Discussion
299
Although mean knowledge levels for HPV and the HPV vaccine were reasonable (with each subset of
300
questions yielding a mean percentage correct score of between 88% and 85%, respectively), only
301
25.2% and 44.3% of respondents scored 100% in each category, respectively. Knowledge about
302
triage and test of cure was lower (mean percentage correct score of 74%) and only 9 1% of
. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. 305
General HPV knowledge was the highest overall but around a quarter of respondents were unaware
306
that having sex at an early age increases the risk of getting HPV. A quarter of respondents were also
307
unaware that HPV is so common that most sexually active people will be exposed to it in their
308
lifetime. Research has shown that considerable stigma can be attached to a positive HPV test7, 8 and
309
that a lower level of education can be associated with an increase in the negative emotions and
310
stigma that patients experience8. Therefore, it is vital that clinical staff are aware of the widespread
311
nature of the virus so that they can reassure patients and reduce stigma. Furthermore, they need to
312
ensure that when terminology such as ‘high risk HPV type’ is used, that this does not further increase
313
patients’ stigma. A third of participants did not know or were unclear that HPV does not usually need
314
treatment. This lack of knowledge has the potential to spread misinformation and cause confusion
315
among patients as they seek treatment that is not available. Perhaps most worryingly, 14% of
316
respondents either believed that HPV causes HIV/AIDS or were unclear that it did not. 305
General HPV knowledge was the highest overall but around a quarter of respondents were unaware
306
that having sex at an early age increases the risk of getting HPV. A quarter of respondents were also
307
unaware that HPV is so common that most sexually active people will be exposed to it in their
308
lifetime. Research has shown that considerable stigma can be attached to a positive HPV test7, 8 and
309
that a lower level of education can be associated with an increase in the negative emotions and
310
stigma that patients experience8. Therefore, it is vital that clinical staff are aware of the widespread
311
nature of the virus so that they can reassure patients and reduce stigma. Furthermore, they need to
312
ensure that when terminology such as ‘high risk HPV type’ is used, that this does not further increase
313
patients’ stigma. A third of participants did not know or were unclear that HPV does not usually need
314
treatment. This lack of knowledge has the potential to spread misinformation and cause confusion
315
among patients as they seek treatment that is not available. 287
Table 5. Logistic regression of the effect of knowledge on feeling adequately informed / confident
288
in answering patient questions 287
Table 5. Logistic regression of the effect of knowledge on feeling adequately informed / confident
288
in answering patient questions 287
Table 5. Logistic regression of the effect of knowledge on feeling adequately informed / confident
288
in answering patient questions Feeling adequately informed 299
Although mean knowledge levels for HPV and the HPV vaccine were reasonable (with each subset of
300
questions yielding a mean percentage correct score of between 88% and 85%, respectively), only
301
25.2% and 44.3% of respondents scored 100% in each category, respectively. Knowledge about
302
triage and test of cure was lower (mean percentage correct score of 74%) and only 9.1% of 299
Although mean knowledge levels for HPV and the HPV vaccine were reasonable (with each subset of
300
questions yielding a mean percentage correct score of between 88% and 85%, respectively), only
301
25.2% and 44.3% of respondents scored 100% in each category, respectively. Knowledge about
302
triage and test of cure was lower (mean percentage correct score of 74%) and only 9.1% of 16 16 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. 328
HPV vaccine knowledge was better, although there was confusion about the protection provided by
329
the vaccine, with a fifth of respondents unclear that the HPV vaccine protects against most cervical
330
cancers (HPV types 16 and 18 are responsible for 70% of all cervical cancers). A third of respondents
331
were unsure or believed that the vaccine does not protect against genital warts. However, all HPV
332
vaccines used in NZ offer protection against low-risk HPV types 6 and 11, which are responsible for
333
90% of genital wart infections. 328
HPV vaccine knowledge was better, although there was confusion about the protection provided by
329
the vaccine, with a fifth of respondents unclear that the HPV vaccine protects against most cervical
330
cancers (HPV types 16 and 18 are responsible for 70% of all cervical cancers). A third of respondents
331
were unsure or believed that the vaccine does not protect against genital warts. However, all HPV
332
vaccines used in NZ offer protection against low-risk HPV types 6 and 11, which are responsible for
333
90% of genital wart infections. 334
Having ever taken a smear independently predicted Triage and TOC knowledge, as did the number
335
of years since training. The number of years since training also predicted HPV knowledge. This
336
pattern was repeated for HPV vaccine knowledge, however the results were not statistically
337
significant, which could be related to the smaller scale on which HPV vaccine knowledge was
338
measured, resulting in less variability in scores and making it difficult to detect small effects. There
339
was some evidence of current role being associated with knowledge scores, with colposcopy
340
workers being more likely to have higher Triage and TOC scores and laboratory staff and other
341
workers being less likely to have higher HPV vaccine knowledge scores. However, some of these
342
groups had very small numbers. The vast majority of respondents indicated that they would
343
recommend the vaccine and they also favoured vaccinating boys and men, with only one individual
344
indicating they would not recommend vaccinating boys and men. This is particularly reassuring since
345
NZ made the vaccine available to boys from January 2017. 334
Having ever taken a smear independently predicted Triage and TOC knowledge, as did the number
335
of years since training. The number of years since training also predicted HPV knowledge. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. Perhaps most worryingly, 14% of
316
respondents either believed that HPV causes HIV/AIDS or were unclear that it did not. 317
In the triage and test of cure questions, while some questions were generally answered accurately,
318
some questions revealed uncertainty and a lack of understanding of the current guidelines. For
319
example, fewer than half of the respondents knew that not all cervical samples showing mild cellular
320
changes are tested for high-risk HPV (only those for women aged 30 and older are tested for HPV
321
under the current NZ guidelines). In addition, almost a quarter of respondents did not know or were
322
unsure that a negative HPV test means that a woman is at low risk from cervical cancer. This
323
uncertainty is likely to be problematic when primary HPV testing is rolled out. Unlike the cell changes
324
that are screened for currently, primary HPV screening is about identifying a woman’s risk factors. 325
Health professionals will need to be confident in talking with women about what their positive test
326
result means. The test of cure questions were also correctly answered by fewer than three quarters
327
of the respondents. 17 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 328
HPV vaccine knowledge was better, although there was confusion about the protection provided by
329
the vaccine, with a fifth of respondents unclear that the HPV vaccine protects against most cervical
330
cancers (HPV types 16 and 18 are responsible for 70% of all cervical cancers). A third of respondents
331
were unsure or believed that the vaccine does not protect against genital warts. However, all HPV
332
vaccines used in NZ offer protection against low-risk HPV types 6 and 11, which are responsible for
333
90% of genital wart infections. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. This
336
pattern was repeated for HPV vaccine knowledge, however the results were not statistically
337
significant, which could be related to the smaller scale on which HPV vaccine knowledge was
338
measured, resulting in less variability in scores and making it difficult to detect small effects. There
339
was some evidence of current role being associated with knowledge scores, with colposcopy
340
workers being more likely to have higher Triage and TOC scores and laboratory staff and other
341
workers being less likely to have higher HPV vaccine knowledge scores. However, some of these
342
groups had very small numbers. The vast majority of respondents indicated that they would
343
recommend the vaccine and they also favoured vaccinating boys and men, with only one individual
344
indicating they would not recommend vaccinating boys and men. This is particularly reassuring since
345
NZ made the vaccine available to boys from January 2017. 347
Research has been conducted in other countries with HPV vaccination programmes to explore
348
healthcare practitioner knowledge about HPV and the vaccination. The studies reviewed below all
349
reveal that, consistent with our NZ results, health care practitioner knowledge about HPV and the
350
HPV vaccination is frequently incomplete. 347
Research has been conducted in other countries with HPV vaccination programmes to explore
348
healthcare practitioner knowledge about HPV and the vaccination. The studies reviewed below all
349
reveal that, consistent with our NZ results, health care practitioner knowledge about HPV and the
350
HPV vaccination is frequently incomplete. 18 18 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 351
An evaluation of knowledge about HPV and HPV vaccination for GP practice nurses in Leicestershire
352
in the UK, where the vaccination has been administered through the NHS since 2008, found that
353
although general HPV knowledge scores were quite high, there were specific gaps or weaknesses in
354
knowledge12 as identified above. There were also gaps in their knowledge about current NHS
355
processes around HPV triage and test of cure. For example, the role of HPV testing post-treatment
356
(TOC) was misinterpreted, with only 66% acknowledging that all normal, borderline nuclear and
357
mildly dyskaryotic samples are tested for high risk HPV post-treatment. Not all nurses felt
358
adequately informed about HPV and a need to improve the provision of training was identified. 359
Nilsen et al explored knowledge of and attitudes to HPV infection and vaccination among public
360
health nurses and GPS in Northern Norway in 2010, one year after the HPV vaccination was
361
introduced for 12 year-old girls in Norway14. Knowledge of HPV infection, vaccine and cervical cancer
362
was measured with 7 open-ended questions (e.g. what is the lifetime risk of a sexually active person
363
getting HPV?). The percentage of GPs getting each question correct ranged from 26-55% while for
364
the nurses it was 35-86%. Self-reported knowledge was considerably higher than actual knowledge. 365
Only 47% of respondents knew that HPV infection is a necessary cause of cervical cancer. 366
In Malaysia there has been a school-based HPV vaccination programme since 2010. Jeyachelvi et al
367
conducted a survey to explore HPV and HPV vaccination knowledge and attitudes in primary health
368
clinic nurses who run the vaccination program in Kelantan, Malaysia15. Nurses were given 11
369
questions to assess their knowledge. The mean score was 5.37 with the minimum score being 0 and
370
the maximum being 9. No question was answered correctly by more than 87.3% of respondents and 366
In Malaysia there has been a school-based HPV vaccination programme since 2010. Jeyachelvi et al
367
conducted a survey to explore HPV and HPV vaccination knowledge and attitudes in primary health
368
clinic nurses who run the vaccination program in Kelantan, Malaysia15. Nurses were given 11
369
questions to assess their knowledge. The mean score was 5.37 with the minimum score being 0 and
370
the maximum being 9. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. No question was answered correctly by more than 87.3% of respondents and
371
the poorest question (External anogenital warts increase the risk of cancer at the same site where
372
the warts are located. True/False) was answered correctly by only 10.6%. 366
In Malaysia there has been a school-based HPV vaccination programme since 2010. Jeyachelvi et al
367
conducted a survey to explore HPV and HPV vaccination knowledge and attitudes in primary health
368
clinic nurses who run the vaccination program in Kelantan, Malaysia15. Nurses were given 11
369
questions to assess their knowledge. The mean score was 5.37 with the minimum score being 0 and
370
the maximum being 9. No question was answered correctly by more than 87.3% of respondents and
371
the poorest question (External anogenital warts increase the risk of cancer at the same site where
372
the warts are located. True/False) was answered correctly by only 10.6%. 373
Rutten et al conducted a survey exploring clinician knowledge, clinician barriers and perceived
374
parental barriers to HPV vaccination in Rochester US16. They found that greater knowledge of HPV 19 19 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 375
and the HPV vaccination (assessed together using an 11-item scale) was associated with higher rates
376
of HPV vaccination initiation and completion of the 3-dose vaccination schedule, suggesting that
377
knowledge is important in order to effectively promote HPV vaccination in addition to reducing
378
stigmatising attitudes of clinicians as mentioned in the Introduction. 375
and the HPV vaccination (assessed together using an 11-item scale) was associated with higher rates
376
of HPV vaccination initiation and completion of the 3-dose vaccination schedule, suggesting that
377
knowledge is important in order to effectively promote HPV vaccination in addition to reducing
378
stigmatising attitudes of clinicians as mentioned in the Introduction. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. 375
and the HPV vaccination (assessed together using an 11-item scale) was associated with higher rates
376
of HPV vaccination initiation and completion of the 3-dose vaccination schedule, suggesting that
377
knowledge is important in order to effectively promote HPV vaccination in addition to reducing
378
stigmatising attitudes of clinicians as mentioned in the Introduction. 380
Our results suggest that education about HPV and particularly the use of HPV testing in the
381
screening programme and test of cure process is urgently needed to address some worrying gaps in
382
knowledge. This is especially important since further changes to the screening programme are due
383
to be implemented, with draft primary HPV screening guidelines recently out for consultation17. As
384
other countries also start to roll out primary HPV screening, the success of primary screening
385
engagement in NZ and the rest of the world may well rest upon the level of knowledge of those
386
people responsible for implementing it. 387
The need for education indicated by the knowledge scores was further reinforced by the fact that
388
over a third of respondents did not agree that they felt adequately informed about HPV and that
389
being adequately informed and feeling confident in responding to patients’ questions were both
390
associated with knowledge. Suggestions for training were proposed by some of the respondents. 391
One promising suggestion, which was also proposed by UK practice nurses,12 was for online training. 392
This would provide a low-cost way to update changes to the vaccination and/or screening
393
programmes and guidelines in a format that would be easily accessible to many staff whilst requiring
394
relatively little time commitment to complete. HPV vaccination online training was developed by the
395
Immunisation Advisory Committee (IMAC) and was released in August 201718. This may address
396
some of the knowledge issues associated with vaccination identified in this study, but additional
397
online training regarding screening and test of cure is needed. 20 20 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 398
Our survey is the first to be conducted in NZ that explores health practitioner knowledge and
399
understanding about HPV, the vaccine and the role of HPV testing in the cervical screening
400
programme and it contributes to the international picture about HPV knowledge that is emerging. It
401
is evident from our findings and those from other countries, that more education is required to
402
ensure that misinformation, stigma and widening inequalities do not inadvertently result from
403
interactions between health practitioners and the public. 398
Our survey is the first to be conducted in NZ that explores health practitioner knowledge and
399
understanding about HPV, the vaccine and the role of HPV testing in the cervical screening
400
programme and it contributes to the international picture about HPV knowledge that is emerging. It
401
is evident from our findings and those from other countries, that more education is required to
402
ensure that misinformation, stigma and widening inequalities do not inadvertently result from
403
interactions between health practitioners and the public. 21 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 404
Data Availability Statement
405
The information sheet, survey and data are deposited publicly on the Open Science Framework
406
website and can be accessed here: osf.io/ub7g2, DOI 10.17605/OSF.IO/UB7G2. 429
References References 429 429
References
430
1. Ministry of Health (2017). Immunisation Handbook. Wellington: Ministry of Health. 431
https://www.health.govt.nz/system/files/documents/publications/immunisation-handbook-
432
2017-may17-v3.pdf Downloaded on 4th January 2018. 433
2. HPV Information Centre (2017). Human Papillomavirus and Related Diseases Report NEW
434
ZEALAND. http://www.hpvcentre.net/statistics/reports/NZL.pdf. Downloaded on 8th June
435
2017. 436
3. National Screening Unit (2017). National Cervical Screenign Programme. 437
https://www.nsu.govt.nz/health-professionals/national-cervical-screening-
438
programme/cervical-screening-coverage/dhb-quarte-21. Downloaded on 12th January 2018. 439
4. National Screening Unit (2008). Guidelines for Cervical Screening in New Zealand:
440
Incorporating the management of women with abnormal cervical smears. Wellington:
441
National Screening Unit, Ministry of Health. 442
5. National Screening Unit (2016). Primary HPV Screening. https://www.nsu.govt.nz/health-
443
professionals/national-cervical-screening-programme/primary-hpv-screening. Downloaded
444
on 8th June 2017. 445
6. Chelimo C, Wouldes TA, Cameron LD. Human papillomavirus (HPV) vaccine acceptance and
446
perceived effectiveness, and HPV infection concern among young New Zealand university
447
students. Sexual health. 2010 Sep 9;7(3):394-6. 448
7. McCaffery K, Waller J, Nazroo J, Wardle J. Social and psychological impact of HPV testing in
449
cervical screening: a qualitative study. Sex Transm Infect. 2006 Apr 1;82(2):169-74. 450
8. Daley EM, Vamos CA, Wheldon CW, Kolar SK, Baker EA. Negative emotions and stigma
451
associated with a human papillomavirus test result: A comparison between human
452
papillomavirus–positive men and women. Journal of health psychology. 2015
453
Aug;20(8):1073-82. 454
9. Kwan TT, Lo SS, Tam KF, Chan KK, Ngan HY. Assessment of knowledge and stigmatizing
455
attitudes related to human papillomavirus among Hong Kong Chinese healthcare providers. 456
International Journal of Gynecology & Obstetrics. 2012 Jan 31;116(1):52-6. 457
10. Thomas DR, Arlidge B, Arroll B, Elder H (2010) General practitioners' views about diagnosing
458
and treating depression in Maori and non-Maori patients. Journal of Primary Health Care 2,
459
2010, 208-216. 460
11. Henninger J. Human papillomavirus and papillomavirus vaccines: knowledge, attitudes and
461
intentions of general practitioners and practice nurses in Christchurch. Journal of primary
462
health care. 2009;1(4):278-85. 463
12. Patel H, Austin-Smith K, Sherman SM, Tincello D, Moss EL. Knowledge, attitudes and
464
awareness of the human papillomavirus amongst primary care practice nurses: an
465
evaluation of current training in England. Journal of Public Health. 2016 Jul 1:1-8. 466
13. Waller J, Ostini R, Marlow LA, McCaffery K, Zimet G. Validation of a measure of knowledge
467
about human papillomavirus (HPV) using item response theory and classical test theory. 468
Preventive medicine. 2013 Jan 31;56(1):35-40. 469
14. 429
References Nilsen K, Aasland OG, Klouman E. The HPV vaccine: knowledge and attitudes among public
470
health nurses and general practitioners in Northern Norway after introduction of the vaccine 30
1. Ministry of Health (2017). Immunisation Handbook. Wellington: Ministry of Health. 31
https://www.health.govt.nz/system/files/documents/publications/immunisation-handbook-
32
2017-may17-v3.pdf Downloaded on 4th January 2018. 2. HPV Information Centre (2017). Human Papillomavirus and Related Diseases Report NEW
ZEALAND. http://www.hpvcentre.net/statistics/reports/NZL.pdf. Downloaded on 8th June
2017. 3. National Screening Unit (2017). National Cervical Screenign Programme. https://www.nsu.govt.nz/health-professionals/national-cervical-screening-
programme/cervical-screening-coverage/dhb-quarte-21. Downloaded on 12th January 2018. 4. National Screening Unit (2008). Guidelines for Cervical Screening in New Zealand:
Incorporating the management of women with abnormal cervical smears. Wellington:
National Screening Unit, Ministry of Health. 4. National Screening Unit (2008). Guidelines for Cervical Screening in New Zealand:
Incorporating the management of women with abnormal cervical smears. Wellington:
National Screening Unit, Ministry of Health. 5. National Screening Unit (2016). Primary HPV Screening. https://www.nsu.govt.nz/health-
professionals/national-cervical-screening-programme/primary-hpv-screening. Downloaded
on 8th June 2017. 6. Chelimo C, Wouldes TA, Cameron LD. Human papillomavirus (HPV) vaccine acceptance and
perceived effectiveness, and HPV infection concern among young New Zealand university
students. Sexual health. 2010 Sep 9;7(3):394-6. 7. McCaffery K, Waller J, Nazroo J, Wardle J. Social and psychological impact of HPV testing in
cervical screening: a qualitative study. Sex Transm Infect. 2006 Apr 1;82(2):169-74. 8. Daley EM, Vamos CA, Wheldon CW, Kolar SK, Baker EA. Negative emotions and stigma
associated with a human papillomavirus test result: A comparison between human
papillomavirus–positive men and women. Journal of health psychology. 2015
Aug;20(8):1073-82. 8. Daley EM, Vamos CA, Wheldon CW, Kolar SK, Baker EA. Negative emotions and stigma
associated with a human papillomavirus test result: A comparison between human
papillomavirus–positive men and women. Journal of health psychology. 2015
Aug;20(8):1073-82. 9. Kwan TT, Lo SS, Tam KF, Chan KK, Ngan HY. Assessment of knowledge and stigmatizing
attitudes related to human papillomavirus among Hong Kong Chinese healthcare providers. International Journal of Gynecology & Obstetrics. 2012 Jan 31;116(1):52-6. 9. Kwan TT, Lo SS, Tam KF, Chan KK, Ngan HY. Assessment of knowledge and stigmatizing
attitudes related to human papillomavirus among Hong Kong Chinese healthcare providers. International Journal of Gynecology & Obstetrics. 2012 Jan 31;116(1):52-6. 10. Thomas DR, Arlidge B, Arroll B, Elder H (2010) General practitioners' views about diagnosing
and treating depression in Maori and non-Maori patients. Journal of Primary Health Care 2,
2010, 208-216. 10. 303
individuals correctly answered all the answers in this section. Some questions in each section
304
revealed worrying knowledge gaps. 407
408
Acknowledgements
409
We would like to thank the following individuals for assistance with data collection:
410
Jane Grant – Cervical Screening Nurse Specialist, Metro Auckland Cervical Screening Coordination
411
Service, Auckland and Waitemata DHBs
412
Lucina Kaukau - Cervical Screening Nurse Specialist, HPV Self-Sampling Feasibility study for Maori
413
women, Research Nurse. 414
Lisbeth Alley – Programme Manager, Immunisation. Auckland and Waitemata DHBs
415
Pam Hewlett – Women’s Health Manager. Auckland and Waitemata DHBs
416
417
418
Author Contributions
419
Conceived and designed the study: SS, CB. Collected the data: KB, CB, HD. Analyzed the data: SS, HD
420
Wrote the paper: SS, KB, HD, HP, EM, JD, CB. 421
422
Ethical Approval
423
Ethics approval was granted by the Massey University Ethics Committee 4000015595
424
The project was registered with Waitemata DHB localities (Reference number RM13518). Both
425
Waitemata and Auckland DHB confirmed that locality authorisation was not required as the researc 22 22 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint 429
References Thomas DR, Arlidge B, Arroll B, Elder H (2010) General practitioners' views about diagnosing
and treating depression in Maori and non-Maori patients. Journal of Primary Health Care 2,
2010, 208-216. 11. Henninger J. Human papillomavirus and papillomavirus vaccines: knowledge, attitudes and
intentions of general practitioners and practice nurses in Christchurch. Journal of primary
health care. 2009;1(4):278-85. 11. Henninger J. Human papillomavirus and papillomavirus vaccines: knowledge, attitudes and
intentions of general practitioners and practice nurses in Christchurch. Journal of primary
health care. 2009;1(4):278-85. 12. Patel H, Austin-Smith K, Sherman SM, Tincello D, Moss EL. Knowledge, attitudes and
awareness of the human papillomavirus amongst primary care practice nurses: an
evaluation of current training in England. Journal of Public Health. 2016 Jul 1:1-8. 12. Patel H, Austin-Smith K, Sherman SM, Tincello D, Moss EL. Knowledge, attitudes and
awareness of the human papillomavirus amongst primary care practice nurses: an
evaluation of current training in England. Journal of Public Health. 2016 Jul 1:1-8. 13. Waller J, Ostini R, Marlow LA, McCaffery K, Zimet G. Validation of a measure of knowledge
about human papillomavirus (HPV) using item response theory and classical test theory. Preventive medicine. 2013 Jan 31;56(1):35-40. 14. Nilsen K, Aasland OG, Klouman E. The HPV vaccine: knowledge and attitudes among public
health nurses and general practitioners in Northern Norway after introduction of the vaccine 14. Nilsen K, Aasland OG, Klouman E. The HPV vaccine: knowledge and attitudes among public
health nurses and general practitioners in Northern Norway after introduction of the vaccine 23 23 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted May 8, 2018. ;
https://doi.org/10.1101/317248
doi:
bioRxiv preprint in the school-based vaccination programme. Scandinavian journal of primary health care. 2017 Oct 2;35(4):387-95. in the school-based vaccination programme. Scandinavian journal of primary health care. 2017 Oct 2;35(4):387-95. in the school-based vaccination programme. Scandinavian journal of primary health care. 2017 Oct 2;35(4):387-95. 15. Jeyachelvi K, Juwita S, Norwati D. 429
References Human papillomavirus Infection and its Vaccines:
Knowledge and Attitudes of Primary Health Clinic Nurses in Kelantan, Malaysia. Asian Pacific
Journal of Cancer Prevention. 2016;17(8):3983-8. 15. Jeyachelvi K, Juwita S, Norwati D. Human papillomavirus Infection and its Vaccines:
Knowledge and Attitudes of Primary Health Clinic Nurses in Kelantan, Malaysia. Asian Pacific
Journal of Cancer Prevention. 2016;17(8):3983-8. 16. Rutten LJ, Sauver JL, Beebe TJ, Wilson PM, Jacobson DJ, Fan C, Breitkopf CR, Vadaparampil
ST, Jacobson RM. Clinician knowledge, clinician barriers, and perceived parental barriers
regarding human papillomavirus vaccination: Association with initiation and completion
rates. Vaccine. 2017 Jan 3;35(1):164-9. 16. Rutten LJ, Sauver JL, Beebe TJ, Wilson PM, Jacobson DJ, Fan C, Breitkopf CR, Vadaparampil
ST, Jacobson RM. Clinician knowledge, clinician barriers, and perceived parental barriers
regarding human papillomavirus vaccination: Association with initiation and completion
rates. Vaccine. 2017 Jan 3;35(1):164-9. 17. National Screening Unit (2017). Updated Guidelines for Cervical Screening in New Zealand. https://www.nsu.govt.nz/health-professionals/national-cervical-screening-
programme/cervical-screening-guidelines/updated. Downloaded on 4th January 2018. 17. National Screening Unit (2017). Updated Guidelines for Cervical Screening in New Zealand. https://www.nsu.govt.nz/health-professionals/national-cervical-screening-
programme/cervical-screening-guidelines/updated. Downloaded on 4th January 2018. 18. The Immunisation Advisory Centre (2017). HPV Vaccination Module. https://www.immune.org.nz/health-professionals/education-training/hpv-vaccination-
module. Downloaded on 4th January 2018. 18. The Immunisation Advisory Centre (2017). HPV Vaccination Module. https://www.immune.org.nz/health-professionals/education-training/hpv-vaccination-
module. Downloaded on 4th January 2018. 24 24
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Numerical Study of Using FRP and Steel Rebars in Simply Supported Prestressed Concrete Beams with External FRP Tendons
|
Polymers
| 2,020
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cc-by
| 12,154
|
Numerical Study of Using FRP and Steel Rebars in
Simply Supported Prestressed Concrete Beams with
External FRP Tendons Miao Pang 1, Zhangxiang Li 2 and Tiejiong Lou 2,* Miao Pang 1, Zhangxiang Li 2 and Tiejiong Lou 2,*
1
Department of Civil Engineering, Zhejiang University, Hangzhou 310058, China; pm@zju.edu.cn
2
Hubei Key Laboratory of Roadway Bridge & Structure Engineering, Wuhan University of Technology,
Wuhan 430070, China; whutlizx@163.com
*
Correspondence: tjlou@whut.edu.cn Miao Pang 1, Zhangxiang Li 2 and Tiejiong Lou 2,*
1
Department of Civil Engineering, Zhejiang University, Hangzhou 310058, China; pm@zju.edu.cn
2
Hubei Key Laboratory of Roadway Bridge & Structure Engineering, Wuhan University of Technology,
Wuhan 430070, China; whutlizx@163.com
*
Correspondence: tjlou@whut.edu.cn Received: 2 November 2020; Accepted: 18 November 2020; Published: 24 November 2020 Received: 2 November 2020; Accepted: 18 November 2020; Published: 24 November 2020 Abstract: This study aimed at examining the feasibility of using fiber-reinforced polymer (FRP) rebars
instead of steel ones in prestressed concrete beams (PCBs) with external FRP tendons. By applying
an experimentally validated program, numerical tests were performed on simply supported PCBs,
with investigated variables including rebars’ type and area. Three types of rebars were considered,
i.e., carbon, glass FRPs (CFRP, GFRP), and reinforcing steel. The ratio of tensile rebars ranged from
0.22% to 2.16%. The results indicated that the beams with CFRP rebars exhibited better crack mode
and higher ultimate load than the beams with GFRP or steel rebars. GFRP rebars led to considerably
higher ultimate deflection and tendon stress increment than steel rebars. In addition, several models
for calculating the ultimate stress in unbonded tendons were assessed. An analytical model was also
proposed to predict the tendon stress at ultimate and flexural strength in externally PCBs with steel
and FRP rebars. The model predictions agreed well with the numerical results. Keywords: fiber-reinforced polymer; beams; rebars; flexural strength; structural analysis Polymers 2020, 12, 2773; doi:10.3390/polym12122773 polymers polymers 1. Introduction Steel corrosion would lead to the deterioration of reinforced or prestressed concrete beams
(RCBs or PCBs) [1]. An effective solution to this problem is to replace steel reinforcement with
fiber-reinforced polymer (FRP). In addition to their non-corrosive property, FRP composites are
high-strength, light-weight, and nonmagnetic. These composite materials are increasingly used for
strengthening structural elements [2–8]. Different types of FRP composites are available, such as aramid,
basalt, carbon, and glass FRPs (AFRP, BFRP, CFRP, and GFRP). Unlike steel reinforcement with ductile
characteristics, FRP composites are linear-elastic materials without yielding [9,10]. In addition, the FRP
modulus of elasticity is usually lower than that of steel reinforcement [9,10]. Hence, some concerns on
the use of FRP reinforcement instead of steel reinforcement may arise, e.g., ductility and deflection
issues due to the lack of yielding and low modulus of elasticity for FRP composites. The bond
performance of FRP reinforcement in concrete under environmental exposure is also a concern. It was
generally demonstrated that harsh environments have adverse effects on FRP reinforcement’s bond
durability [11–13]. External prestressing has been widely used in the retrofit or construction of various concrete
structures. Extensive research has been conducted concerning PCBs with external FRP tendons. Grace and Abdel-Sayed [14] carried out tests on four specimens to examine the behavior of PCBs
with combined externally unbonded and internally bonded CFRP tendons. Their test results
showed that the ductility could be improved by strengthening using draped external CFRP tendons. Polymers 2020, 12, 2773; doi:10.3390/polym12122773 www.mdpi.com/journal/polymers www.mdpi.com/journal/polymers 2 of 16
several
ing the Polymers 2020, 12, 2773
Beeby [15] presente
factors influencing Ghallab and Beeby [15] presented the test results of 16 PCBs with external AFRP tendons. They studied
several factors influencing the ultimate stress in external FRP or steel tendons and proposed modifying
the BS8110 equation. Abdel Aziz et al. [16] developed an analytical method to predict the load-deflection
behavior of PCBs with external CFRP tendons. Wang et al. [17] tested four specimens, including 3 PCBs
with external BFRP tendons and one control reinforced concrete beam (RCBs). Their study showed that
using BFRP composites as external tendons for strengthening RCBs can effectively improve structural
performance. Zhu et al. [18] experimentally investigated the influence of bending angle and radius on
external FRP tendons’ performance. They concluded that FRP tendons’ load capacities are reduced
as the bending angle increases or the bending radius decreases. 1. Introduction Both numerical and experimental
investigations [19,20] showed that behaviors of PCBs with external CFRP and steel tendons are similar. behavior of PCBs with external CFRP tendons. Wang et al. [17] tested four specimens, including 3
PCBs with external BFRP tendons and one control reinforced concrete beam (RCBs). Their study
showed that using BFRP composites as external tendons for strengthening RCBs can effectively
improve structural performance. Zhu et al. [18] experimentally investigated the influence of bending
angle and radius on external FRP tendons’ performance. They concluded that FRP tendons’ load
capacities are reduced as the bending angle increases or the bending radius decreases. Both numerical
and experimental investigations [19,20] showed that behaviors of PCBs with external CFRP and steel
tendons are similar. In PCBs with external tendons, some bonded rebars need to be provided to avoid behaving as a
tied arch and limit the crack width and spacing [21]. Previous studies demonstrated that externally
PCB
ith
t b
d d
b
hibit i
ifi
t
k
t
ti
hi h
b
ff
ti
l
li
d In PCBs with external tendons, some bonded rebars need to be provided to avoid behaving as a
tied arch and limit the crack width and spacing [21]. Previous studies demonstrated that externally
PCBs without bonded rebars exhibit significant crack concentration, which can be effectively relieved
by providing bonded rebars [19]. In existing works on PCBs with external FRP tendons, steel rebars
were usually provided. The performance of conventional RCBs with FRP rebars instead of steel ones
has been extensively studied [22–30]. However, the findings obtained from the study of RCBs with
FRP/steel rebars may not be valid for PCBs with external FRP tendons as the latter is a different
structural system from the former due to external prestressing. The influence of using FRP rebars
instead of steel ones on the behavior of PCBs with external FRP tendons has yet to be investigated. PCBs without bonded rebars exhibit significant crack concentration, which can be effectively relieved
by providing bonded rebars [19]. In existing works on PCBs with external FRP tendons, steel rebars
were usually provided. The performance of conventional RCBs with FRP rebars instead of steel ones
has been extensively studied [22–30]. 1. Introduction However, the findings obtained from the study of RCBs with
FRP/steel rebars may not be valid for PCBs with external FRP tendons as the latter is a different
structural system from the former due to external prestressing. The influence of using FRP rebars
instead of steel ones on the behavior of PCBs with external FRP tendons has yet to be investigated. This paper presents a numerical and analytical study to evaluate the feasibility of providing FRP
rebars instead of steel ones in PCBs with external FRP tendons. A comprehensive numerical
l
d
d
l
d PCB
ll
l d
d This paper presents a numerical and analytical study to evaluate the feasibility of providing
FRP rebars instead of steel ones in PCBs with external FRP tendons. A comprehensive numerical
evaluation was conducted on simply supported PCBs using an experimentally validated program,
where investigated variables included the type and amount of bonded rebars. An analytical model
was also proposed to predict the tendon stress at ultimate and flexural strength in externally PCBs
with steel and FRP rebars. evaluation was conducted on simply supported PCBs using an experimentally validated program,
where investigated variables included the type and amount of bonded rebars. An analytical model
was also proposed to predict the tendon stress at ultimate and flexural strength in externally PCBs
with steel and FRP rebars. 2. Numerical Program and Verification 2.1. Numerical Program
A numerical progr
finite element assumes A numerical program employing the Euler–Bernoulli beam element was developed [31]. The finite
element assumes negligible shear deformation, small strains with large displacements, and moderate
rotations. The plane section hypothesis was also adopted. The element has six degrees of freedom as
shown in Figure 1, where u, v, and θ are the axial, transverse displacements, and rotation, respectively,
and the subscripts 1 and 2 refer to the two end nodes. A linear variation for u and a cubic variation for
v along the x-axis is assumed. By applying an updated Lagrangian approach, the governing equations
were developed, where the stiffness matrix consists of the material and geometric stiffness matrices. The effects of external tendons are transformed into equivalent loads acting on the finite elements. A detailed formulation of the numerical program can be seen in Reference [31]. g g
g
p
moderate rotations. The plane section hypothesis was also adopted. The element has six degrees of
freedom as shown in Figure 1, where u, v, and θ are the axial, transverse displacements, and rotation,
respectively, and the subscripts 1 and 2 refer to the two end nodes. A linear variation for u and a
cubic variation for v along the x-axis is assumed. By applying an updated Lagrangian approach, the
governing equations were developed, where the stiffness matrix consists of the material and
geometric stiffness matrices. The effects of external tendons are transformed into equivalent loads
acting on the finite elements. A detailed formulation of the numerical program can be seen in
Reference [31]. Figure 1. Beam element, u, v, and θ are the axial, transverse displacements, and rotation, respectively. Figure 1. Beam element, u, v, and θ are the axial, transverse displacements, and rotation, respectively. 3 of 16
re 2. The
adopted Polymers 2020, 12, 2773
The stress s
stress–strain relat The stress–strain relationships for materials are demonstrated schematically in Figure 2. The stress–strain relationship for concrete in compression recommended in Eurocode 2 [32] was
adopted. It is shown in Figure 2a and expressed by σc/ fcm = (kη −η2)/[1 + (k −2)η], where η = εc/εc0;
σc and εc are the concrete stress and strain, respectively; εc0(%) = 0.7 f 0.31
cm
< 2.8; fcm = fck + 8 in
which fck is the concrete characteristic cylinder compressive strength, in MPa; k = 1.05Ecεc0/ fcm;
and Ec = 22( fcm/10)0.3, in GPa. 2.1. Numerical Program
A numerical progr
finite element assumes As shown in Figure 2b, a bilinear elastic and strain-softening law is
adopted for concrete in tension. FRP prestressing tendons are linearly elastic, as shown in Figure 2c. FRP rebars are also linearly elastic, while steel rebars are elastic-perfectly plastic, as shown in Figure 2d. It is shown in Figure 2a and expressed by
(
) [
(
) ]
c
cm
f
, where
0
c
c
;
c
and
c
are the concrete stress and strain, respectively;
0.31
0(
)
0.7
2.8
c
cm
f
‰
8
cm
ck
f
f
in which
ckf
is the concrete characteristic cylinder compressive strength, in MPa;
0
1.05
/
c
c
cm
k
E
f
; and
0.3
22(
/10)
c
cm
E
f
, in GPa. As shown in Figure 2b, a bilinear elastic and
strain-softening law is adopted for concrete in tension. FRP prestressing tendons are linearly elastic
as shown in Figure 2c. FRP rebars are also linearly elastic, while steel rebars are elastic-perfectly
plastic, as shown in Figure 2d. Figure 2. Stress–strain curves of materials. (a) concrete in compression recommended in Eurocode 2
[32]; (b) concrete in tension; (c) Fiber-reinforced polymer (FRP) tendons; (d) FRP and steel rebars. Figure 2. Stress–strain curves of materials. (a) concrete in compression recommended in Eurocode 2 [32];
(b) concrete in tension; (c) Fiber-reinforced polymer (FRP) tendons; (d) FRP and steel rebars. Figure 2. Stress–strain curves of materials. (a) concrete in compression recommended in Eurocode 2
[32]; (b) concrete in tension; (c) Fiber-reinforced polymer (FRP) tendons; (d) FRP and steel rebars. Figure 2. Stress–strain curves of materials. (a) concrete in compression recommended in Eurocode 2 [32];
(b) concrete in tension; (c) Fiber-reinforced polymer (FRP) tendons; (d) FRP and steel rebars. A typical analysis consists of two steps: load-control analysis at prestress transfer an
displacement-control analysis at loading up to the ultimate. The above-mentioned numeric
procedure can simulate the nonlinear behavior of PCBs with combined external FRP tendons an
internal steel/FRP rebars under short-term loading up to failure. A typical analysis consists of two steps:
load-control analysis at prestress transfer and
displacement-control analysis at loading up to the ultimate. The above-mentioned numerical procedure
can simulate the nonlinear behavior of PCBs with combined external FRP tendons and internal steel/FRP
rebars under short-term loading up to failure. 2 2 Verification with Experimental Result
2.2. Verification with Experimental Results e ifi a io
i
E pe i e
a
esu s
Bennitz et al. [20] conducted a series of laboratory tests to investigate the flexural behavior o
PCBs with external CFRP tendons. The main test variables were the tendon eccentricity, effective
prestress, and deviator arrangement. Four of the beams (B2, B3, B6, and B7) were analyzed herein to
verify the proposed model. The beams were of T-shaped section, simply supported over a span of 3.0
m, and were under third-point loading up to failure, as shown in Figure 3. The beams were post
tensioned with two horizontal straight external CFRP tendons (8 mm in diameter each) with an initia
effective depth of 200 mm. Beams B2 and B3 were provided with one deviator at the midspan, while
Bennitz et al. [20] conducted a series of laboratory tests to investigate the flexural behavior of
PCBs with external CFRP tendons. The main test variables were the tendon eccentricity, effective
prestress, and deviator arrangement. Four of the beams (B2, B3, B6, and B7) were analyzed herein
to verify the proposed model. The beams were of T-shaped section, simply supported over a span
of 3.0 m, and were under third-point loading up to failure, as shown in Figure 3. The beams were
post-tensioned with two horizontal straight external CFRP tendons (8 mm in diameter each) with an
initial effective depth of 200 mm. Beams B2 and B3 were provided with one deviator at the midspan,
while Beams B6 and B7 had no deviator. The bottom reinforcement consisted of two deformed steel
rebars, each having 16 mm in diameter, while the top reinforcement consisted of four deformed steel
rebars, each having 8 mm in diameter. The material parameters of the specimens are given in Table 1. Polymers 2020, 12, 2773
each having 16 mm
h h
each having 8 mm 4 of 16
rebars,
1. Figure 3. Prestressed concrete beam (PCB) specimens with external CFRP tendons [20], As,
'
sA and
A are the areas of tensile compressive steel rebars and prestressing tendons respectively
Figure 3. Prestressed concrete beam (PCB) specimens with external CFRP tendons [20], As, A′s and Ap
are the areas of tensile, compressive steel rebars and prestressing tendons, respectively. Figure 3. Prestressed concrete beam (PCB) specimens with external CFRP tendons [20], As,
'
sA and
Ap are the areas of tensile, compressive steel rebars and prestressing tendons, respectively. 'A Figure 3. 2 2 Verification with Experimental Result
2.2. Verification with Experimental Results Prestressed concrete beam (PCB) specimens with external CFRP tendons [20], As,
'
sA and
A are the areas of tensile compressive steel rebars and prestressing tendons respectively
Figure 3. Prestressed concrete beam (PCB) specimens with external CFRP tendons [20], As, A′s and Ap
are the areas of tensile, compressive steel rebars and prestressing tendons, respectively. Figure 3. Prestressed concrete beam (PCB) specimens with external CFRP tendons [20], As,
sA and
Ap are the areas of tensile, compressive steel rebars and prestressing tendons, respectively. According to the proposed analysis, crushing failure occurs at the midspan, after significant
yielding of tensile rebars (formation of a plastic hinge with sufficient rotation). This failure mode is
consistent with the experimental observation. The load-displacement curves and the tendon force’s
development generated by the proposed analysis are compared to the experimental data in Figure 4. It was observed that although the proposed analysis overestimated the tendon force at ultimate in
some specimens (B2, B3, and B7), the numerical results were generally in satisfactory agreement with
the experimental ones over the entire ranges of loading
According to the proposed analysis, crushing failure occurs at the midspan, after significant
yielding of tensile rebars (formation of a plastic hinge with sufficient rotation). This failure mode is
consistent with the experimental observation. The load-displacement curves and the tendon force’s
development generated by the proposed analysis are compared to the experimental data in Figure 4. It was observed that although the proposed analysis overestimated the tendon force at ultimate in
some specimens (B2, B3, and B7), the numerical results were generally in satisfactory agreement with
the experimental ones over the entire ranges of loading. According to the proposed analysis, crushing failure occurs at the midspan, after significant
yielding of tensile rebars (formation of a plastic hinge with sufficient rotation). This failure mode is
consistent with the experimental observation. The load-displacement curves and the tendon force’s
development generated by the proposed analysis are compared to the experimental data in Figure 4. It was observed that although the proposed analysis overestimated the tendon force at ultimate in
some specimens (B2, B3, and B7), the numerical results were generally in satisfactory agreement with
the experimental ones over the entire ranges of loading. p
g
g
Figure 4. Verification with experimental results. (a) load versus midspan deflection; (b) load versus
tendon force. Figure 4. Verification with experimental results. 2 2 Verification with Experimental Result
2.2. Verification with Experimental Results (a) load versus midspan deflection; (b) load versus
tendon force. Polymers 2020, 12, 2773 5 of 16
ncrete Table 1. Material parameters for specimens [20]. Pa)
(mm2)
(MPa)
(GPa)
(mm2)
(GPa)
72
201
510
187
100 5
158 Table 1. Material parameters for specimens [20]. Pa)
(mm2)
(MPa)
(GPa)
(mm )
(GPa)
72
201
510
187
100 5
158 Beam
Steel Rebars
FRP Tendons
Concrete
As
(mm2)
fy
(MPa)
Es
(GPa)
A
′
s
(mm2)
f
′
y
(MPa)
E
′
s
(GPa)
Ap
(mm2)
Ef
(GPa)
ff
(MPa)
σpe
(MPa)
fck
(MPa)
B2
402
560
172
201
510
187
100.5
158
2790
895
35.2
B3
402
560
172
201
510
187
100.5
158
2790
1382
33.4
B6
402
560
172
201
510
187
100.5
158
2790
917
40.6
B7
402
560
172
201
510
187
100.5
158
2790
1407
35.9
Note: As, fy and Es = area, yield strength, and elastic modulus of tensile steel rebars, respectively; A′
s, f ′
y and E′
s =
area, yield strength, and elastic modulus of compressive steel rebars, respectively; Ap = tendon area; Ef and ff =
elastic modulus and tensile strength of FRP composites, respectively; σpe = effective prestress. B2
402
560
172
201
510
187
100.5
158
2790
895
35.2
B3
402
560
172
201
510
187
100.5
158
2790
1382
33.4
B6
402
560
172
201
510
187
100.5
158
2790
917
40.6
B7
402
560
172
201
510
187
100.5
158
2790
1407
35.9
Note: As, fy and Es = area, yield strength, and elastic modulus of tensile steel rebars, respectively;
'
sA
,
'
yf and
'
sE = area, yield strength, and elastic modulus of compressive steel rebars, respectively;
Ap = tendon area; Ef and ff = elastic modulus and tensile strength of FRP composites, respectively; σpe
= effective prestress. 3 Numerical Evaluation 3. Numerical Evaluation
A simply supported P A simply supported PCB with external tendons draped at two deviators, as shown in Figure 5,
is used herein for the study. The depths of external tendons at the end supports and deviators were 300
and 500 mm, respectively. The prestressing tendons were 1000 mm2 in area, made of CFRP composites
having the tensile strength of 1840 MPa and the modulus of elasticity of 147 GPa. The initial prestress
prior to prestress transfer was 1104 MPa, namely 60% of the tensile strength. The area of tensile rebars,
Ar, varied from 360 to 3560 mm2. Thus, the value of ρr (ratio of tensile rebars) ranged from 0.22% to
2.16%, where ρr = Ar/(bdr) in which b is the cross-sectional width; and dr is the depth of tensile rebars. The area of compressive rebars was 360 mm2. Three types of bonded rebars were considered, namely,
steel, CFRP, and GFRP. The mechanical properties of the rebars are given in Table 2. The concrete
strength fck is 60 MPa. is used herein for the study. The depths of external tendons at the end supports and deviators were
300 and 500 mm, respectively. The prestressing tendons were 1000 mm2 in area, made of CFRP
composites having the tensile strength of 1840 MPa and the modulus of elasticity of 147 GPa. The
initial prestress prior to prestress transfer was 1104 MPa, namely 60% of the tensile strength. The area
of tensile rebars, Ar, varied from 360 to 3560 mm2. Thus, the value of ρr (ratio of tensile rebars) ranged
from 0.22% to 2.16%, where ρr = Ar/(bdr) in which b is the cross-sectional width; and dr is the depth of
tensile rebars. The area of compressive rebars was 360 mm2. Three types of bonded rebars were
considered, namely, steel, CFRP, and GFRP. The mechanical properties of the rebars are given in
Table 2. The concrete strength fck is 60 MPa. Figure 5. Simply supported beam for numerical investigation, Ar and
'
rA are the areas of tensile and
i
b
i
l
Figure 5. Simply supported beam for numerical investigation, Ar and A′r are the areas of tensile and
compressive rebars, respectively. Figure 5. Simply supported beam for numerical investigation, Ar and
'
rA are the areas of tensile and
Figure 5. Simply supported beam for numerical investigation, Ar and A′r are the areas of tensile and
compressive rebars, respectively. vely. Table 2. 3. Numerical Evaluation
A simply supported P Figure 6. Concrete strain distribution at ultimate. (a) ρr = 0.0%; (b) ρr = 0.22%; (c) ρr = 2.16%. Cracks of concrete occur once the tensile strain reaches its cracking strain. Over the cracking
zone, the crack width may be represented by the ultimate tensile strain. In the case for ρr = 0%, there
appears a huge tensile strain at the midspan against marginal ones over the other zones. This
indicates an unfavorable crack mode, i.e., the beam has only one large crack at the midspan, and the
concrete over other zones is nearly uncracked. By providing a minimum content of tensile rebars (ρr
= 0.22%), the crack mode was substantially improved, i.e., the crack width at the midspan was
reduced, and more cracks occurred at the noncritical zones. The use of CFRP rebars is more effective
than the use of GFRP or steel rebars to improve the crack mode. At a high ρr level of 2.16%, the crack
modes for the beams with CFRP and steel rebars are similar, while GFRP rebars lead to smaller crack
zone and larger crack width over the flexural span than steel rebars. Cracks of concrete occur once the tensile strain reaches its cracking strain. Over the cracking zone,
the crack width may be represented by the ultimate tensile strain. In the case for ρr = 0%, there appears
a huge tensile strain at the midspan against marginal ones over the other zones. This indicates an
unfavorable crack mode, i.e., the beam has only one large crack at the midspan, and the concrete
over other zones is nearly uncracked. By providing a minimum content of tensile rebars (ρr = 0.22%),
the crack mode was substantially improved, i.e., the crack width at the midspan was reduced, and more
cracks occurred at the noncritical zones. The use of CFRP rebars is more effective than the use of GFRP
or steel rebars to improve the crack mode. At a high ρr level of 2.16%, the crack modes for the beams
with CFRP and steel rebars are similar, while GFRP rebars lead to smaller crack zone and larger crack
width over the flexural span than steel rebars. 3. Numerical Evaluation
A simply supported P Mechanical properties of rebars. vely. Table 2. Mechanical properties of rebars. compressive rebars, respectively. Table 2. Mechanical properties of rebars. Rebars
Tensile Strength (MPa)
Yield Strength (MPa)
Elastic Modulus (GPa)
Steel
450
450
200
CFRP
1840
-
147
GFRP
750
-
40
Table 2. Mechanical properties of rebars. Rebars
Tensile Strength (MPa)
Yield Strength (MPa)
Elastic Modulus (GPa)
Steel
450
450
200
CFRP
1840
-
147
GFRP
750
-
40
3.1. Failure and Cracking Modes GFRP
750
3.1. Failure and Cracking Modes Figure 6 shows the ultimate strain distribution in the top and bottom fibers of the beams with
different ρr levels. The failure and cracking modes can be seen in the graphs of this figure. Failure of
all the investigated beams occurs due to concrete crushing at midspan when the specified ultimate
compressive strain of 0.003 is reached. For a beam with no rebars (ρr = 0.0%), the noncritical sections
were still far below their ultimate strain capacity at failure. When a minimum content of tensile rebars 6 of 16
ltimate
i Polymers 2020, 12, 2773
all the investigated was provided (ρr = 0.22%), the exploitation of noncritical sections was improved. Such improvement
is pronounced for FRP (especially CFRP) rebars and relatively not so notable for steel rebars. However,
at a high ρr level of 2.16%, the exploitation of noncritical sections for steel rebars is nearly comparable
to that for FRP rebars. At failure, both FRP tendons and rebars did not reach their rupture strength
while the tensile steel rebars had yielded. were still far below their ultimate strain capacity at failure. When a minimum content of tensile rebars
was provided (ρr = 0.22%), the exploitation of noncritical sections was improved. Such improvement
is pronounced for FRP (especially CFRP) rebars and relatively not so notable for steel rebars. However, at a high ρr level of 2.16%, the exploitation of noncritical sections for steel rebars is nearly
comparable to that for FRP rebars. At failure, both FRP tendons and rebars did not reach their rupture
strength while the tensile steel rebars had yielded. Figure 6. Concrete strain distribution at ultimate. (a) ρr = 0.0%; (b) ρr = 0.22%; (c) ρr = 2.16%. Figure 6. Concrete strain distribution at ultimate. (a) ρr = 0.0%; (b) ρr = 0.22%; (c) ρr = 2.16%. Figure 6. Concrete strain distribution at ultimate. (a) ρr = 0.0%; (b) ρr = 0.22%; (c) ρr = 2.16%. 3.2. Tendon Stress Development
3.2. Tendon Stress Development Figure 7a shows the stress increase in external tendons versus midspan deflection curves for the
beams with different types of rebars (ρr = 1.19%). There was a roughly linear relationship between the
tendon stress and the deflection. The slopes for the beams with steel, CFRP, and GFRP rebars were
2.12, 2.2, and 2.46 MPa/mm, respectively. Figure 7b shows the stress increase in external tendons with
the applied load. The beams with FRP rebars exhibited bilinear behavior with a turning point due 7 of 16
endons
g point Polymers 2020, 12, 2773
were 2.12, 2.2, and 2
with the applied lo to concrete cracking, while the beam with steel rebars exhibit trilinear behavior with turning points
due to concrete cracking and steel yielding, respectively. Since the elastic behavior is dominated by
concrete, the type of rebars appears to have practically no influence on the tendon stress evolution in
the elastic range of loading. Beyond cracking, GFRP rebars develop substantially lower rebar stress
because of the smaller elastic modulus and, therefore, a higher stress increase in external tendons is
required at a given load level to satisfy the force equilibrium when compared to CFRP or steel rebars. due to concrete cracking, while the beam with steel rebars exhibit trilinear behavior with turning
points due to concrete cracking and steel yielding, respectively. Since the elastic behavior is
dominated by concrete, the type of rebars appears to have practically no influence on the tendon
stress evolution in the elastic range of loading. Beyond cracking, GFRP rebars develop substantially
lower rebar stress because of the smaller elastic modulus and, therefore, a higher stress increase in
external tendons is required at a given load level to satisfy the force equilibrium when compared to
CFRP or steel rebars. Figure 7. Tendon stress development for the beams with different types of rebars. (a) midspan
deflection versus tendon stress increment; (b) load versus tendon stress increase; (c) variation of
ultimate tendon stress increment with varying ρr; (d) variation of ultimate load with varying ρr. Figure 7. Tendon stress development for the beams with different types of rebars. (a) midspan deflection
versus tendon stress increment; (b) load versus tendon stress increase; (c) variation of ultimate tendon
stress increment with varying ρr; (d) variation of ultimate load with varying ρr. Figure 7. Tendon stress development for the beams with different types of rebars. 3.2. Tendon Stress Development
3.2. Tendon Stress Development (a) midspan
deflection versus tendon stress increment; (b) load versus tendon stress increase; (c) variation of
ultimate tendon stress increment with varying ρr; (d) variation of ultimate load with varying ρr. Figure 7. Tendon stress development for the beams with different types of rebars. (a) midspan deflection
versus tendon stress increment; (b) load versus tendon stress increase; (c) variation of ultimate tendon
stress increment with varying ρr; (d) variation of ultimate load with varying ρr. The variations of the ultimate stress increment in external tendons (Δσp) and the ultimate load
(Pu) with the ρr level are illustrated in Figure 7c,d, respectively. As ρr increases, the value of Δσp
quickly decreases for the beams with CFRP rebars while it decreases slightly for the beams with GFRP
or steel rebars. As expected, GFRP rebars resulted in substantially higher (around 70% higher) Δσp
than steel rebars. At ρr = 0.22%, the Δσp ratio between the beams with CFRP and steel rebars was as
high as 1.73, attributed to the fact that CFRP rebars led to much better exploitation of noncritical
sections than steel rebars. This ratio was reduced to 1.12 at ρr = 2.16%. This could be explained by the
comparable exploitation of noncritical sections of the beams with CFRP and steel rebars, as
mentioned in the previous section. The ultimate load is dependent upon the ultimate stresses in
external tendons and tensile rebars. Due to higher reinforcement stresses at ultimate, CFRP rebars
led to substantially higher (37.1% higher) ultimate load than steel rebars at ρr = 0.22%. The difference
The variations of the ultimate stress increment in external tendons (∆σp) and the ultimate load
(Pu) with the ρr level are illustrated in Figure 7c,d, respectively. As ρr increases, the value of ∆σp
quickly decreases for the beams with CFRP rebars while it decreases slightly for the beams with GFRP
or steel rebars. As expected, GFRP rebars resulted in substantially higher (around 70% higher) ∆σp
than steel rebars. At ρr = 0.22%, the ∆σp ratio between the beams with CFRP and steel rebars was
as high as 1.73, attributed to the fact that CFRP rebars led to much better exploitation of noncritical
sections than steel rebars. This ratio was reduced to 1.12 at ρr = 2.16%. This could be explained by the
comparable exploitation of noncritical sections of the beams with CFRP and steel rebars, as mentioned
in the previous section. 3.3. Deformation Behavior
3.3. Deformation Behavior The moment-curvature and load-deflection curves for the beams with different types of rebars
(ρr = 1.19%) are shown in Figure 8a,b, respectively. In the precracking stage, the effect of rebars on
the response characteristics is negligible due to slight stress increments in rebars. The responses for
the beams with FRP and steel rebars differ after cracking because the rebar contribution becomes
increasingly important. The reduction in flexural stiffness due to cracking is highly dependent on the
rebar modulus of elasticity, i.e., the higher the rebar modulus of elasticity, the less the reduction in
member stiffness. Therefore, at a given load level, GFRP rebars lead to higher post cracking deformation
than CFRP or steel rebars. The post cracking deformation for the beams with FRP rebars develops
linearly until failure. For the beam with steel rebars, a significant further reduction in flexural stiffness
occurs on steel yielding. The moment-curvature and load-deflection curves for the beams with different types of rebars
(ρr = 1.19%) are shown in Figure 8a,b, respectively. In the precracking stage, the effect of rebars on
the response characteristics is negligible due to slight stress increments in rebars. The responses for
the beams with FRP and steel rebars differ after cracking because the rebar contribution becomes
increasingly important. The reduction in flexural stiffness due to cracking is highly dependent on the
rebar modulus of elasticity, i.e., the higher the rebar modulus of elasticity, the less the reduction in
member stiffness. Therefore, at a given load level, GFRP rebars lead to higher post cracking
deformation than CFRP or steel rebars. The post cracking deformation for the beams with FRP rebars
develops linearly until failure. For the beam with steel rebars, a significant further reduction in
flexural stiffness occurs on steel yielding. Figure 8. Deformation behavior for the beams with different types of rebars. (a) moment versus
curvature at midspan; (b) load versus midspan deflection; (c) variation of ultimate curvature with
varying ρr; (d) variation of ultimate deflection with varying ρr. Figure 8. Deformation behavior for the beams with different types of rebars. (a) moment versus
curvature at midspan; (b) load versus midspan deflection; (c) variation of ultimate curvature with
varying ρr; (d) variation of ultimate deflection with varying ρr. Figure 8. Deformation behavior for the beams with different types of rebars. 3.2. Tendon Stress Development
3.2. Tendon Stress Development The ultimate load is dependent upon the ultimate stresses in external tendons
and tensile rebars. Due to higher reinforcement stresses at ultimate, CFRP rebars led to substantially
higher (37.1% higher) ultimate load than steel rebars at ρr = 0.22%. The difference tends to decrease
with increasing ρr due to the reduced difference between reinforcement stresses in the beams with
CFRP and steel rebars. At a low ρr level of 0.22%, GFRP rebars lead to a slightly higher ultimate load
than steel rebars. As ρr increases, the increase in ultimate load for the beams with GFRP rebars is
slower than that for the beams with steel rebars. As a result, when ρr increases to a level greater than 8 of 16
y higher
h GFRP Polymers 2020, 12, 2773
the beams with CF
l i
l
d h 0.77%, the ultimate load for the beams with GFRP rebars turns to be lower than that for the beams with
steel rebars. rebars is slower than that for the beams with steel rebars. As a result, when ρr increases to a leve
greater than 0.77%, the ultimate load for the beams with GFRP rebars turns to be lower than that fo
the beams with steel rebars. 0.77%, the ultimate load for the beams with GFRP rebars turns to be lower than that for the beams with
steel rebars. rebars is slower than that for the beams with steel rebars. As a result, when ρr increases to a leve
greater than 0.77%, the ultimate load for the beams with GFRP rebars turns to be lower than that for
the beams with steel rebars. 3.4. Neutral Axis Depth and Rebar Strain
3.4. Neutral Axis Depth and Rebar Strain Since the neutral axis depth and rebar strain are key parameters describing flexural ductility [33],
it is important to understand their behavior well. The movement of the neutral axis, after it rises to
the midspan section’s bottom fiber, with the moment for the beams with different types of rebars
(ρr = 1.19%) is illustrated in Figure 9a. For the beams with FRP rebars, the neutral axis shifts rapidly
with the increasing moment, and then the shift gradually slows down. Similar behavior was observed
for the beam with steel rebars until yielding. After that, a fast movement of the neutral axis is resumed
due to steel yielding. Figure 9b shows the strain development in FRP and steel rebars with the bending
moment (ρr = 1.19%). The behavior for the beam with steel rebars exhibited three stages with transitions
caused by cracking and yielding, respectively. Two-stage behavior was observed for the beams with
FRP rebars because of the lack of yielding. Due to a lower modulus of elasticity, GFRP rebars exhibited
a significantly faster increase in strain after cracking but a slower stress development when compared
to steel or CFRP rebars. Since the neutral axis depth and rebar strain are key parameters describing flexural ductility
[33], it is important to understand their behavior well. The movement of the neutral axis, after it rises
to the midspan section’s bottom fiber, with the moment for the beams with different types of rebars
(ρr = 1.19%) is illustrated in Figure 9a. For the beams with FRP rebars, the neutral axis shifts rapidly
with the increasing moment, and then the shift gradually slows down. Similar behavior was observed
for the beam with steel rebars until yielding. After that, a fast movement of the neutral axis is resumed
due to steel yielding. Figure 9b shows the strain development in FRP and steel rebars with the
bending moment (ρr = 1.19%). The behavior for the beam with steel rebars exhibited three stages with
transitions caused by cracking and yielding, respectively. Two-stage behavior was observed for the
beams with FRP rebars because of the lack of yielding. Due to a lower modulus of elasticity, GFRP
rebars exhibited a significantly faster increase in strain after cracking but a slower stress development
when compared to steel or CFRP rebars. Figure 9. Neutral axis depth and bar strain for different types of rebars. 3.3. Deformation Behavior
3.3. Deformation Behavior CFRP rebars register considerably higher Δu than steel rebars at low ρr levels, while the difference is
reduced with increasing ρr. This could also be attributed to the rebar effect on the exploitation of
noncritical sections, as explained previously. This was because the ultimate curvature was directly proportional to the ultimate concrete tensile strain
in terms of the plane section hypothesis. As shown in Figure 8d, GFRP rebars register substantially
higher ∆u than steel rebars because of a significantly lower modulus of elasticity. CFRP rebars
register considerably higher ∆u than steel rebars at low ρr levels, while the difference is reduced with
increasing ρr. This could also be attributed to the rebar effect on the exploitation of noncritical sections,
as explained previously. previously. This was because the ultimate curvature was directly proportional to the ultimate
concrete tensile strain in terms of the plane section hypothesis. As shown in Figure 8d, GFRP rebars
register substantially higher Δu than steel rebars because of a significantly lower modulus of elasticity. CFRP rebars register considerably higher Δu than steel rebars at low ρr levels, while the difference is
reduced with increasing ρr. This could also be attributed to the rebar effect on the exploitation of
noncritical sections, as explained previously. 3.3. Deformation Behavior
3.3. Deformation Behavior (a) moment versus
curvature at midspan; (b) load versus midspan deflection; (c) variation of ultimate curvature with
varying ρr; (d) variation of ultimate deflection with varying ρr. Figure 8. Deformation behavior for the beams with different types of rebars. (a) moment versus
curvature at midspan; (b) load versus midspan deflection; (c) variation of ultimate curvature with
varying ρr; (d) variation of ultimate deflection with varying ρr. Figure 8c,d illustrates the variation of ultimate midspan curvature (κu) and deflection (Δu) with
varying ρr, respectively. It is seen in Figure 8c that the value of κu decreases as ρr increases. GFRP
rebars mobilize a smaller κu at low ρr levels whereas a larger κu at high ρr levels in comparison with
steel rebars. CFRP rebars mobilize lower κu than steel rebars; the difference is substantial at low ρ
Figure 8c,d illustrates the variation of ultimate midspan curvature (κu) and deflection (∆u) with
varying ρr, respectively. It is seen in Figure 8c that the value of κu decreases as ρr increases. GFRP rebars
mobilize a smaller κu at low ρr levels whereas a larger κu at high ρr levels in comparison with steel
rebars. CFRP rebars mobilize lower κu than steel rebars; the difference is substantial at low ρr levels
but reduced with increasing ρr. It was noted that the above observation was similar to the effect of
rebars on the crack width (represented by the ultimate concrete tensile strain), as discussed previously. 9 of 16
e effect Polymers 2020, 12, 2773
levels but reduced This was because the ultimate curvature was directly proportional to the ultimate concrete tensile strain
in terms of the plane section hypothesis. As shown in Figure 8d, GFRP rebars register substantially
higher ∆u than steel rebars because of a significantly lower modulus of elasticity. CFRP rebars
register considerably higher ∆u than steel rebars at low ρr levels, while the difference is reduced with
increasing ρr. This could also be attributed to the rebar effect on the exploitation of noncritical sections,
as explained previously. previously. This was because the ultimate curvature was directly proportional to the ultimate
concrete tensile strain in terms of the plane section hypothesis. As shown in Figure 8d, GFRP rebars
register substantially higher Δu than steel rebars because of a significantly lower modulus of elasticity. 3.4. Neutral Axis Depth and Rebar Strain
3.4. Neutral Axis Depth and Rebar Strain (a) moment versus neutral
axis depth at midspan; (b) moment versus rebar strain at midspan; (c) variation of ultimate neutral
axis depth with varying ρr; (d) variation of ultimate tensile rebar strain with varying ρr. Figure 9. Neutral axis depth and bar strain for different types of rebars. (a) moment versus neutral axis
depth at midspan; (b) moment versus rebar strain at midspan; (c) variation of ultimate neutral axis
depth with varying ρr; (d) variation of ultimate tensile rebar strain with varying ρr. Figure 9. Neutral axis depth and bar strain for different types of rebars. (a) moment versus neutral
axis depth at midspan; (b) moment versus rebar strain at midspan; (c) variation of ultimate neutral
axis depth with varying ρr; (d) variation of ultimate tensile rebar strain with varying ρr. Figure 9. Neutral axis depth and bar strain for different types of rebars. (a) moment versus neutral axis
depth at midspan; (b) moment versus rebar strain at midspan; (c) variation of ultimate neutral axis
depth with varying ρr; (d) variation of ultimate tensile rebar strain with varying ρr. Polymers 2020, 12, 2773 10 of 16 Polymers 2020, 12, 2773 Figure 9c,d shows the variation of neutral axis depth (cu) and tensile bar strain at ultimate (εr)
with varying ρr, respectively. Comparing these graphs to the graph of Figure 8c, it is seen that the effect
of rebars on κu is opposite to that on cu while coincident to that on εr. Their theoretical relationships
can explain this observation. According to the plane section hypothesis, cu and εr are related to κu by:
cu = εu/κu; εr = κu(dr −cu) = κudr −εu, where dr is the depth of tensile rebars, equal to 550 mm; εu is
the ultimate concrete compressive strain, equal to 0.003. Because of a smaller value of κu, CFRP rebars
mobilize a higher cu value and a lower εr value than steel rebars. Likewise, the values of cu and εr
mobilized by GFRP rebars could be higher or lower than those by steel rebars, depending on the
ρr level. 4. Analytical Modeling 4.1. Existing Models Using Combined Reinforcing Index for Prediction of Ultimate Stress in Unbonded Tend Owing to strain incompatibility, the stress in external or unbonded tendons is member-dependent. The quantification of the tendon stress is a key task in design practice. The combined reinforcing
index is considered one of the best parameters used for calculating the tendon stress, as this parameter
involves several important factors, including the tendon area and depth, effective prestress, rebar area,
and concrete strength. The combined reinforcing index for the beams with steel rebars is defined by ω0 =
Apσpe + Ar fy
bdp fck
(1) (1) where dp is the tendon depth. At ultimate, FRP rebars often reach a stress level far below their
rupture strength. Therefore, the combined reinforcing index for the beams with FRP rebars could be
expressed by ω0 =
Apσpe + Arσr
bdp fck
(2) (2) where σr is the stress in tensile rebars at ultimate. where σr is the stress in tensile rebars at ultimate. Figure 10a shows the numerical results regarding the relationship between ∆σp and ω0 for the
beams with FRP and steel rebars. It is interesting to note that the variations of ∆σp with varying ω0 for
the beams with CFRP and GFRP rebars appear to be consistent. Since CFRP represents the highest
modulus of elasticity and GFRP the lowest one amongst FRP groups, it can be concluded that the
beams with different types of FRP rebars follow approximately the same ∆σp-ω0 response. At a given
ω0 value, the beams with FRP rebars led to substantially higher tendon stress than the beams with
steel rebars. The tendon stress tends to decrease with increasing ω0, while the decrease for the beams
with FRP rebars is much quicker than that for the beams with steel rebars. 11 of 16 Polymers 2020, 12, 2773 Figure 10. Relationship between tendon stress increment and combined reinforcing index. (a)
numerical results; (b) comparison between simplified models and numerical data for the beams with
steel rebars; (c) comparison between simplified models and numerical data for the beams with FRP
Figure 10. Relationship between tendon stress increment and combined reinforcing index. (a) numerical
results; (b) comparison between simplified models and numerical data for the beams with steel rebars;
(c) comparison between simplified models and numerical data for the beams with FRP rebars. Figure 10. Relationship between tendon stress increment and combined reinforcing index. 4. Analytical Modeling (a)
numerical results; (b) comparison between simplified models and numerical data for the beams with
steel rebars; (c) comparison between simplified models and numerical data for the beams with FRP
Figure 10. Relationship between tendon stress increment and combined reinforcing index. (a) numerical
results; (b) comparison between simplified models and numerical data for the beams with steel rebars;
(c) comparison between simplified models and numerical data for the beams with FRP rebars. rebars. By performing laboratory tests of 22 PCBs with unbonded tendons, Du and Tao [34] found an
approximately linear relationship between the tendon stress and the combined reinforcing index. By performing laboratory tests of 22 PCBs with unbonded tendons, Du and Tao [34] found an
approximately linear relationship between the tendon stress and the combined reinforcing index. They recommended the following expression for calculating ∆σp: pression for calculating Δσp:
786 1920
(3)
∆σp = 786 −1920ω0
(3) (3) 0
p
JGJ/T 92-93 [35] suggested the following Equation for the computation of Δσp:
JGJ/T 92-93 [35] suggested the following Equation for the computation of ∆σp: 0
p
JGJ/T 92-93 [35] suggested the following Equation for the computation of Δσp:
JGJ/T 92-93 [35] suggested the following Equation for the computation of ∆σp: g
q
p
0
500 770
p
(4)
0
250 380
p
(5)
∆σp = 500 −770ω0
(4)
∆σp = 250 −380ω0
(5) 0
500 770
p
(4)
∆σp = 500 −770ω0
(4) (4)
(4) p
0
250 380
p
(5)
∆σp = 250 −380ω0
(5) for L/dp ≤ 35; and
for L/dp ≤35; and 0
250 380
p
(5)
∆σp = 250 −380ω0
(5) p
0
250 380
p
(5)
∆σp = 250 −380ω0
(5) (5)
(5) L/dp > 35. 4.2. Proposed Model
According to t
illustrated in Figure According to the linear fit to the numerical data of the beams with steel and FRP rebars as
illustrated in Figure 11, the following Equation is proposed for predicting the stress increment in
external tendons at ultimate:
illustrated in Figure 11, the following Equation is proposed for predicting the stress increment in
external tendons at ultimate:
0
303 220
p
(7) ∆σp = 303 −220ω0
(7)
∆σp = 626 −1032ω0
(8)
0
626 1032
p
(8) ∆σp = 303 −220ω0
(7) ∆σp = 303 −220ω0
(7)
for the beams with steel rebars; and
∆σp = 626 −1032ω0
(8)
for the beams with steel rebars; and
0
626 1032
p
(8)
for the beams with FRP rebars (7) ∆σp = 303 −220ω0
(7)
for the beams with steel rebars; and
∆σp = 626 −1032ω0
(8)
for the beams with steel rebars; and
0
626 1032
p
(8)
for the beams with FRP rebars ∆σp = 626 −1032ω0
(8)
0
626 1032
p
(8) (8)
(8) ∆σp = 626 −1032ω0
(8)
0
p
(8) for the beams with FRP rebars. for the beams with FRP rebars. for the beams with FRP rebars. for the beams with FRP rebars. Figure 11. Linear fit to the numerical data. (a) beams with steel rebars; (b) beams with FRP rebars. Figure 11. Linear fit to the numerical data. (a) beams with steel rebars; (b) beams with FRP rebars. Figure 11 Linear fit to the numerical data (a) beams with steel rebars; (b) beams with FRP rebars
Figure 11. Linear fit to the numerical data. (a) beams with steel rebars; (b) beams with FRP rebars. g
( )
( )
It is worth mentioning that at given cross-sectional and material properties, the value of ω0 in
the beams with steel rebars is known (see Equation (1)), whereas that in the beams FRP rebars is
unknown (see Equation (2)). 4. Analytical Modeling JGJ 92-2016 [36] proposed a modification of the above Equation, which is expressed as follows
p
JGJ 92-2016 [36] proposed a modification of the above Equation, which is expressed as follows: 2
0
0
1
(240
335
)(0.45
5.5 /
)
p
L
h L
L
(6)
∆σp = (240 −335ω0)(0.45 + 5.5h/L0)L2
L1
(6) (6)
(6) 1
where ω0 is not greater than 0.4; h is the cross-sectional depth; L0 is the span length; L1 is the tendon
length between end anchorages for continuous beams, and L2 is the total length of loaded spans. where ω0 is not greater than 0.4; h is the cross-sectional depth; L0 is the span length; L1 is the tendon
length between end anchorages for continuous beams, and L2 is the total length of loaded spans. 1
where ω0 is not greater than 0.4; h is the cross-sectional depth; L0 is the span length; L1 is the tendon
length between end anchorages for continuous beams, and L2 is the total length of loaded spans. where ω0 is not greater than 0.4; h is the cross-sectional depth; L0 is the span length; L1 is the tendon
length between end anchorages for continuous beams, and L2 is the total length of loaded spans. Polymers 2020, 12, 2773 12 of 16 12 of 16 It should be noted that the above models were developed for the beams with steel rebars. To evaluate the applicability of the models, the predictions by the simplified equations against the
numerical data for the beams with steel rebars are presented in Figure 10b, while those for the beams
with FRP rebars are presented in Figure 10c. It was observed that, in general, the Du and Tao model
and JGJ/T 92-93 are unsafe, while JGJ 92-2016 is conservative when predicting the tendon stress in the
beams with steel rebars. In addition, the Du and Tao model substantially overestimates the influence
of ω0. For the beams with FRP rebars, the effect of ω0 on the tendon stress is overestimated by the Du
and Tao model while underestimated by JGJ/T 92-93 and JGJ 92-2016. Moreover, JGJ 92-2016 appears
to be overly conservative. Polymers 2020, 12, x FOR PEER REVIEW
12 of 16
with FRP rebars are presented in Figure 10c. 4.2. Proposed Model
According to t
illustrated in Figure Consequently, the value of Δσp in the beams with steel rebars can be
calculated directly by using Equation (7), while the computation of Δσp in the beams with FRP rebars
It is worth mentioning that at given cross-sectional and material properties, the value of ω0 in the
beams with steel rebars is known (see Equation (1)), whereas that in the beams FRP rebars is unknown
(see Equation (2)). Consequently, the value of ∆σp in the beams with steel rebars can be calculated
directly by using Equation (7), while the computation of ∆σp in the beams with FRP rebars needs to
combine Equation (8) with the section equilibrium equation. needs to combine Equation (8) with the section equilibrium equation. The a ial e uilib iu
e uatio
of the
iti al e tio
of the bea
ith teel eba
i
The axial equilibrium equation of the critical section of the beams with steel rebars is equa io
o
e
i i a se io
o
e
ea
s
i
s ee
e a s is
'
'
1
0.85
(
)
ck
u
p
pe
p
r
y
r
y
f b c
A
A f
A f
(9)
0.85 fckbβ1cu = Ap(σpe + ∆σp) + Ar fy −A′
r f ′
y
(9) (9)(9) where β1 is the stress-block factor for concrete, taken equal to 0.85; Ar and
'
rA are the area of tensile
and compressive rebars, respectively Substituting Equation (7) into Equation (9) results in
where β1 is the stress-block factor for concrete, taken equal to 0.85; Ar and A′
r are the area of tensile and
compressive rebars, respectively. 4. Analytical Modeling It was observed that, in general, the Du and Tao model
and JGJ/T 92-93 are unsafe, while JGJ 92-2016 is conservative when predicting the tendon stress in the
beams with steel rebars. In addition, the Du and Tao model substantially overestimates the influence
of ω0. For the beams with FRP rebars, the effect of ω0 on the tendon stress is overestimated by the Du
and Tao model while underestimated by JGJ/T 92-93 and JGJ 92-2016. Moreover, JGJ 92-2016 appears
to be overly conservative. 4.2. Proposed Model 4.2. Proposed Model
According to t
illustrated in Figure p
g
On the other hand, the axial equilibrium of the critical section of the beams with FRP rebars is
given by On the other hand, the axial equilibrium of the critical section of the beams with FRP rebars is
given by 0.85 fckbβ1cu = Ap(σpe + ∆σp) + Arσr −A′
rσ′
r
(15) g
y
0.85 fckbβ1cu = Ap(σpe + ∆σp) + Arσr −A′
rσ′
r (15) 0.85 fckbβ1cu = Ap(σpe + ∆σp) + Arσr −A′
rσ′
r 0.85 fckbβ1cu = Ap(σpe + ∆σp) + Arσr −A′
rσ′
r where σr = E f εu
dr
cu
−1
! (16)
σ′
r = E′
f εu
1 −d′
r
cu
! (17) σr = E f εu
dr
cu
−1
! (16)
σ′
r = E′
f εu
1 −d′
r
cu
! (17) σr = E f εu
dr
cu
−1
! (16)
d′ ! (16) σ′
r = E′
f εu
1 −d′
r
cu
! (17) (17) where Ef and E′
f are the elastic modulus of tensile and compressive FRP rebars, respectively; εu is taken
equal to 0.003. Combining Equations (2), (8), (16), and (17) with Equation (15) leads to where Ef and E′
f are the elastic modulus of tensile and compressive FRP rebars, respectively; εu is taken
equal to 0.003. Combining Equations (2), (8), (16), and (17) with Equation (15) leads to cu = −B +
√
B2 −4AC
2A
(18) cu = −B +
√
B2 −4AC
2A
(18) (18) where where A = 0.85 fckbβ1
B = ArE f εu(1 −1032ρp/ fck) + A′
rE′
f εu −Ap(σpe + 626 −1032ωp)
C = −ArE f εudr(1 −1032ρp/ fck) −A′
rE′
f εud′
r where where
ρp =
Ap
bdp
(19)
ωp =
Apσpe
bdp fck
(20) ρp =
Ap
bdp
(19)
ωp =
Apσpe
bdp fck
(20) ρp =
Ap
bdp
(19) (19) ωp =
Apσpe
bdp fck
(20) (20) Hence, the flexural strength for the beams with FRP rebars is obtained by Mu = Ap(σpe + ∆σp)de + Arσrdr −A′
rσ′
rd′
r −0.85 fckb(β1cu)2/2
(21) (21) A comparison of the ultimate tendon stress increment and flexural strength predicted by the
proposed analytical model with the numerical results for the beams with steel and FRP rebars is
presented in Table 3 and Figure 12. It is seen that there is a good agreement between the analytical
predictions and numerical data. 4.2. Proposed Model
According to t
illustrated in Figure Substituting Equation (7) into Equation (9) results in p
y
g
q
( )
q
( )
'
'
0
1
(
303
220
)
0.85
p
pe
r
y
r
y
u
ck
A
A f
A f
c
f b
(10)
cu =
Ap(σpe + 303 −220ω0) + Ar fy −A′
r f ′
y
0.85 fckbβ1
(10) (10)
(10) Hence, the flexural strength for the beams with steel rebars is calculated from
Hence, the flexural strength for the beams with steel rebars is calculated from '
'
'
2
1
(
)
0.85
(
) / 2
u
p
pe
p
e
r
y
r
r
y
r
ck
u
M
A
d
A f d
A f d
f b
c
(11)
Mu = Ap(σpe + ∆σp)de + Ar fydr −A′
r f ′
yd′
r −0.85 fckb(β1cu)2/2
(11) (11)
(11) Polymers 2020, 12, 2773
13 13 of 16 Polymers 2020, 12, 2773 Polymers 2020, 12, 2773 where dr and d′
r are the effective depth of tensile and compressive rebars, respectively; de is the effective
depth at ultimate of external tendons. The value of de can be obtained by where dr and d′
r are the effective depth of tensile and compressive rebars, respectively; de is the effective
depth at ultimate of external tendons. The value of de can be obtained by de = Rddp
(12) (12) where Rd is the depth reduction factor as a result of second-order effects of externally PCBs, which may
be calculated from where Rd is the depth reduction factor as a result of second-order effects of externally PCBs, which may
be calculated from Rd = 1.14 −0.005(L/dp) −0.19(Sd/L) ≤1.0
(13) (13) for center-point loading; and Rd = 1.25 −0.01(L/dp) −0.38(Sd/L) ≤1.0
(14) (14) for third-point loading. 4.2. Proposed Model
According to t
illustrated in Figure The mean discrepancy for the ultimate tendon stress increment is
1.03% with a standard deviation of 4.08%, while that for the ultimate moment is −4.33% with a standard
deviation of 2.32%. olymers 2020, 12, 2773
14 of 16
lymers 2020, 12, x FOR PEER REVIEW
14 of 16
Figure 12. Comparison between analytical predictions and numerical results. (a) tendon stress
increment at ultimate; (b) ultimate moment. Figure 12. Comparison between analytical predictions and numerical results. (a) tendon stress
increment at ultimate; (b) ultimate moment. 14 of 16 14 of 16 Polymers 2020, 12, 2773 Figure 12. Comparison between analytical predictions and numerical results. (a) tendon stress
increment at ultimate; (b) ultimate moment. Figure 12. Comparison between analytical predictions and numerical results. (a) tendon stress
increment at ultimate; (b) ultimate moment. Table 3. Comparison of ultimate tendon stress increment and moment predicted by the analytical
model with numerical results. Table 3. Comparison of ultimate tendon stress increment and moment predicted by the analytical
model with numerical results. Table 3. Comparison of ultimate tendon stress increment and moment predicted by the analytical
model with numerical results. Table 3. Comparison of ultimate tendon stress increment and moment predicted by the analytical
model with numerical results. Rebars
ρr (%)
Δσp (MPa)
Mu (kN m)
Analytical
Numerical
Error (%)
Analytical
Numerical
Error (%)
Steel
0.22
272.05
267.60
1.66
654.40
653.12
0.20
0.70
263.25
269.05
−2.15
812.72
831.63
−2.27
1.19
254.45
252.44
0.80
962.98
997.08
−3.42
1.67
245.65
246.93
−0.52
1105.17
1154.67
−4.29
2.16
236.85
234.63
0.95
1239.31
1315.42
−5.79
CFRP
0.22
448.74
463.02
−3.09
837.89
874.32
−4.17
0.70
382.53
400.19
−4.41
1054.40
1129.98
−6.69
1.19
338.41
324.23
4.37
1189.31
1256.83
−5.37
1.67
304.47
287.09
6.05
1288.01
1366.46
−5.74
2.16
276.65
261.86
5.65
1365.57
1466.04
−6.85
GFRP
0.22
483.51
435.02
11.15
713.43
706.31
1.01
0.70
455.67
451.08
1.02
808.85
840.51
−3.77
1.19
433.69
437.72
−0.92
882.11
928.20
−4.97
1.67
415.19
428.42
−3.09
942.27
1004.81
−6.22
model with numerical results. 4.2. Proposed Model
According to t
illustrated in Figure Rebars
ρr (%)
∆σp (MPa)
Mu (kN m)
Analytical
Numerical
Error (%)
Analytical
Numerical
Error (%)
Steel
0.22
272.05
267.60
1.66
654.40
653.12
0.20
0.70
263.25
269.05
−2.15
812.72
831.63
−2.27
1.19
254.45
252.44
0.80
962.98
997.08
−3.42
1.67
245.65
246.93
−0.52
1105.17
1154.67
−4.29
2.16
236.85
234.63
0.95
1239.31
1315.42
−5.79
CFRP
0.22
448.74
463.02
−3.09
837.89
874.32
−4.17
0.70
382.53
400.19
−4.41
1054.40
1129.98
−6.69
1.19
338.41
324.23
4.37
1189.31
1256.83
−5.37
1.67
304.47
287.09
6.05
1288.01
1366.46
−5.74
2.16
276.65
261.86
5.65
1365.57
1466.04
−6.85
GFRP
0.22
483.51
435.02
11.15
713.43
706.31
1.01
0.70
455.67
451.08
1.02
808.85
840.51
−3.77
1.19
433.69
437.72
−0.92
882.11
928.20
−4.97
1.67
415.19
428.42
−3.09
942.27
1004.81
−6.22
2.16
399.09
407.03
−1.95
993.58
1063.79
−6.60 2.16
5. Conclusions 5. Conclusions
A numerical and analytical study was conducted on simply supported PCBs with external CFRP
tendons, aimed at identifying the effect of providing FRP bonded rebars instead of steel ones. Based
A numerical and analytical study was conducted on simply supported PCBs with external CFRP
tendons, aimed at identifying the effect of providing FRP bonded rebars instead of steel ones. Based on
the results of the study, the following conclusions can be drawn: y
g
p
g
on the results of the study, the following conclusions can be drawn:
FRP (especially CFRP) rebars lead to better exploitation of noncritical sections than steel rebars,
particularly notable at a low ρr level. The crack mode is improved by providing a minimum
amount of rebars while the improvement is more effective using CFRP rebars than using GFRP
•
FRP (especially CFRP) rebars lead to better exploitation of noncritical sections than steel rebars,
particularly notable at a low ρr level. The crack mode is improved by providing a minimum
amount of rebars, while the improvement is more effective using CFRP rebars than using GFRP or
steel rebars. amount of rebars, while the improvement is more effective using CFRP rebars than using GFRP
or steel rebars.
CFRP rebars lead to larger ultimate load and neutral axis depth but smaller ultimate curvature
and tensile rebar strain than steel rebars. Such values registered by GFRP rebars could be larger
•
CFRP rebars lead to larger ultimate load and neutral axis depth but smaller ultimate curvature
and tensile rebar strain than steel rebars. Such values registered by GFRP rebars could be larger or
smaller than those by steel rebars, depending on the ρr level. and tensile rebar strain than steel rebars. Such values registered by GFRP rebars could be larger
or smaller than those by steel rebars, depending on the ρr level.
GFRP rebars mobilize substantially higher ultimate deflection and tendon stress increment than
steel rebars. CFRP rebars lead to similar observation at a low ρr level, while the difference
•
GFRP rebars mobilize substantially higher ultimate deflection and tendon stress increment than
steel rebars. CFRP rebars lead to similar observation at a low ρr level, while the difference between
the values for the beams with CFRP and steel rebars diminishes as ρr increases. 2.16
5. Conclusions Polymers 2020, 12, 2773 15 of 16 •
Both JGJ/T 92-93 and JGJ 92-2016 underestimated the influence of combined reinforcing index on
external tendons’ stress at ultimate in the beams with FRP rebars. Moreover, JGJ 92-2016 appears
to be overly conservative for predicting the ultimate tendon stress. •
An analytical model was proposed to predict the tendon stress at ultimate and flexural strength in
externally PCBs with steel and FRP rebars. The model predictions are in good agreement with the
numerical results. •
An analytical model was proposed to predict the tendon stress at ultimate and flexural strength in
externally PCBs with steel and FRP rebars. The model predictions are in good agreement with the
numerical results. The present study is limited to simply supported conditions. Further studies on the use of FRP
rebars instead of steel ones in continuous PCBs with external tendons shall be performed in the future. Author Contributions: Conceptualization, M.P. and T.L.; Methodology, M.P.; Software, T.L.; Validation, Z.L.;
Formal Analysis, Z.L.; Investigation, Z.L. and M.P.; Resources, T.L.; Data Curation, Z.L.; Writing—Original Draft
Preparation, M.P.; Writing—Review & Editing, Z.L.; Visualization, Z.L.; Supervision, T.L. All authors have read
and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ahmad, S. Reinforcement corrosion in concrete structures, its monitoring and service life prediction—A
review. Cem. Concr. Compos. 2003, 25, 459–471. 1. Ahmad, S. Reinforcement corrosion in concrete structures, its monitoring and service life prediction—A
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(ACI 318R-14); American Concrete Institute: Farmington Hills, MI, USA, 2014. 22. Abed, F.; Oucif, C.; Awera, Y.; Mhanna, H.H.; Alkhraisha, H. FE modeling of concrete beams and columns
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slabs reinforced with BFRP bars. Compos. Struct. 2014, 107, 664–674. 31. Lou, T.; Xiang, Y. Finite element modeling of concrete beams prestressed with external tendons. Eng. Struct. 2006, 28, 1919–1926. 32. CEN. Eurocode 2: Design of Concrete Structures—Part 1-1: General Rules and Rules for Buildings. EN 1992-1-1;
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30, 72–91. 35. JGJ/T 92-93. Technical Specification for Concrete Structures Prestressed with Unbonded Tendons; China Planning
Press: Beijing, China, 1993. 36. JGJ 92-2016. Technical Specification for Concrete Structures Prestressed with Unbonded Tendons; China Architecture
& Building Press: Beijing, China, 2016. References 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/).
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Substantial improvements not seen in health behaviors following corner store conversions in two Latino food swamps
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BMC public health
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cc-by
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UCLA
UCLA Previously Published Works
Title
Substantial improvements not seen in health behaviors following corner store
conversions in two Latino food swamps
Permalink
https://escholarship.org/uc/item/4pk8k8nt
Journal
BMC Public Health, 16(1)
ISSN
1471-2458
Authors
Ortega, Alexander N
Albert, Stephanie L
Chan-Golston, Alec M
et al.
Publication Date
2016-12-01
DOI
10.1186/s12889-016-3074-1
Peer reviewed UCLA
UCLA Previously Published Works
Title
Substantial improvements not seen in health behaviors following corner store
conversions in two Latino food swamps
Permalink
https://escholarship.org/uc/item/4pk8k8nt
Journal
BMC Public Health, 16(1)
ISSN
1471-2458
Authors
Ortega, Alexander N
Albert, Stephanie L
Chan-Golston, Alec M
et al. Publication Date
2016-12-01
DOI
10.1186/s12889-016-3074-1
Peer reviewed UCLA
UCLA Previously Published Works © 2016 Ortega 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. Substantial improvements not seen in
health behaviors following corner store
conversions in two Latino food swamps Alexander N. Ortega1*, Stephanie L. Albert2, Alec M. Chan-Golston3, Brent A. Langellier1, Deborah C. Glik2,
Thomas R. Belin3, Rosa Elena Garcia2, Ron Brookmeyer3, Mienah Z. Sharif2 and Michael L. Prelip2 Abstract Background: The effectiveness of food retail interventions is largely undetermined, yet substantial investments
have been made to improve access to healthy foods in food deserts and swamps via grocery and corner store
interventions. This study evaluated the effects of corner store conversions in East Los Angeles and Boyle Heights,
California on perceived accessibility of healthy foods, perceptions of corner stores, store patronage, food
purchasing, and eating behaviors. Methods: Household data (n = 1686) were collected at baseline and 12- to 24-months post-intervention
among residents surrounding eight stores, three of which implemented a multi-faceted intervention and five
of which were comparisons. Bivariate analyses and logistic and linear regressions were employed to assess
differences in time, treatment, and the interaction between time and treatment to determine the effectiveness of this
intervention. Results: Improvements were found in perceived healthy food accessibility and perceptions of corner stores. No
changes were found, however, in store patronage, purchasing, or consumption of fruits and vegetables. Conclusions: Results suggest limited effectiveness of food retail interventions on improving health behaviors. Future
research should focus on other strategies to reduce community-level obesity. Keywords: Corner store, Food deserts, Food environment, Food supply, Healthy food availability, Obesity, Urban health,
Latinos, Hispanic Americans, Food policy Recently, there has been an increase in interventions
to improve eating behaviors through supportive changes
to the built environment. These initiatives have largely
been concentrated in low-income neighborhoods that
have a disproportionate prevalence of chronic diseases,
as well as poor access to affordable healthy food. Strat-
egies to improve access to healthy foods have included
introducing farmers’ markets to communities, changing
restaurant menu offerings, and improving access to gro-
cery and corner stores that sell affordable fresh fruits
and vegetables [7–9]. * Correspondence: ano37@drexel.edu
1Department of Health Management and Policy, Dornsife School of Public
Health, Drexel University, 3215 Market Street, Nesbitt Hall, Room 335,
Philadelphia, PA 19104, USA
Full list of author information is available at the end of the article Powered by the California Digital Library
University of California eScholarship.org Ortega et al. BMC Public Health (2016) 16:389
DOI 10.1186/s12889-016-3074-1 Background Disparities have been documented in the prevalence of
overweight and obesity among low-income and ethnic
minority populations. For example, in the United States,
78 % of Latinos are overweight or obese compared with
67 % of non-Latino whites [1]. Several public health
intervention
strategies
have been
implemented
and
tested to reduce disparities in obesity. These include
individual-level programs to improve physical activity
and dietary habits [2–4], as well as community-level so-
cial marketing and education campaigns [5, 6]. Findings on the impact of corner store interventions
to improve the food environment in low-income com-
munities have been mixed [10, 11]. For example, an
intervention to improve healthy food access and market-
ing in corner stores and markets in Baltimore found * Correspondence: ano37@drexel.edu
1Department of Health Management and Policy, Dornsife School of Public
Health, Drexel University, 3215 Market Street, Nesbitt Hall, Room 335,
Philadelphia, PA 19104, USA
Full list of author information is available at the end of the article Ortega et al. BMC Public Health (2016) 16:389 Page 2 of 10 positive changes in healthy food purchasing and prepar-
ation among patrons and individuals from local commu-
nity
organizations
[12]. A
review
of
small-store
intervention studies found that interventions were able
to increase the availability of healthy items and improve
knowledge about health and nutrition [10]. Findings
were mixed, however, in terms of sales and purchasing
of healthy food items, perceived availability of healthy
items, and behavioral intentions to purchase healthy
items [10]. Similarly, a small store intervention in North
Carolina aimed at promoting sales of fruits and vegeta-
bles had mixed success. Results indicated that stores in-
creased the availability of vegetables but not fruit, and
there were no differences in consumption of either vege-
tables or fruits as a result of the intervention [8]. In an-
other intervention, the nutritional content of purchases
remained unchanged following a large-scale effort to in-
crease the availability of healthier products in Philadel-
phia corner stores [13]. evaluation time frames, and examination only of changes
among store patrons without attention to potential changes
at the community level [10, 11]. The intervention: Proyecto MercadoFRESCO
The intervention, Proyecto MercadoFRESCO, has been
described in greater detail elsewhere [16]. In brief, the
intervention was based in two urban communities in
Los Angeles (LA) County, California, East LA and Boyle
Heights. These neighboring communities are comprised
of majority Mexican-American populations. Background In particu-
lar, 97.1 % of the 2010 Census population in East LA
was Latino, making it the proportionally largest Latino
population in the US [17]. Both of these communities
have a high prevalence of overweight and obesity. For
example, nearly 77 % of East LA residents are over-
weight or obese [18]. Both communities have been char-
acterized as food swamps, within which residents are
faced with a disproportionate number of places to pur-
chase unhealthy foods (e.g., fast food restaurants, taco
stands, and corner stores) and have fewer options for
healthy food purchasing than in more affluent areas. Despite these mixed findings, food environment inter-
ventions have gained considerable traction among fed-
eral and state policymakers, as well as private funders. For example, in 2011, the Federal Government an-
nounced the Healthy Food Financing Initiative, a $400
million program to improve access to healthy foods and
to eliminate food deserts in the U.S. [14]. Similarly, the
California FreshWorks fund is a $272 million public-
private partnership loan fund that finances new and
upgraded grocery stores to improve healthy food access
to low-income communities [15]. The high cost of food
environment interventions underscores the need for
rigorous studies to evaluate best practices for reducing
community-level obesity. y
g
The project was community-engaged in order to cre-
ate a sense of “ownership” within the community. A
broad range of community partners including business
owners, schools, community-based organizations, local
politicians, and a community health center participated
on the community advisory board (CAB) and helped
guide the project on all aspects of the intervention de-
sign, implementation and evaluation. Additionally, the
study included a formative research phase in which ten
focus groups, with a total of 92 community members,
were held to elicit residents’ perceptions about corner
stores and their views about the food environment and
facilitators and barriers to purchasing, preparing, and
consuming healthy foods. In general, formative research
demonstrated that community residents had negative
perceptions regarding the quality, healthfulness, and af-
fordability of foods sold in local corner stores. Thus,
from the outset, the intervention sought to improve
community perceptions regarding corner stores in order
to increase patronage, purchasing, and consumption of
healthy foods sold at the stores. Overview of corner store conversions The components of corner store interventions are var-
ied, but they typically include changing the interior and
exterior of stores, modifying floor plans, installing new
shelving and refrigeration units, and increasing availabil-
ity of affordable fresh fruits and vegetables. Some inter-
ventions, however, have not included core elements of
formative research, such as assessing community health
practices and needs, determining residents’ perceptions
of food environments, and understanding neighborhood
contexts. This allows for program developers to implement
interventions that are accepted by community residents,
while also facilitating the development of community-
tailored social marketing campaigns, both of which can
help sustain the demand for the newly available products. Moreover, few interventions have explicitly included store
owner training on important practices such as managing
inventories and negotiating with wholesalers. In addition to
programmatic limitations, evaluations have been hampered
by small sample sizes, lack of comparison stores, short Four stores were recruited to be intervention stores,
and four additional stores were selected as comparisons. One of the intervention stores stopped implementation
activities during the early stages of the planned conver-
sion effort at the request of the store owner. At the ana-
lysis stage, this
store
was treated
as part
of the
comparison group, resulting in three intervention stores
and five comparison stores. The comparison stores were
located at least a mile away from intervention stores and
were separated by a major freeway, in order to limit the
potential for contamination effects. Ortega et al. BMC Public Health (2016) 16:389 Page 3 of 10 Page 3 of 10 question, “can community-level changes be achieved by
improving the food environment through corner store
conversions.” Based on a priori power calculations, the
target sample size was 1000 residential addresses at
baseline (i.e., 125 around each store) and an additional
1000 at follow-up. Sampling was conducted using a
multi-stage process. First, catchment areas were con-
structed for each of the intervention and comparison
stores. Catchment areas included the blocks closest in
proximity to each of the stores. Blocks were selected
until there were roughly 300 households in a catchment
area, which would allow for enough participants based
on expected occupancy, screening, and response rates. Next, households were randomly sampled from each
catchment area and approached for participation. Fi-
nally, the adult identified as the primary food purchaser
and preparer for the household was invited to partici-
pate. Overview of corner store conversions Respondents from homes surrounding converted
stores comprise the intervention group, while respon-
dents from homes around comparison stores make up
the comparison group. Intervention stores received a comprehensive “make-
over” to both the interior and exterior of the locations
including new signage and paint, security upgrades, store
layout alterations, product placement, and social market-
ing promotions for healthful eating. Each store was also
provided refrigeration equipment at no cost to display a
variety of newly available fresh fruits and vegetables. The
intervention specifically focused on increasing the avail-
ability of fruits and vegetables; however, storeowners
were autonomous in decisions regarding the variety and
quantity of produce in an effort to match these items to
the preferences and needs of local residents. The in-
crease was substantial, with most stores adding at least a
dozen new fruits and vegetables to their inventory. g
y
In addition to the store renovation, owners received
training on business practices provided by a former cor-
ner store owner with experience and expertise in con-
verting stores into healthier outlets. They received
training on issues relevant to the procurement and
handling of affordable fresh fruits and vegetables includ-
ing minimizing waste, meeting community demand, re-
moving
spoiled
inventory,
and
developing
business
relationships with produce wholesalers and local farmers'
markets. This training was included to increase store
owner engagement, maintain profitability, and enhance
sustainability. There was also a multi-component social
marketing campaign implemented in the study neighbor-
hoods. After stores were converted, they were monitored
for fidelity to ensure that stores stocked fresh fruits and
vegetables and that those items were of good quality. Comparison stores were also monitored throughout the
study period to ensure that no comparable improve-
ments were made to the appearance of the store or their
merchandise. At baseline, 1035 community residents completed a
household survey, for an American Association for Pub-
lic Opinion Research (AAPOR) response rate (i.e. num-
ber of completed interviews divided by the number of
individuals who were eligible) of 80 %. Twelve to 24-
months following baseline interviews, the same residen-
tial addresses were again approached for participation. In order to be eligible at follow-up, household residents
had to live in the neighborhood for at least one year. This minimum residency requirement necessitated add-
itional households to be sampled in order to achieve
roughly equal number of interviews. Overview of corner store conversions During the second
round of data collection, 1052 individuals completed the
survey for an AAPOR household follow-up response rate
of 75 %. Sixty-three percent of baseline respondents
were re-interviewed at follow-up. This paper will present findings from this community-
engaged, multi-level corner store intervention project. Baseline and follow-up findings from a survey of
community residents are examined with regard to
perceptions
of
the
food
environment
and
corner
stores as well as patronage, food purchasing, and con-
sumption behaviors. The interviews were administered face-to-face using
computer-assisted personal interviewing (CAPI) and
took approximately one and a half hours to complete. Respondents had the choice of completing the interview
in Spanish or English and received a $25 gift card to a
large local market for participation. Methods A household survey of neighborhood residents was ad-
ministered to assess the impact of the intervention at
the community-level. In-depth information regarding
the study protocol can be found elsewhere [16]. The
study was approved by the Institutional Review Board at
UCLA, and all study participants provided informed
consent. Measures Demographic measures included in the study were sex,
age (in years), marital status, nativity status (US-born/
foreign-born), Mexican heritage (yes/no), primary lan-
guage
spoken
at
home
(English-only,
bilingual,
or
Spanish-only), education (in years), and participation in
the
Special
Supplemental
Nutrition
Program
for
Women, Infants, and Children or Supplemental Nutri-
tion Assistance Program (yes/no). The study employed a repeated cross-sectional evalu-
ation design. In-person household interviews were con-
ducted in the neighborhoods surrounding the converted
and
comparison
stores
to
test
the
main
research Ortega et al. BMC Public Health (2016) 16:389 Page 4 of 10 Page 4 of 10 Page 4 of 10 This study focuses on five outcomes of interest: (1)
perceived accessibility of healthy foods, (2) perception of
corner stores, (3) corner store patronage, (4) food pur-
chasing, and (5) eating behaviors. Eating behaviors Participants were also asked to estimate how many serv-
ings of fruits and how many servings of vegetables they
eat each day. These two questions were taken from the
validated
Food
Behavior
Checklist
[20]
and
were
summed to reflect total daily fruit and vegetable intake. Perceptions of corner stores All
analyses
were
performed
using
STATA
14.0. Summary statistics were performed for all variables. Chi-
squared tests were used to assess whether the intervention
and comparison groups improved from baseline to follow-
up for the following binary outcome variables: healthy food
accessibility, corner store characteristics, and store patron-
age. Independent sample t-tests were used to identify statis-
tically significant differences in the summary perception
score, food purchasing, and food consumption between
time points. Differences between baseline and follow-up
were calculated for both the comparison group and the
intervention group for all variables of interest. To test for
statistically significant differences in these change scores,
the interaction of intervention status and time in regression
models was used. Fifteen items assessed participants’ perceptions about
corner stores. These items were developed by the re-
search team de novo and represent major themes identi-
fied during the formative research phase of the study,
such as convenience, price, quality, variety, and cus-
tomer service. Individuals were first asked if there is a
local corner store where they usually shop. Those indi-
viduals responding affirmatively were subsequently asked
to name the store, and the 15 items measuring percep-
tions were then asked about that specific store. Individ-
uals that did not have a usual corner store were asked
about their general perceptions of corner stores. Respon-
dents were asked to indicate whether they believed state-
ments about corner stores were true or false. Responses
indicating a positive perception towards a corner store
were coded as 1 while negative and “don’t know” re-
sponses were coded as 0. A score summing the number
of positive perceptions across the 15 questions was also
calculated (range: 0–15; Cronbach alpha: 0.83). Multiple logistic regression models were fitted to esti-
mate odds ratios for perceptions of food accessibility for
time, intervention status, and the interaction between
time and intervention status while controlling for sex,
age (years), nativity status, language spoken at home,
education (years), and food assistance. Multiple linear
regressions modeling the summary corner store percep-
tion score, food purchasing, and food consumption were
performed controlling for the same variables as previ-
ously described. Statistical analyses The analyses conducted for this study included partici-
pants with data at either or both baseline and follow-up. Additionally, only those individuals with data for all vari-
ables of interest were included in the final analyses,
yielding an analytic sample of 1686 household interviews
(baseline n = 795; follow-up n = 891). Comparability in
demographic characteristics between the full sample and
the subsample of participants with no missing data was
assessed. There were no statistically significant differ-
ences between the groups, suggesting that the subsample
is a reasonably good representation of the sample as a
whole. Accessibility of healthy foods Four
questions
measured
perceived
accessibility
of
healthy foods: (a) you have a convenient place to buy
healthy food, (b) healthy food is too expensive, (c) it is
hard to find places in your neighborhood where you can
buy healthy foods, and (d) the healthy foods sold in your
neighborhood are of low quality. Respondents were
asked to indicate if these statements were true or false. Although these questions were newly developed, they
were consistent in content with a previous study [19]. These items were explored as separate outcomes esti-
mating individuals’ perceptions of neighborhood healthy
food accessibility. Store patronage Store patronage was counted as affirmative when a re-
spondent reported having purchased food on at least
one occasion at an intervention or comparison store. Finally, sensitivity analyses were conducted to con-
trast the findings using the sample of all available
respondents with the findings from the subset of indi-
viduals who completed interviews at both baseline and
follow-up (n = 650). Using conditional logistic regres-
sions for binary variables and linear regressions with
random effects for continuous variables, these analyses
accounted for the within-person associations that arise
from repeated measures on an individual. Results percentage were of Mexican heritage and a smaller per-
centage were Spanish-only speakers than the comparison
sample. At baseline for both the intervention and comparison
samples, the majority of participants were female, youn-
ger than 50, married or living with partner, foreign-born,
of Mexican-heritage, had less than a high school educa-
tion, and did not receive food assistance (see Table 1). The only difference between the groups was that a
smaller proportion of respondents in the intervention
sample spoke only Spanish as compared to the compari-
son sample. Food purchasing In order to assess food purchasing behaviors, partici-
pants were asked to estimate the total number of dollars
spent per week on all food, as well as how much per
week was spent on canned, frozen, or fresh fruits and
vegetables. A percentage of the total amount spent on
fruits and vegetables was then calculated by dividing the
amount spent on produce by the total spent on all food. Ortega et al. BMC Public Health (2016) 16:389 Page 5 of 10 Perceptions of healthy food accessibility As Table 2 shows, there was a non-statistically signifi-
cant increase between baseline and follow-up in the per-
centage of intervention neighborhood residents who
reported having a convenient place to buy healthy food,
while there was a statistically significant improvement
among residents of comparison neighborhoods. Partici-
pants from both communities were less likely to perceive
challenges in finding places within their neighborhoods At follow-up participants had similar demographic
characteristics. The only differences between the samples
at that time were in the intervention sample a larger Table 1 Characteristics of Proyecto MercadoFRESCO Sample (N = 1686)
Baseline Intervention
Percent or Mean (SD)
Baseline Comparison
Percent or Mean (SD)
Follow-up Intervention
Percent or Mean (SD)
Follow-up Compariso
Percent or Mean (SD)
N = 313
N = 482
N = 323
N = 568
Characteristics
Sex
Male
21.7
22.4
17.3
21.0
Female
78.3
77.6
82.7
79.0
Age (Years)
44.4 (15.5)
44.3 (15.9)
47.1 (14.8)
46.4 (15.4)
Marital Statusa
Single
23.6
21.8
22.4
21.9
Married/With Partner
56.2
58.5
58.1
60.9
Separated/Divorced/Widowed
20.1
19.7
19.6
17.3
Nativity
U.S. Born
37.4
35.9
34.7
33.5
Foreign Born
62.6
64.1
65.3
66.5
Mexican Heritageb
Yes
89.2
84.9
95.3
88.5**
No
10.8
15.1
4.7
11.5
Language Spoken at Home
English Only
13.7
14.9**
12.7
8.6**
English and Spanish
56.2
45.2
67.5
62.1
Spanish Only
32.0
39.8
19.8
29.2
Language of Interview
English
43.1
39.6
43.0
40.3
Spanish
56.9
60.4
57.0
59.7
Education (years)
9.9 (4.1)
10.3 (3.9)
10.1 (4.3)
10.0 (4.1)
Food Assistancec
Yes
26.8
31.5
28.2
31.5
No
73.2
68.5
71.8
68.5
NOTE: Some percentages may not sum to 100 due to rounding. Significant differences between intervention and comparison in categorical variables were tested
at baseline and follow-up using chi-squared tests. Perceptions of corner stores Improvements were found in perceptions of corner
stores from baseline to follow-up for both the interven-
tion and comparison samples (see Table 3). A larger pro-
portion of participants at follow-up reported that corner
stores sell a wide variety of fresh, frozen, and canned
fruits and vegetables, as well as healthy foods in general. Only in the comparison sample did perceptions of fruit
quality improve, while only in the intervention sample
did perceptions of vegetable quality improve. Fruit and vegetable consumption Table 4 shows that consumption of fruits and vegetables
did not change over time among respondents from ei-
ther sample. A test for an intervention effect was also
non-significant (see Additional file 3: Table S3). when testing for an interaction between time and inter-
vention status. when testing for an interaction between time and inter-
vention status. to buy healthy foods at follow-up. No changes were
found between baseline and follow-up regarding per-
ceived expense or quality of healthy food in either neigh-
borhood, even though the majority of respondents
indicated that healthy food was too expensive and that
the quality of food sold was poor. Additionally, no inter-
vention effects were found when testing for an inter-
action
between
time
and
intervention
status
(see
Additional file 1: Table S1). to buy healthy foods at follow-up. No changes were
found between baseline and follow-up regarding per-
ceived expense or quality of healthy food in either neigh-
borhood, even though the majority of respondents
indicated that healthy food was too expensive and that
the quality of food sold was poor. Additionally, no inter-
vention effects were found when testing for an inter-
action
between
time
and
intervention
status
(see
Additional file 1: Table S1). Store patronage The proportion who reported patronizing one of the
study stores remained consistent between baseline and
follow-up (see Table 3) for both intervention and com-
parison samples. A larger percentage of respondents
from the intervention neighborhoods reported shopping
at these study stores at both times. Food purchasing
d ff No differences were observed between baseline and
follow-up for the intervention or comparison samples in
the percent of total dollars spent on fruits and vegetables
per week (see Table 4). Moreover, the interaction be-
tween intervention condition and time was not statisti-
cally significant, suggesting that the intervention had no
effect on food purchasing (see Additional file 3: Table
S3). Corner store characteristics associated with store con-
versions improved over time for the intervention group
only. Specifically, perceptions regarding the cleanliness
of corner stores, safety of corner stores, quality of cus-
tomer service, and ability to get information about
healthy eating and nutrition all improved between base-
line and follow-up. Participants from both samples re-
ported increased availability of culturally appropriate
Latino ingredients and language concordance between
customers and staff at follow-up. Perceptions of healthy food accessibility Significant differences between intervention and comparison in continuous variables were tested at baseline
and follow-up independent sample t-tests tests
**p <0 .01
aData were only available for 332 (99.8 %) for the follow-up intervention group and 567 (99.8 %) observations for the follow-up control group due to missing dat
bData were only available for 306 (97.8 %) for the baseline intervention group, 469 (97.3 %) observations for the baseline control group, 318 (98.4 %) for the
follow-up intervention group, and 555 (97.7 %) for the follow-up control group due to missing data
cParticipation in Special Supplemental Nutrition Program for Women, Infants or Children (WIC) or Supplemental Nutrition Assistance Program (SNAP) Table 1 Characteristics of Proyecto MercadoFRESCO Sample (N = 1686) Table 1 Characteristics of Proyecto MercadoFRESCO Sample (N = 1686) cParticipation in Special Supplemental Nutrition Program for Women, Infants or Children (WIC) or Supplemental Nutrition Assistance Ortega et al. BMC Public Health (2016) 16:389 Ortega et al. BMC Public Health (2016) 16:389 Page 6 of 10 Table 2 Perceptions about Food Accessibility by Intervention Status and Time
Intervention Percent
Comparison Percent
Percent Difference
(Follow-up – Baseline)
Baseline
Follow-up
Baseline
Follow-up
Intervention
Comparison
(N = 313)
(N = 323)
(N = 482)
(N = 568)
You have a convenient place where you can buy healthy food
83.4
88.2
77.8
88.2***
4.8
10.4
Healthy food is too expensive
52.4
53.6
55.2
53.0
1.2
−2.2
It’s hard to find places in your neighborhood where you can buy
healthy foods
39.3
31.3*
39.0
33.1*
−8.0
−5.9
The healthy foods sold in your neighborhood are low quality
56.2
51.7
63.5
57.6
−4.5
−5.9
NOTES: Significant differences were tested between intervention baseline and follow-up using chi-squared tests, comparison baseline and follow-up using
chi-squared tests, and percent difference (follow-up – baseline) for intervention and comparison using a Wald test on the interaction term of a logistic regression
(more details can be seen in Additional file 1: Table S1). Perceptions of healthy food accessibility This Wald test can be thought of as testing whether the relative change (on an odds ratio scale) is the
same in the intervention and comparison groups
*p < 0.05,***p < 0.001 Table 2 Perceptions about Food Accessibility by Intervention Status and Time NOTES: Significant differences were tested between intervention baseline and follow-up using chi-squared tests, comparison baseline and follow-up using
chi-squared tests, and percent difference (follow-up – baseline) for intervention and comparison using a Wald test on the interaction term of a logistic regression
(more details can be seen in Additional file 1: Table S1). This Wald test can be thought of as testing whether the relative change (on an odds ratio scale) is the
same in the intervention and comparison groups
*p < 0 05 ***p < 0 001 Sensitivity analyses The intervention effects of the individual items meas-
uring corner store perceptions were mixed, with three
significant interactions showing more improvements in
comparison store, but they mainly showed patterns of
non-significance (See Additional file 2: Table S2). The
summary index score, which is a more robust measure
than the individual items, showed marked improve-
ments over time in both samples (See Additional file 3:
Table S3). No significant intervention effect was found In separate sensitivity analyses that are not shown, re-
sults indicated that the main findings presented above
were robust regardless if the data were limited to those
participants who had responses at both time points, ac-
counting for correlation between repeated measure-
ments from the same respondent. The fact that different
analytical strategies yielded qualitatively similar results
provide reassurance that the main findings of the study
were not artifacts of analytical choices. Ortega et al. Sensitivity analyses BMC Public Health (2016) 16:389 Page 7 of 10 Table 3 Perceptions about Corner Stores and Patronage by Intervention Status and Time
Intervention
Comparison
Percent or Mean Difference
(Follow-up – Baseline)
Percent or Mean (SD)
Percent or Mean (SD)
Baseline
Follow-up
Baseline
Follow-up
Intervention
Comparison
(N = 313)
(N = 323)
(N = 482)
(N = 568)
Corner Store Characteristics
Corner stores sell a wide variety of fresh fruits
8.9
26.3***
18.5
26.6**
17.4
8.1
Corner stores sell a wide variety of fresh vegetables
10.9
35.6***
19.5
29.6***
24.7
10.1
Corner stores sell a wide variety of frozen or canned fruits
27.2
36.2*
31.1
42.3***
9.0
11.2***
Corner stores sell a wide variety of frozen or canned vegetables
25.9
35.3*
30.5
41.7***
9.4
11.2**
Fresh fruits sold at corner stores are not of poor qualitya
40.0
45.5
44.2
51.4*
5.5
7.2
The fresh vegetables sold at corner stores are of good quality
22.7
44.0***
25.1
30.3
21.3
5.2
Corner stores sell healthy food
34.2
51.4***
37.6
45.2*
17.2
7.6
Corner stores are not dirtyb
60.1
77.4***
66.2
68.8
17.3
2.6
Corner stores are not dangerousc
70.0
83.9***
75.5
75.2
13.9
−0.3
Corner stores have good customer service
73.5
85.4***
70.5
75.0
11.9
4.5
I can get information about nutrition and healthy eating at
corner stores
10.2
28.5***
12.9
16.2
18.3
3.3
Corner stores sell traditional Latino food ingredients
77.0
83.6*
76.6
83.5**
6.6
6.9***
The staff at corner stores speaks my language
86.3
92.0*
82.8
90.7***
5.7
7.9
Food sold at corner stores is not expensived
20.4
22.6
28.2
28.0
2.2
−0.2
It is convenient to shop at corner stores
47.6
50.5
49.4
52.3
2.9
2.9
Overall Beliefs About Corner Stores Score (Range: 0-15
6.1 (3.4)
8.0 (3.6)***
6.7 (3.4)
7.6 (3.6)***
1.9
1.0
Corner Store Patronage
Shops at 1 or more study stores
41.5
46.7
28.2
23.4
5.2
−4.8***
NOTES: Significant differences in binary variables were tested between intervention baseline and follow-up using chi-squared tests, comparison baseline and
follow-up using chi-squared tests, and percent difference (follow-up – baseline) for intervention and comparison using a Wald test on the interaction term of a
logistic regression (more details can be seen in Additional file 2: Table S2). This Wald test can be thought of as testing whether the relative change (on an odds
ratio scale) is the same in the intervention and comparison groups. Sensitivity analyses Significant differences in continuous variables were tested between intervention baseline and
follow-up using independent sample t-tests, comparison baseline and follow-up using independent sample t-tests tests, and mean difference (follow-up – baseline) for
intervention and comparison using a F-test on the interaction term of a linear regression (more details can be seen in Additional file 3: Table S3)
*p < 0.05, **p < 0.01, ***p < 0.001
aQuestion was reverse coded. Original statement was “Fresh fruits sold at corner stores are of poor quality”
bQuestion was reverse coded. Original statement was “Corner stores are dirty”
cQuestion was reverse coded. Original statement was “Corner stores are dangerous”
dQuestion was reverse coded. Discussion most prior studies. This study was among the most
rigorously
designed
and
implemented
interventions
aimed at modifying the built environment, yet no signifi-
cant changes in dietary behavior were found. Corner store conversions, particularly in urban and
rural food deserts and swamps, have emerged as a
popular strategy to improve healthy food access, des-
pite limited evidence on their effectiveness [10, 11]. This study, in two neighboring Latino food swamps,
found that regardless of intervention status, percep-
tions of food accessibility and corner stores improved
over time, but effects on patronage, purchasing and
consumption of healthy foods were non-significant. In
general, the mixed findings are consistent with the re-
sults of other studies [8, 10, 11, 13]. Our findings are consistent with similar types of inter-
ventions that have also found that altering the food en-
vironment does not necessarily lead to improved healthy
eating behaviors. For example, a grocery store interven-
tion in North Philadelphia raised
awareness about
healthy eating but did not impact fruit and vegetable in-
take [19]. Similarly, a Whole Foods grocery store that
was introduced into a Detroit food desert did not im-
prove patronage or consumption of healthy food items
among local residents; rather, the store was largely sup-
ported by professionals who worked in the area [23]. A
recent study detailing the results of a new supermarket
in the South Bronx, New York, found no observable
changes in fruit and vegetable consumption or overall
diet quality one year after the store opening [24]. These
and other findings suggest that simply improving access
to healthy foods does not necessarily translate into im-
proved dietary behaviors within the community. While there were modest improvements in percep-
tions of the food environment, these changes did not ap-
pear to be attributable to the intervention alone. One
explanation is that other local initiatives were underway
in the two communities during the study period. Initia-
tives have included providing health education and pro-
moting access to healthy food [21, 22]. The combination
of initiatives might have collectively improved percep-
tions of the food environment over time. Nevertheless,
because improvements were not found in key behavioral
outcomes associated with obesity, these findings chal-
lenge the notion that simply changing the food environ-
ment will improve eating behaviors. Discussion Alternatively, urban
food swamps may be so densely-populated with low-
cost, unhealthy food options that modest interventions
at a limited number of retailers may be insufficient to
improve health behaviors and outcomes. Similarly, a 2008 LA zoning regulation banned new or re-
modeled fast food restaurants in South LA, another urban
food swamp; this ban did not result in lowering the preva-
lence of obesity as expected [25]. The policy directive, how-
ever, lacked the inclusion of important public health
intervention initiatives such as raising awareness about
healthy eating, reducing the number of venues that sell
unhealthy foods, and offering economic disincentives, such
as taxes on unhealthy food, to promote community health. This study makes a number of contributions to the
science of food environment interventions. Most not-
ably, the study used community engagement to under-
stand perceptions, needs and demands about food
accessibility and healthy eating, which informed the de-
sign, implementation, and evaluation of the intervention. Moreover, the intervention had a variety of community
partners who participated in the conversions, marketing
campaigns, and evaluation [16]. y
p
y
Reducing disparities in obesity has proven to be diffi-
cult. The public health field continues to grapple with
the best strategies for using the ecological framework
in health disparities interventions, including imple-
menting multi-level intervention projects that have
large and sustainable population impact. Albeit limited,
evidence has demonstrated that these approaches can
in fact be effective. For example, a community-based
participatory research intervention in Somerville, Mas-
sachusetts resulted in significant reductions in child
and parent obesity [26, 27]. This intervention had com-
prehensive partnerships that allowed for effective planning,
implementation, and dissemination. Food environment in-
terventions need to be comprehensive in scope and address
multiple components of the social determinants of health
framework [28] through public-private partnerships, with
local, regional, and national policy support, in order to im-
prove healthy eating and ultimately decrease the prevalence
of obesity among the most vulnerable populations. In addition to the community-wide social marketing,
the study benefited from the ability to provide store
owners with technical assistance to facilitate purchasing
and selling of fresh produce. In short, because the inter-
vention supplemented the physical transformations of
the stores with these additional resources and support
systems, the conversions approximate the ‘best case sce-
nario’ of what might be expected of interventions in a
limited number of small food stores’ offerings. Discussion Another advantage was that this study utilized com-
parison stores, which helped to answer the larger policy
question as to whether this type of intervention will lead
to community-level changes. The study also allowed suf-
ficient time to detect changes, with up to two years be-
tween baseline and follow-up data collection. Finally, the
survey had a relatively large sample size compared with Sensitivity analyses Original statement was “Food sold at corner stores is expensive” Table 3 Perceptions about Corner Stores and Patronage by Intervention Status and Time Table 4 Food Purchasing and Fruit and Vegetable Consumption by Time and Intervention Status
Intervention
Comparison
Mean Difference
(Follow-up – Baseline)
Mean (SD)
Mean (SD)
Baseline
Follow-up
Baseline
Follow-up
Intervention
Comparison
(N = 313)
(N = 323)
(N = 482)
(N = 568)
Dollars spent on food per weeka
131.6 (64.0)
140.6 (126.0)
123.1 (66.4)
134.8 (74.8)**
9.0
11.7
Dollars spent on fruits and vegetables per weeka
46.2 (28.4)
49.8 (33.9)
44.6 (29.7)
47.2 (32.6)
3.6
2.6
Percent of dollars spent on fruits and vegetables
36.5 (16.7)
38.1 (17.6)
37.7 (18.0)
36.9 (18.7)
1.6
−0.8
Servings of fruits and vegetables consumed each day
4.4 (2.0)
4.2 (2.1)
4.5 (2.4)
4.8 (2.3)
−0.2
0.3
NOTES: Significant differences in continuous variables were tested between intervention baseline and follow-up using independent sample t-tests, comparison
baseline and follow-up using independent sample t-tests tests, and mean difference (follow-up – baseline) for intervention and comparison using a F-test on the
interaction term of a linear regression (more details can be seen in Additional file 3: Table S3)
**p < 0.01
aThe significance of the mean difference was not tested, as this variable was only of interest to calculate percent of dollars spent on fruits and vegetables Table 4 Food Purchasing and Fruit and Vegetable Consumption by Time and Intervention Status NOTES: Significant differences in continuous variables were tested between intervention baseline and follow-up using independent sample t-tests, comparison
baseline and follow-up using independent sample t-tests tests, and mean difference (follow-up – baseline) for intervention and comparison using a F-test on the
interaction term of a linear regression (more details can be seen in Additional file 3: Table S3)
**
0 01 p < 0.01
aThe significance of the mean difference was not tested, as this variable was only of interest to calculate percent of dollars spent on fruits and vegetables Ortega et al. BMC Public Health (2016) 16:389 Page 8 of 10 Page 8 of 10 Competing interests 17. U.S. Census Bureau. The Hispanic Population: 2010. 2011; Available from:
http://www.census.gov/prod/cen2010/briefs/c2010br-04.pdf. Accessed
July 29, 2015. 17. U.S. Census Bureau. The Hispanic Population: 2010. 2011; Available from:
http://www.census.gov/prod/cen2010/briefs/c2010br-04.pdf. Accessed
July 29, 2015. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding
Th
h The authors received support from the National Heart, Lung and Blood
Institute (NHLBI) (grants P50 HL105188 and R25 HL108854) of the National
Institutes of Health (NIH). Received: 13 January 2016 Accepted: 4 May 2016 Received: 13 January 2016 Accepted: 4 May 2016 Availability of data and materials Participant level data are available from the last author
MLP. 10. Gittelsohn J, Rowan M, Gadhoke P. Interventions in small food stores to
change the food environment, improve diet, and reduce risk of chronic
disease. Prev Chronic Dis. 2012;9:E59. 11. Langellier BA et al. Corner Store Inventories, Purchases, and Strategies for
Intervention: A Review of the Literature. Calif J Health Promot. 2013;11(3):1–13. 11. Langellier BA et al. Corner Store Inventories, Purchases, and Strategies for
Intervention: A Review of the Literature. Calif J Health Promot. 2013;11(3):1–13. Ethics approval and consent to participate 7. Hanni KD et al. Targeting the taqueria: implementing healthy food options
at Mexican American restaurants. Health Promot Pract. 2009;10(2 Suppl):
91S–9S. All study protocols were approved by the UCLA Insti-
tutional Review Board. Written informed consent was
obtained from all study participants. 8. Ayala GX et al. Efficacy of a store-based environmental change intervention
compared with a delayed treatment control condition on store customers’
intake of fruits and vegetables. Public Health Nutr. 2013;16(11):1953–60. 9. Ayala GX et al. A store-based intervention to increase fruit and vegetable
consumption: The El Valor de Nuestra Salud cluster randomized controlled
trial. Contemp Clin Trials. 2015;42:228–38. Limitations The study had a range of limitations. First, this study re-
lied on self-reports of behaviors that are subject to recall Ortega et al. BMC Public Health (2016) 16:389 Page 9 of 10 Page 9 of 10 Page 9 of 10 Page 9 of 10 and reporting biases. Second, due to factors not under
the control of the investigative team, one of the inter-
vention stores dropped out during the early stages of its
conversion, leading to it being considered as a compari-
son store in the analyses. Third, we did not have sales
data, as it was much more difficult than anticipated to
collect cash-register data from these small independently
owned stores. Fourth, because the study prioritized
interviewing the primary food purchaser and preparer,
the community sample is majority female, which may
limit the generalizability of the findings beyond primary
household food purchasers and preparers. Abbreviations
AAPOR A AAPOR: American Association for Public Opinion Research; CAPI: computer-
assisted personal interviewing.; LA: Los Angeles. AAPOR: American Association for Public Opinion Research; CAPI: computer-
assisted personal interviewing.; LA: Los Angeles. 16. Ortega AN et al. Proyecto MercadoFRESCO: A Multi-level, Community-
Engaged Corner Store Intervention in East Los Angeles and Boyle Heights. J Community Health. 2015;40(2):347–56. Additional files 12. Gittelsohn J et al. An urban food store intervention positively affects food-
related psychosocial variables and food behaviors. Health Educ Behav. 2010;37(3):390–402. Additional file 1: Table S1. Logistic Regression Predicting Food
Accessibility (N = 1686). (DOC 34 kb)
Additional file 2: Table S2. Logistic Regression Predicting Perceptions
about Corner Stores and Patronage (N = 1686). (DOC 47 kb)
Additional file 3: Table S3. Regression Models Predicting Purchasing,
Consumption and Corner Store Perceptions (N = 1686). (DOC 38 kb) Additional file 1: Table S1. Logistic Regression Predicting Food
Accessibility (N = 1686). (DOC 34 kb)
Additional file 2: Table S2. Logistic Regression Predicting Perceptions
about Corner Stores and Patronage (N = 1686). (DOC 47 kb)
Additional file 3: Table S3. Regression Models Predicting Purchasing,
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characteristics of adult, adolescent and child urban corner store purchases
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f 1Department of Health Management and Policy, Dornsife School of Public
Health, Drexel University, 3215 Market Street, Nesbitt Hall, Room 335,
Philadelphia, PA 19104, USA. 2Department of Community Health Sciences,
Fielding School of Public Health, University of California, Los Angeles, Los
Angeles, CA 90095, USA. 3Department of Biostatistics, Fielding School of
Public Health, University of California, Los Angeles, Los Angeles, CA 90095,
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4, 2015. ANO oversaw all aspects of the study’s design, implementation, analyses, and
manuscript development. SLA and AMCG led the data analyses,
interpretation, and drafting of the manuscript. BAL and MZS contributed to
the interpretation of study results and drafting of the manuscript. DCG, TRB,
RB, and MLP contributed to the study design, interpretation of data, and
edited the manuscript. REG led the acquisition of data efforts and edited the
manuscript. All authors read and approved the final manuscript. ANO oversaw all aspects of the study’s design, implementation, analyses, and
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from: http://www.choosehealthla.com/. Accessed 4 Aug 2015. . California Department of Public Health. Choose Health LA. 2015; A from: http://www.choosehealthla.com/. Accessed 4 Aug 2015. 22. The California Endowment. Available from: http://www.bhccon 22. The California Endowment. Available from: http://www.bhcconnect.org/
health-happens-here/boyle-heights. Accessed 4 Aug 2015. health-happens-here/boyle-heights. Accessed 4 Aug 2015. 23. McMillan, T. Can Whole Foods Change the Way Poor People Eat? Slate. 2014; Available from: http://www.slate.com/articles/life/food/2014/11/
whole_foods_detroit_can_a_grocery_store_really_fight_elitism_racism_and. html. Accessed August 4, 2015 24. Elbel B et al. The Introduction of a Supermarket via Tax-Credits in a Low-Income
Area: The Influence on Purchasing and Consumption. Am J Health Promot. 2015 Sep 21. [Epub ahead of print] doi:10.4278/ajhp.150217-QUAN-733. 25. Sturm R, Hattori A. Diet and obesity in Los Angeles County 2007–2012: Is
there a measurable effect of the 2008 “Fast-Food Ban”? Soc Sci Med. 2015;
133:205–11. 26. Economos CD et al. A community intervention reduces BMI z-score in
children: Shape up Somerville first year results. Obesity. 2007;15(5):1325–36. 27. Coffield E et al. Shape Up Somerville: Change in Parent Body Mass In
During a Child-Targeted, Community-Based Environmental Change
Intervention. Am J Public Health. 2015;105(2):E83–9. 28. Koh HK et al. Healthy People: A 2020 Vision for the Social Determinants
Approach. Health Educ Behav. 2011;38(6):551–7. 28. Koh HK et al. Healthy People: A 2020 Vision for the Social Determinants
Approach. Health Educ Behav. 2011;38(6):551–7. • We accept pre-submission inquiries
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Processing and Mechanical Properties of Macro Polyamide Fiber Reinforced Concrete
|
Materials
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cc-by
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Materials 2014, 7, 7634-7652; doi:10.3390/ma7127634 Materials 2014, 7, 7634-7652; doi:10.3390/ma7127634 Materials 2014, 7, 7634-7652; doi:10.3390/ma7127634
materials
ISSN 1996-1944
www.mdpi.com/journal/materials Review
Article
Processing and Mechanical Properties of Macro Polyamide
Fiber Reinforced Concrete
Joong Kyu Jeon 1,†, WooSeok Kim 2,†,*, Chan Ki Jeon 3,† and Jin Cheol Kim 4,†
1 Research and Business Development Center, Kolon Global Corp, Pogok-eup, Cheoin-gu,
Yongin-si, Gyeonggi-do 449-815, Korea; E-Mail: jkjeon31@kolon.comd
2 Department of Civil Engineering, Chungnam National University, 99, Daehak-ro, Yuseong-gu,
Daejeon 305-764, Korea
3 Department of Urban Construction Engineering, Incheon National University, 119, Academy-ro,
Yeonsu-gu, Incheon 406-772, Korea; E-Mail: johnland@incheon.ac.kr
4 Expressway and Transportation Research Institute, Korea Expressway Corp. 208-96,
Dongbu-daero 922 beon-gil, Dongtan-myeon, Hwaseong-si, Gyeonggi-do 445-810, Korea;
E-Mail: jckim@ex.co.kr
† These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: wooseok@cnu.ac.kr;
Tel.: +82-42-821-6584; Fax: +82-42-825-0318. External Editor: Luciano Feo
Received: 28 August 2014; in revised form: 20 October 2014 / Accepted: 17 November 2014 /
Published: 26 November 2014
Abstract: This study developed a macro-sized polyamide (PA) fiber for concrete
reinforcement and investigated the influence of the PA fiber on flexural responses in
d
ith ASTM t
d d
PA fib
d
t
d t
t
l fib
th t
OPEN ACCESS Processing and Mechanical Properties of Macro Polyamide
Fiber Reinforced Concrete Joong Kyu Jeon 1,†, WooSeok Kim 2,†,*, Chan Ki Jeon 3,† and Jin Cheol Kim 4,† 1. Introduction Randomly distributed short fiber reinforcements are used to improve the brittle characteristics of
concrete. The reinforcements, including steel fibers [1–5], carbon fibers and polymer fibers [6–10],
are used to control the initiation and propagation of cracks [11–15]. The fibers hold together the cracks
and reduce potential problems such as water permeating causing steel corrosion and deterioration. The resulting fiber reinforced concrete (FRC) exhibits superior performance compared to plain concrete
due to its high tensile strength and ductile tensile behavior. Application of FRCs is increasing recently
in areas requiring extreme mechanical and environmental loadings. Steel fiber reinforced concrete has both high tensile strength and bending strength and controls
cracks. However, gravitation and corrosion of the steel fiber may occur, and rebounding during tunnel
shotcrete application due to low adhesion characteristics causes lower than expected strength and higher
costs [16]. Organic fiber usually has lower elastic modulus and tensile strength compared to steel fiber
and is easily tangled, causing low workability. Thus, for practical use in reinforced concrete, organic
fiber requires improved mechanical characteristics and workability. This study developed polyamide fibers with optimized features for reinforcing concrete, to improve
the mechanical properties and workability of organic fiber reinforced concrete. Typically, concrete is
comprised of multiple phases, including micron-scale phases for the C-S-H gel, millimeter-scale for
sands, and centimeter-scale for aggregates, and this multiphase composition results in complex-cracking
behavior [17,18]. However, since cracks usually occur along the aggregates, it is consequently necessary
for reinforcing fiber to perform in the macro-scale. Thus, this study developed and investigated
macro-sized, or bundled, polyamide (PA) fiber, tested and optimized the PA fiber bundles, and
investigated the properties of PA fiber reinforced concrete (PAFRC). The polyamide fiber bundles were made of approximately 384 micro-size polyamide fibers
(diameter = 19.5 μm), and the bundled format improved adhesion to cement paste. Polyamide fiber has
improved mechanical properties compared to polypropylene (PP) fiber, and lower weight and density
and no corrosion compared to steel fiber. Polypropylene fiber also gets popularity due to concrete
performance improvement under crack opening and slippage [4]. These improved mechanical properties
for compression, tension, flexure, impact blows and plastic shrinkage cracking [5] and adhesion
characteristics lead to improved workability and less rebounding when spouting shotcrete. Joong Kyu Jeon 1,†, WooSeok Kim 2,†,*, Chan Ki Jeon 3,† and Jin Cheol Kim 4,† p
p
g
p
g
Yongin-si, Gyeonggi-do 449-815, Korea; E-Mail: jkjeon31@kolon.comd
2 Department of Civil Engineering, Chungnam National University, 99, Daehak-ro, Yuseong-gu,
Daejeon 305-764, Korea 2 Department of Civil Engineering, Chungnam National University, 99, Daehak-ro, Yuseong-gu,
Daejeon 305-764, Korea 2 Department of Civil Engineering, Chungnam National University, 99, Daehak-ro, Yuseong-gu,
Daejeon 305-764, Korea 3 Department of Urban Construction Engineering, Incheon National University, 119, Academy-ro,
Yeonsu-gu, Incheon 406-772, Korea; E-Mail: johnland@incheon.ac.kr 3 Department of Urban Construction Engineering, Incheon National University, 119, Academy-ro,
Yeonsu-gu, Incheon 406-772, Korea; E-Mail: johnland@incheon.ac.kr 4 Expressway and Transportation Research Institute, Korea Expressway Corp. 208-96,
Dongbu-daero 922 beon-gil, Dongtan-myeon, Hwaseong-si, Gyeonggi-do 445-810, Korea;
E-Mail: jckim@ex.co.kr 4 Expressway and Transportation Research Institute, Korea Expressway Corp. 208-96,
Dongbu-daero 922 beon-gil, Dongtan-myeon, Hwaseong-si, Gyeonggi-do 445-810, Korea;
E-Mail: jckim@ex.co.kr † These authors contributed equally to this work. † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: wooseok@cnu.ac.kr;
Tel.: +82-42-821-6584; Fax: +82-42-825-0318. * Author to whom correspondence should be addressed; E-Mail: wooseok@cnu.ac.kr;
Tel.: +82-42-821-6584; Fax: +82-42-825-0318. Received: 28 August 2014; in revised form: 20 October 2014 / Accepted: 17 November 2014 /
Published: 26 November 2014 Received: 28 August 2014; in revised form: 20 October 2014 / Accepted: 17 November 2014 /
Published: 26 November 2014 Abstract: This study developed a macro-sized polyamide (PA) fiber for concrete
reinforcement and investigated the influence of the PA fiber on flexural responses in
accordance with ASTM standards. PA fibers are advantageous compared to steel fibers that
are corrosive and gravitated. The macro-sized PA fiber significantly improved concrete
ductility and toughness. Unlike steel fibers, the PA fibers produced two peak bending
strengths. The first-peaks occurred near 0.005 mm of deflection and decreased up to 0.5 mm
of deflection. Then the bending strength increased up to second-peaks until the deflections
reached between 1.0 and 1.5 mm. The averaged flexural responses revealed that PA fiber
content did not significantly influence flexural responses before L/600, but had significant
influence thereafter. Toughness performance levels were also determined, and the results
indicated more than Level II at L/600 and Level IV at others. Materials 2014, 7 7635 Materials 2014, 7 Materials 2014, 7 Keywords: fiber; polyamide; mechanical properties; mechanical testing; extrusion 1. Introduction The objective of this study is to provide the PA fiber processing technique and to report the physical
and mechanical properties of PAFRC in terms of compressive and flexural responses in accordance with
ASTM C1609/C 1609M-05 and C1018-97 [19,20]. 7636 Materials 2014, 7 Materials 2014, 7 2.1. Macro PA Fiber Development and Processing Polyamide (PA) fiber is one of the synthetic fibers commonly used in textiles. A micro-size polyamide
fiber is presented in Figure 1. Polyamide fibers are produced through a shaped nozzle, and the thickness
of the fiber is constant throughout its length. A dispersant is used during the production of PA filaments
to prevent the fibers tangling, as shown in Figure 2. The dispersant is composed of hydrophilic and
lipophilic groups made of 40% to 50% polyalcohol ester lubricant, 30% to 40% nonionic surfactant
solution and 10% to 30% anti-static agent. In concrete, the hydrophilic group, having a negative charge,
causes repulsion between fibers, prevents the fibers tangling, and improves fiber dispersion. The hydrophilic group induces hydrogen bonding and leads to improved adhesion between the fibers
and cement paste. Also, the dispersant coating during the filament production decreases fiber elongation
and increases fiber strength. Figure 1. Picture of micro polyamide fibers. (a) Single polyamide fiber production;
(b) magnification of single polyamide fiber. e 1. Picture of micro polyamide fibers. (a) Single polyamide fiber production;
agnification of single polyamide fiber. (a)
(b)
Figure 2. PA fiber (Diameter: 23 μm) with dispersant coating. 23 ㎛ (a)
(b)
Figure 2. PA fiber (Diameter: 23 μm) with dispersant coating. 23 ㎛ (a) (b)
23 ㎛ (b) (a) Figure 2. PA fiber (Diameter: 23 μm) with dispersant coating. This study used a fiber fabrication method of air-textured yarn to develop bundled PA fibers in
a macro size. In the production process two-polyamide filaments, one for the core yarn and one for
the effect yarn (see Figure 3a), are injected into the nozzle with oblique high-pressure air-flow (see
Figure 3b). The injection speed of the effect yarn is slower than the core yarn and a number of loops are
subsequently formed due to the difference in injection air-flow speed. After passing the air nozzle, the
effect yarn length is longer than the core yarn, and the fiber is then stabilized with a proper temperature
treatment, preventing separation of the yarn. Polyamide fiber produced by the air textured yarn method
is shown in Figure 4. The loops and bulking of the textured yarn allows cement paste to pass through 7637 Materials 2014, 7 Materials 2014, 7 the yarn, and produces an increased surface area for contact between the cement paste and yarns,
resulting in better adhesion. It is expected that this improved adhesion property will produce higher
flexural responses compared to polypropylene (PP) fibers, which are mono-filaments. Figure 3. Production process of bundled PA fiber using air textured yarn. (a) Air texturing
process; (b) air nozzle; (c) PA fiber (30 mm × 0.47 mm). (a)
(b)
(c)
Figure 4. PA fiber processed by air textured yarn (Optical microscope ×8). (a) PA fiber
before temperature treatment; (b) PA fiber after temperature treatment. (b) (c) (a) (b) (c) (a) Figure 4. PA fiber processed by air textured yarn (Optical microscope ×8). (a) PA fiber
before temperature treatment; (b) PA fiber after temperature treatment. (a)
(b)
The bundled PA fibers developed by this study have a larger fiber size and greater number of
filaments compared to PP fibers and other organic fibers. To determine the number of PA filaments
needed to fit the desired mechanical properties, this study tested and optimized the number of PA
filaments at 1,800 Denier and 384 filaments of PA fiber. The mechanical properties of the PA fiber are
tabulated in Table 1 and compared to polypropylene (PP) fibers. For PP fibers, BarChip Macro produced
by Elasto Plastic Concrete [21], which is a synthetic fiber frequently used in Europe [18], was selected. (a) (b) (a) (b) The bundled PA fibers developed by this study have a larger fiber size and greater number of
filaments compared to PP fibers and other organic fibers. To determine the number of PA filaments
needed to fit the desired mechanical properties, this study tested and optimized the number of PA
filaments at 1,800 Denier and 384 filaments of PA fiber. The mechanical properties of the PA fiber are
tabulated in Table 1 and compared to polypropylene (PP) fibers. For PP fibers, BarChip Macro produced
by Elasto Plastic Concrete [21], which is a synthetic fiber frequently used in Europe [18], was selected. The bundled PA fibers developed by this study have a larger fiber size and greater number of
filaments compared to PP fibers and other organic fibers. To determine the number of PA filaments
needed to fit the desired mechanical properties, this study tested and optimized the number of PA
filaments at 1,800 Denier and 384 filaments of PA fiber. Materials 2014, 7 The mechanical properties of the PA fiber are
tabulated in Table 1 and compared to polypropylene (PP) fibers. For PP fibers, BarChip Macro produced
by Elasto Plastic Concrete [21], which is a synthetic fiber frequently used in Europe [18], was selected. Table 1. Material properties of PA and PP fibers. Table 1. Material properties of PA and PP fibers. Fiber
Type
Density
(g/cm3)
Length
(mm)
Diameter
(mm)
Elastic Modulus
(GPa)
Tensile Strength
(MPa)
PA
1.14
30
0.47
Min. 3
650
*PP
0.9–0.92
42
**Rec. 8.2
550
* PP: BarChip Macro fiber; ** Rec.: rectangular section of 1.0 mm × 0.5 mm. Table 1. Material properties of PA and PP fibers. 2.2. Experimental Program for Pull-Out Test Materials 2014, 7 An appropriate
amount of WRA was used to meet the target slump of 120 ± 25 mm, which is standard for tunnel
shotcrete. The mixture was mixed with PA fibers and placed in a mold for three days. After demolding,
the specimens were cured at room temperature. All specimens were tested on the 28th day. Materials 2014, 7 fibers used in the test had Ф0.47 mm and the length of the fiber embedded in the matrix
(25 × 25 mm) was 15 mm (half of the total PA fiber length). A total of four specimens were prepared
for the test. Similar to the method used in previous research [22], the pull-out load was directly measured
from the load cell in the cross head, and the displacement or slip of the PA fiber was measured using a
linear variable differential transformer (LVDT). Figure 5. Pictures of pull-out test specimen and mold. (a) Pull-out test specimen;
(b) pull-out test mold. (a)
(b)
The weight ratios of cement matrix used in the pull-out test were 0.628, 1.0, 0.25, 2.3, 0.00089 and
0.0079 for water, ordinary portland cement (OPC), fly ash, sand, air-entraining agents (AEA) and
water-reducing admixture (WRA). The compressive strength of the specimen on the 28th day was
58.09 MPa. In the cement matrix, only fine aggregates with AEA were used with OPC. An appropriate
amount of WRA was used to meet the target slump of 120 ± 25 mm, which is standard for tunnel
shotcrete. The mixture was mixed with PA fibers and placed in a mold for three days. After demolding,
the specimens were cured at room temperature. All specimens were tested on the 28th day. (b) (a) (a) (b) The weight ratios of cement matrix used in the pull-out test were 0.628, 1.0, 0.25, 2.3, 0.00089 and
0.0079 for water, ordinary portland cement (OPC), fly ash, sand, air-entraining agents (AEA) and
water-reducing admixture (WRA). The compressive strength of the specimen on the 28th day was
58.09 MPa. In the cement matrix, only fine aggregates with AEA were used with OPC. An appropriate
amount of WRA was used to meet the target slump of 120 ± 25 mm, which is standard for tunnel
shotcrete. The mixture was mixed with PA fibers and placed in a mold for three days. After demolding,
the specimens were cured at room temperature. All specimens were tested on the 28th day. The weight ratios of cement matrix used in the pull-out test were 0.628, 1.0, 0.25, 2.3, 0.00089 and
0.0079 for water, ordinary portland cement (OPC), fly ash, sand, air-entraining agents (AEA) and
water-reducing admixture (WRA). The compressive strength of the specimen on the 28th day was
58.09 MPa. In the cement matrix, only fine aggregates with AEA were used with OPC. 2.2. Experimental Program for Pull-Out Test A pull-out test of a macro-size PA fiber was conducted to determine the fiber-to-matrix bond
behavior, as shown in Figure 5. The bundled PA fibers have a larger specific surface area, which
accommodates attachment to cement, compared to PP fibers and other fiber reinforcements. The PA 7638 Materials 2014, 7 2.3. Experimental Program for Flexural Bending Test The schematic and picture of the flexural test set-up are presented in Figure 6. An INSTRON 5582
testing machine (DatapointLabs, Ithaca, NY, USA) was used in a laboratory for this flexural test. The
rate of deflection was controlled to 0.1 mm/min after L/900 (=0.333 mm) with the sampling rate of 16
Hz (≥2.5 Hz). The boundary conditions follow ASTM Testing Method C78 [23], which allows supporting
rollers rotating on their axes. The beam specimen size of 100 × 100 × 400 mm with a span length (L) of
300 mm was determined in accordance with ASTM C1609/C 1609M-05 [20]. The specimen was built
with dimensions of 150 × 150 × 550 mm and then cut into the size 100 × 100 × 400 mm (L = 300 mm)
to prevent the fiber aligning parallel to the direction of the beam length, and to place the fiber randomly. A steel frame was attached at the neutral axis of the beam specimen to measure the net deflection of the
beam using two LVDTs at both sides. (Only one LVDT is shown in Figure 6 and the other one is located
at the other side of the beam.) Applied load (P) was measured directly from the load cell in the crosshead. Materials 2014, 7 7639 Materials 2014, 7 Figure 6. Bending specimen geometry and test set-up. (a) Specimen geometry; (b) Test
set-up picture. e 6. Bending specimen geometry and test set-up. (a) Specimen geometry; (b) Test
picture. Figure 6. Bending specimen geometry and test set-up. (a) Specimen geometry; (b) Test
set-up picture. (a)
(b)
The specimens were prepared in a laboratory. The concrete mix design used in this study is
summarized in Table 2. The maximum gravel size (Gmax) was controlled to be 10 mm, the ratio of sand
to aggregate (S/a) was fixed at 60% for all specimens. Ordinary Portland cement (OPC) was 480 to
482 kg/m3. An appropriate amount of water-reducing admixture (AD) was used to meet the target slump
of 120 ± 25 mm, which was determined for tunnel shotcrete usage. The mixture was then mixed with
PA fibers and placed in a mold for three days. After demolding, the specimens were cured at room
temperature to represent PAFRC used in tunnel shotcrete. All specimens were tested on the 28th day. The 28-day compressive strength of the PAFRC was measured based on Ф100 × 200 mm cylindrical
specimens. 2.3. Experimental Program for Flexural Bending Test The slump and compressive strength of the specimens are presented in Table 3. (a) (b) (a) (b) The specimens were prepared in a laboratory. The concrete mix design used in this study is
summarized in Table 2. The maximum gravel size (Gmax) was controlled to be 10 mm, the ratio of sand
to aggregate (S/a) was fixed at 60% for all specimens. Ordinary Portland cement (OPC) was 480 to
482 kg/m3. An appropriate amount of water-reducing admixture (AD) was used to meet the target slump
of 120 ± 25 mm, which was determined for tunnel shotcrete usage. The mixture was then mixed with
PA fibers and placed in a mold for three days. After demolding, the specimens were cured at room
temperature to represent PAFRC used in tunnel shotcrete. All specimens were tested on the 28th day. The 28-day compressive strength of the PAFRC was measured based on Ф100 × 200 mm cylindrical
specimens. The slump and compressive strength of the specimens are presented in Table 3. The specimens were prepared in a laboratory. The concrete mix design used in this study is
summarized in Table 2. The maximum gravel size (Gmax) was controlled to be 10 mm, the ratio of sand
to aggregate (S/a) was fixed at 60% for all specimens. Ordinary Portland cement (OPC) was 480 to
482 kg/m3. An appropriate amount of water-reducing admixture (AD) was used to meet the target slump
of 120 ± 25 mm, which was determined for tunnel shotcrete usage. The mixture was then mixed with
PA fibers and placed in a mold for three days. After demolding, the specimens were cured at room
temperature to represent PAFRC used in tunnel shotcrete. All specimens were tested on the 28th day. The 28-day compressive strength of the PAFRC was measured based on Ф100 × 200 mm cylindrical
specimens. The slump and compressive strength of the specimens are presented in Table 3. Table 2. Concrete mix proportions. Table 2. Concrete mix proportions. 2.3. Experimental Program for Flexural Bending Test Specimen
Gmax
(mm)
W/C
(%)
S/a
(%)
Unit Weight (kg/m3)
WRA
(C%)
W
OPC
S
CS
G
Fiber
PA7-RFA50 *
10
43.1
60
207
480
480
484
640
7.0
0.7
PA8-RFA50 *
10
43.1
60
207
480
480
484
640
8.0
0.8
PA9-RFA50 *
10
43.1
60
207
480
480
484
640
9.0
0.9
PA7-RFA60 *
10
43.8
60
211
482
381
576
635
7.0
0.8
PA8-RFA60 *
10
43.8
60
211
482
381
576
635
8.0
0.9
PA9-RFA60 *
10
43.8
60
211
482
381
576
635
9.0
1.0
PA10-RFA60 *
10
43.8
60
211
482
381
576
635
10.0
1.1
* RFA xx: ratio of recycled fine aggregate to fine aggregate. Table 2. Concrete mix proportions. Table 2. Concrete mix proportions. Table 3. PAFRC slump and compressive strength. Table 3. PAFRC slump and compressive strength. Specimen
Slump (mm)
28-day Compressive strength (MPa)
SP-1
SP-2
SP-3
Avg. PA7-RFA50
120
51.0
49.8
50.5
50.4
PA8-RFA50
115
51.5
50.2
51.8
51.0
PA9-RFA50
120
52.8
51.5
53.1
52.5
PA7-RFA60
115
47.5
49.6
49.5
48.9
PA8-RFA60
110
50.4
51.1
48.0
49.8
PA9-RFA60
120
49.7
48.9
51.4
50.0
PA10-RFA60
110
49.1
52.0
47.5
49.5 7640 Materials 2014, 7 Materials 2014, 7 Materials 2014, 7 3.1. Pull-Out Test Results Although the PA fiber was failed within the fibers, 7641 Materials 2014, 7 Materials 2014, 7 the fiber-to-matrix bonding strength was expected to be larger than or equal to the PA fiber tensile
strength. Thus, Figure 7a was derived using Equation (1). 3.1. Pull-Out Test Results Pull-out test results are presented in Figure 7. The PA fiber strength increased almost linearly as the
displacement increased before peak loading. After peak loading, the PA fiber that was exposed outside the
cement matrix started to untangle, and elongated. At this point, the micro PA fiber began to fracture. Contrary to other organic reinforcing fibers and steel fibers, the macro-size PA fiber broke without
separating from the cement matrix. Steel fiber pull-out tests in other research [22] reported that the steel
fiber was pulled out from the matrix at increasing loads. However, the macro PA fiber was not pulled out
and instead fractured at increasing loads. Thus, the pull-out stress was computed to be for fiber fractured: 2
f
max
min
π
4
d
P
σ
(1) (1) where, σmin is the minimum fiber-to-matrix bonding strength, which is greater than or equal to the PA
fiber tensile strength; Pmax is the maximum pull-out load; and df is the diameter of the PA fiber. Generally, when the fiber is pulled out, such as the steel fiber during the pull-out test, the pull-out
stress is computed as: embed
f
max
max
π
τ
L
d
P
(2) (2) where, τmax is the maximum fiber-to-matrix bonding strength based on the surface shear around
the fiber; Lembed is the fiber embedment length in the matrix when debonding starts to initiate. Figure 7. Pull-out test results. (a) Load-slip curve; (b) Fiber fracture. Figure 7. Pull-out test results. (a) Load-slip curve; (b) Fiber fracture. Figure 7. Pull-out test results. (a) Load-slip curve; (b) Fiber fracture. (a)
(b)
As shown in Figure 7, this PA pull-out phenomenon originates with the improved bonding strength
of PA fiber attachment to the cement matrix. Because the macro PA fiber is made of 384 micro PA
filaments and the specific surface area is significantly increased, the bonding strength between the macro
PA fiber and cement matrix is greatly increased. Although the PA fiber was failed within the fibers, (a) (b) (a) (b) As shown in Figure 7, this PA pull-out phenomenon originates with the improved bonding strength
of PA fiber attachment to the cement matrix. Because the macro PA fiber is made of 384 micro PA
filaments and the specific surface area is significantly increased, the bonding strength between the macro
PA fiber and cement matrix is greatly increased. 3.2. Flexural Bending Test Results The flexural responses of PAFRCs with varying fiber volumes of 7, 8, 9 and 10 kg/m3 were tested in
accordance with ASTM C1609/C 1609M-05 [20]. As in Figure 8, this study identified two peak loads:
a (1) first-peak load (P1) on the load-deflection, caused by the initiation of cracks in the specimen, and
(2) second-peak load (P2) on the load-deflection when the PA fibers reached their ultimate strength. Corresponding strengths and deflections were denoted as f1 and δ1 for P1 and f2 and δ2 for P2,
respectively. Residual loads (P
D
600 and P
D
150) and strength (
D
f600 and P
D
150) at a net deflection of L/600 and
L/150 were measured for a beam with a depth of D. Toughness (T
D
150) and equivalent flexural strength
ratio (R
D
T,150) at a net deflection of L/150 were also identified as follows: 2
2
or
1
2
or
1
bd
L
P
f
(3)
(%)
100
150
2
1
150
150
,
d
b
f
T
R
D
D
T
(4) 2
2
or
1
2
or
1
bd
L
P
f
(3) (3) (%)
100
150
2
1
150
150
,
d
b
f
T
R
D
D
T
(4) (4) Based on the deflection (δ1) at the first-peak load, three additional points were investigated at 3δ1,
5.5δ1 and 10.5δ1 as per ASTM C1018 [19]. Current ASTM standard C1609 specifies δ1, δp (deflection
at the peak load regardless of the first-peak or second-peak), L/600 and L/150. However, PAFRC
occasionally exhibits a second peak that is larger than the first peak depending on the PA fiber volume
content. This study intended to clearly identify whether pre- or post-cracking strength was larger. Also,
PAFRC yields after cracking before the PA fiber reaches its ultimate strength, similar to hyper-elastic
materials. Thus, additional points at 3δ1, 5.5δ1 and 10.5δ1 were used. Figure 8. Typical load-deflection response of FRC. Figure 8. Typical load-deflection response of FRC. Figure 8. Typical load-deflection response of FRC. 3.2.1. Bending and Equivalent Bending Strength
Flexural bending tests of three specimens for each fiber volume content were conducted, as shown in
Table 4. The bending strength (fr) and equivalent bending strength (fe) of each specimen was calculated
based on the load-deflection curves in the following section (Figure 9), using the following equations: Figure 8. Materials 2014, 7 Materials 2014, 7 7642 2
r
bd
PL
f
(5)
2
150
b
e
δ bd
L
A
f
(6) (5) (6) where, P = peak load at δ1; L = span length (=300 mm); b and h = beam cross-section width and depth
at the fracture surface, respectively; Ab = area under the load-deflection curves up to δ150 (N·mm) and
δ150 = deflection of L/150 (=2.0 mm). The equivalent bending strength (fe) is derived from
the equivalent flexural strength ratio,
D
T
R
150
,
, in Equation (4), which is equal to (fr/fe) × 100%. Figure 9. Load-deflection curves. (a) PA-7-RFA50; (b) PA-8-RFA50; (c) PA-9-RFA50;
(d) PA-7-RFA60; (e) PA-8-RFA60; (f) PA-9-RFA60; (g) PA-10-RFA60. Figure 9. Load-deflection curves. (a) PA-7-RFA50; (b) PA-8-RFA50; (c) PA-9-RFA50;
(d) PA-7-RFA60; (e) PA-8-RFA60; (f) PA-9-RFA60; (g) PA-10-RFA60. 3.2. Flexural Bending Test Results Typical load-deflection response of FRC
3.2.1. Bending and Equivalent Bending Strength 3.2.1. Bending and Equivalent Bending Strength Flexural bending tests of three specimens for each fiber volume content were conducted, as shown in
Table 4. The bending strength (fr) and equivalent bending strength (fe) of each specimen was calculated
based on the load-deflection curves in the following section (Figure 9), using the following equations: Materials 2014, 7 (a)
(b)
(c)
(d)
(e)
(f)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA50-1
PA7-RFA50-2
PA7-RFA50-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA50-1
PA8-RFA50-2
PA8-RFA50-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA50-1
PA9-RFA50-2
PA9-RFA50-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA60-1
PA7-RFA60-2
PA7-RFA60-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA60-1
PA8-RFA60-2
PA8-RFA60-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA60-1
PA9-RFA60-2
PA9-RFA60-3 0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA50-1
PA8-RFA50-2
PA8-RFA50-3 0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA50-1
PA7-RFA50-2
PA7-RFA50-3 Deflection (mm) Deflection (mm) (b)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA60-1
PA7-RFA60-2
PA7-RFA60-3 (a)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA50-1
PA9-RFA50-2
PA9-RFA50-3 (b) (a) Deflection (mm) (d)
(
)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA60-1
PA9-RFA60-2
PA9-RFA60-3 (c)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA60-1
PA8-RFA60-2
PA8-RFA60-3 (d) (c) Deflection (mm) Deflection (mm) (e)
(f)
Deflection (mm)
Deflection (mm) (f) (e) 7643 Materials 2014, 7 Figure 9. Cont. (g)
For both RFA50 and RFA60, it is clearly observed that PA fiber improved the beam flexural strength,
as in Table 4. Materials 2014, 7 Both bending strength and equivalent bending strength increased as fiber content increased
from 7 to 10 kg/m3. However, the equivalent bending strength of PA9-FRA50 was smaller than
PA7-RFA50 and PA8-RFA50. This phenomenon often occurs in other FRCs when the volume content
of the reinforcing fiber is larger than a certain limit. Bending strengths of the RFA60 series were more
significantly improved as PA fiber content was increased, compared to the RFA50 series. The RFA50
series produced larger bending strengths for PA7 and PA8, but the RFA60 series produced larger
bending strengths for PA9 and PA10. To confirm that an excessive amount of PA fiber may drop the
bending strength, similar to the RFA50 series, the PA10-RFA60 specimen was tested, but the bending
strength continuously and significantly increased. 0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA10-RFA60-1
PA10-RFA60-2
PA10-RFA60-3 Figure 9. Cont. (g)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA10-RFA60-1
PA10-RFA60-2
PA10-RFA60-3 Figure 9. Cont. (g) For both RFA50 and RFA60, it is clearly observed that PA fiber improved the beam flexural strength,
as in Table 4. Both bending strength and equivalent bending strength increased as fiber content increased
from 7 to 10 kg/m3. However, the equivalent bending strength of PA9-FRA50 was smaller than
PA7-RFA50 and PA8-RFA50. This phenomenon often occurs in other FRCs when the volume content
of the reinforcing fiber is larger than a certain limit. Bending strengths of the RFA60 series were more
significantly improved as PA fiber content was increased, compared to the RFA50 series. The RFA50
series produced larger bending strengths for PA7 and PA8, but the RFA60 series produced larger
bending strengths for PA9 and PA10. To confirm that an excessive amount of PA fiber may drop the
bending strength, similar to the RFA50 series, the PA10-RFA60 specimen was tested, but the bending
strength continuously and significantly increased. For both RFA50 and RFA60, it is clearly observed that PA fiber improved the beam flexural strength,
as in Table 4. Both bending strength and equivalent bending strength increased as fiber content increased
from 7 to 10 kg/m3. However, the equivalent bending strength of PA9-FRA50 was smaller than
PA7-RFA50 and PA8-RFA50. Materials 2014, 7 This phenomenon often occurs in other FRCs when the volume content
of the reinforcing fiber is larger than a certain limit. Bending strengths of the RFA60 series were more
significantly improved as PA fiber content was increased, compared to the RFA50 series. The RFA50
series produced larger bending strengths for PA7 and PA8, but the RFA60 series produced larger
bending strengths for PA9 and PA10. To confirm that an excessive amount of PA fiber may drop the
bending strength, similar to the RFA50 series, the PA10-RFA60 specimen was tested, but the bending
strength continuously and significantly increased. Table 4. Bending and equivalent bending strength. Specimen
Bending strength (MPa)
Equivalent bending strength (MPa)
SP1
SP2
SP3
Average
SP1
SP2
SP3
Average
PA7-RFA50
5.31
5.18
5.69
5.39
3.28
4.11
3.86
3.75
PA8-RFA50
5.21
6.11
5.30
5.54
3.42
4.20
3.68
3.77
PA9-RFA50
5.59
5.71
5.46
5.59
3.32
3.68
3.34
3.45
PA7-RFA60
4.82
5.09
4.05
4.65
3.63
3.72
3.01
3.45
PA8-RFA60
4.04
4.39
4.42
4.28
3.77
3.36
3.24
3.46
PA9-RFA60
5.11
5.60
4.59
5.10
4.07
4.47
4.31
4.29
PA10-RFA60
4.51
5.10
4.41
4.97
5.60
5.09
4.78
5.16
3.2.2. Load-Deflection Relationship of PAFRC Table 4. Bending and equivalent bending strength. Table 4. Bending and equivalent bending strength. 3.2.2. Load-Deflection Relationship of PAFRC 3.2.2. Load-Deflection Relationship of PAFRC Load-deflection curves of three specimens were generated, as presented in Figure 9. Overall
load-deflection curves were similar to polypropylene FRC [24–29] and synthetic FRC [30–32]. The
load-deflection curves linearly increased up to the first-peak. After the first-peak, the loads were dropped
approximately 50%. Then the curves exhibited convex shapes increasing and decreasing after the
second-peak. The PA fibers did not notably change the first-peak, but the post-cracking behaviors of all specimens
were significantly improved. All specimens produced two peak bending strengths. The first-peaks Materials 2014, 7 7644 Materials 2014, 7 apparently occurred near 0.005 mm of deflection and decreased up to 0.5 mm of deflection, which was
approximately the lowest bending strength after the first-peaks. The load-deflection curves increased up
to second-peaks until the deflections reached between 1.0 and 1.5 mm after the lowest peaks. The curves
moderately decreased after the second-peaks, compared to the lines after the first-peaks. The first-peak loads were 17975, 18468 and 18624 N for the RFA50 series and 15514, 14280, 16993
and 16572 N for the RFA60 series, respectively. Materials 2014, 7 The results show that the first-peak loads tended to
increase as PA fiber content increased. However, the first-peak loads for PA8-RFA60 and PA10-RFA60
were smaller than for PA7-RFA60 and PA9-RFA60, respectively, because the first-peak loads are related
to the concrete strength. Also, it should be noted that the first-peaks of the RFA50 series were larger
than those of the RFA60 series, which was contrary to the bending strength, although the second-peaks
of the RFA60 series were larger than those of the RFA50 series. Initial PA fiber elongation was observed after the first-peak load. Since PA fiber tends to exhibit
hyper-elasticity, a deflection occurs up to the inflection point and thereafter the load-deflection curve
rapidly increases. In Figure 9, all PAFRC specimens exhibit the kinked (inflection) points after
the first-peak near the L/600 and PA fiber elongation. The second-peaks of the RFA50 series specimens were similar to the first-peaks. However, as
discussed in Section 4.1, the second-peaks of the RFA60 series specimens exceeded the first-peaks. The
ratios of the first to second-peak loads were 81.7, 85.6 and 74.5% for the RFA50 series and 100.2, 116.5,
113.8 and 153.0% for the RFA60 series. Detailed discussion regarding the flexural responses is provided
in the following section. 4.1. Flexural Responses of PAFRC The flexural responses of PAFRC at δ1, δ2, 3δ1, 5.5δ1, 10.5δ1, L/600 and L/150 were investigated for
loads, bending strength and toughness. The results are tabulated in Table 5 in the order of increasing
deflections. The flexural responses in terms of bending strength, deflection and toughness are presented
in Table 5 and Figure 10. Bending strength decreased from f1 to f600, and then increased up to f2 for all
specimens. These bending strength fluctuations correspond to the load-deflection curves in Figure 9. PA fiber content significantly influenced the second-peaks, as seen in Figure 10a,c. This results
implies that the first-peaks are mainly dependent on the concrete strength. However, the deflections at
the first-peaks were insignificantly influenced by PA fiber content as in Figure 10b,d. Toughness after
the second-peak was significantly influenced by PA fiber content as seen in Figure 10e,f,g), while
toughness before the second-peak was not. The PA fiber produces two peaks, as in Figure 9. Up to L/600
(=0.5 mm), which is an approximate lowest point, deflections are smaller than the second-peak and PA
fiber bending strength is not exerted. However, PA fiber significantly improves the bending strength
beyond L/600. Also, the toughness results of both the RFA 50 and 60 series at L/150 (=2.0 mm) lay
between the maximum and minimum toughness (T
100
150) suggested by ASTM C1609/C1609M-10 [20]. Materials 2014, 7 7645 Materials 2014, 7 Figure 10. Average flexural responses of PAFRC. (a) Strength for first- and second-peak;
(b) deflection for first- and second- peak; (c) strength for L/600 and L/150; (d) deflection for
3δ1, 5.5δ1 and 10.5δ1; (e) toughness for first- and second-peak; (f) toughness for 3δ1, 5.5δ1
and 10.5δ1; (g) PA-10-RFA60. Figure 10. Average flexural responses of PAFRC. (a) Strength for first- and second-peak;
(b) deflection for first- and second- peak; (c) strength for L/600 and L/150; (d) deflection for
3δ1, 5.5δ1 and 10.5δ1; (e) toughness for first- and second-peak; (f) toughness for 3δ1, 5.5δ1
and 10.5δ1; (g) PA-10-RFA60. p
; ( )
g
; ( )
5δ1 and 10.5δ1; (e) toughness for first- and second-peak; (f) toughness for 3
.5δ1; (g) PA-10-RFA60. 4.1. Flexural Responses of PAFRC (a)
(b)
(c)
(d)
(e)
(f)
(g)
0
5
10
15
20
25
30
0
2
4
6
8
10
PA-7
PA-8
PA-9
PA-10
Load (kN)
Bending Strength (MPa)
PA Fiber Content (kg)
RFA50-1P
RFA60-1P
RFA50-2P
RFA60-2P
0.0
0.5
1.0
1.5
2.0
PA-7
PA-8
PA-9
PA-10
Deflection (mm)
PA Fiber Content (kg)
RFA50-1P
RFA60-1P
RFA50-2P
RFA60-2P
0
5
10
15
20
25
30
0
2
4
6
8
10
PA-7
PA-8
PA-9
PA-10
Load (kN)
Bending Strength (MPa)
PA Fiber Content (kg)
RFA50-L/600
RFA60-L/600
RFA50-L/150
RFA60-L/150
0.0
0.5
1.0
1.5
2.0
PA-7
PA-8
PA-9
PA-10
Deflection (mm)
PA Fiber Content (kg)
RFA50-3delta
RFA60-3delta
RFA50-5.5delta
RFA60-5.5delta
RFA50-10.5delta
RFA60-10.5delta
0
5
10
15
20
25
PA-7
PA-8
PA-9
PA-10
Toughness (Joule)
PA Fiber Content (kg)
RFA50-1P
RFA60-1P
RFA50-2P
RFA60-2P
0
5
10
15
20
25
PA-7
PA-8
PA-9
PA-10
Toughness (Joule)
PA Fiber Content (kg)
RFA50-3delta
RFA60-3delta
RFA50-5.5delta
RFA60-5.5delta
RFA50-10.5delta
RFA60-10.5delta
0
10
20
30
40
50
1
2
3
4
Toughness (Joule)
PA Fiber Content (kg)
RFA50-L/600
RFA60-L/600
RFA50-L/150
RFA60-L/150
T150
100 0.0
0.5
1.0
1.5
2.0
PA-7
PA-8
PA-9
PA-10
Deflection (mm)
PA Fiber Content (kg)
RFA50-1P
RFA60-1P
RFA50-2P
RFA60-2P 0
5
10
15
20
25
30
0
2
4
6
8
10
PA-7
PA-8
PA-9
PA-10
Load (kN)
Bending Strength (MPa)
PA Fiber Content (kg)
RFA50-1P
RFA60-1P
RFA50-2P
RFA60-2P (a)
( g)
0
5
10
15
20
25
30
0
2
4
6
8
10
PA-7
PA-8
PA-9
PA-10
Load (kN)
Bending Strength (MPa)
PA Fiber Content (kg)
RFA50-L/600
RFA60-L/600
RFA50-L/150
RFA60-L/150 (b)
( g)
0.0
0.5
1.0
1.5
2.0
PA-7
PA-8
PA-9
PA-10
Deflection (mm)
PA Fiber Content (kg)
RFA50-3delta
RFA60-3delta
RFA50-5.5delta
RFA60-5.5delta
RFA50-10.5delta
RFA60-10.5delta (b) (a) Bending Strength (MPa) (d)
( g)
0
5
10
15
20
25
PA-7
PA-8
PA-9
PA-10
Toughness (Joule)
PA Fiber Content (kg)
RFA50-3delta
RFA60-3delta
RFA50-5.5delta
RFA60-5.5delta
RFA50-10.5delta
RFA60-10.5delta (c)
( g)
0
5
10
15
20
25
PA-7
PA-8
PA-9
PA-10
Toughness (Joule)
PA Fiber Content (kg)
RFA50-1P
RFA60-1P
RFA50-2P
RFA60-2P (c) (d) (e)
(
(g)
0
10
20
30
40
50
1
2
3
4
Toughness (Joule)
PA Fiber Content (kg)
RFA50-L/600
RFA60-L/600
RFA50-L/150
RFA60-L/150
T150
100 (f) (e) (g) 7646 Materials 2014, 7 Table 5. Average value of flexural responses of PAFRC. 4.2. Toughness Performance Levels The toughness performance levels (TPL) by Chen (1995) [32] were used to specify the PAFRC
toughness information. As discussed in the last paragraph of Section 4.1, the PAFRC toughness lay
between the maximum and minimum of ASTM C1609/C1609M-10 [20]. However, this information is
not enough to explain the toughness characteristics of PAFRC and therefore, a detailed description is
required to verify compliance with construction specifications, or quality control, of in-service PAFRC. In tunneling practice, the TPL by Chen [33] (1995) is commonly used for steel fiber reinforced shotcrete. The TPL is based on nominal residual flexural strengths based on the load-deflection curves at δ1,
L/600 and L/150. The TPL defines five different toughness levels and corresponding residual loads,
as in Table 6. Equivalent flexural toughness parameters to TPL levels can be found in Chen (1995). The residual loads here were computed based on the 100 × 100 × 300 mm specimen size. A design
flexural strength of 4.0 MPa, which is equivalent to a design flexural load of 13,333 N, was adopted in
this study as it is a common practice in tunnel shotcrete. It should be noted that the TPL does not consider
the unstable region in the load-deflection curves, i.e. from the first-crack (δ1) to L/600 (=0.5 mm). Load-deflection curves of each specimen were investigated to determine the TPL levels presented in
Figure 11. Figure 11. Toughness performance level. (a) PA-7-RFA50; (b) PA-8-RFA50;
(c) PA-9-RFA50; (d) PA-7-RFA60; (e) PA-8-RFA60; (f) PA-9-RFA60; (g) PA-10-RFA60;
(h) TPL Levels. Materials 2014, 7 7647 Materials 2014, 7 Materials 2014, 7 4.1. Flexural Responses of PAFRC Location
Response
PA-7-RFA50
PA-8-RFA50
PA-9-RFA50
PA-7-RFA60
PA-8-RFA60
PA-9-RFA60
PA-10-RFA60
First-peak
P (N)
17975
18468
18624
15514
14280
16993
16572
δ (mm)
0.045
0.045
0.042
0.052
0.044
0.057
0.042
f (MPa)
5.39
5.54
5.59
4.65
4.28
5.10
4.97
T (Joule)
0.520
0.508
0.481
0.580
0.396
0.659
0.415
3δ1
P (N)
15678
16403
16538
13204
12087
14096
14653
δ (mm)
0.134
0.135
0.125
0.156
0.132
0.170
0.126
f (MPa)
4.70
4.92
4.96
3.96
3.63
4.23
4.40
T (Joule)
2.110
2.135
2.019
2.039
1.567
2.408
1.751
5.5δ1
P (N)
13336
14095
14397
10257
9498
11402
12351
δ (mm)
0.246
0.248
0.230
0.286
0.241
0.311
0.231
f (MPa)
4.00
4.23
4.32
3.08
2.85
3.42
3.71
T (Joule)
3.594
3.780
3.608
3.634
2.785
4.138
3.081
10.5δ1
P (N)
8681
10530
10213
6405
6686
10263
10781
δ (mm)
0.469
0.473
0.439
0.546
0.461
0.594
0.441
f (MPa)
2.70
3.16
3.06
1.92
2.01
3.08
3.23
T (Joule)
6.037
6.530
6.135
5.666
4.420
4.518
5.395
L/600
(=0.50 mm)
P
100
600(N)
8799
9972
8933
6647
7094
9658
10863
f
100
600 (MPa)
2.64
2.99
2.68
1.99
2.13
2.90
3.26
T600(Joule)
6.369
6.782
6.776
5.393
4.699
6.833
6.089
Second-peak
P2 (N)
14685
15807
13954
15540
15843
19333
25357
δ2 (mm)
1.261
1.419
1.321
1.429
1.298
1.284
1.456
f2 (MPa)
4.41
4.74
4.19
4.66
4.75
5.80
7.61
T2 (Joule)
15.596
17.003
14.902
15.225
13.568
16.695
22.616
L/150
(=2.00 mm)
P
100
150 (N)
11589
12107
10129
12059
11320
13554
18755
f
100
150 (MPa)
3.48
3.63
3.04
3.62
3.40
4.07
5.63
T150 (Joule)
24.981
25.114
22.982
23.019
23.046
28.569
34.401
R
100
T,150(%)
69.6
67.9
61.7
74.2
81
84.5
104.0 Table 5. Average value of flexural responses of PAFRC. Materials 2014, 7 4.2. Toughness Performance Levels TPL Level
Residual loads (% of Design flexural strength)
at δ1 (=0.047 mm)
at L/600 (=0.5 mm)
at L/150 (=2.0 mm)
0
No fiber reinforced
I
13333 (100%)
2000 (15%)
667 (5%)
II
13333 (100%)
4000 (30%)
2000 (15%)
III
13333 (100%)
6667 (50%)
4000 (30%)
IV
13333 (100%)
10000 (15%)
6000 (45%)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA60-1
PA8-RFA60-2
PA8-RFA60-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA60-1
PA9-RFA60-2
PA9-RFA60-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA10-RFA60-1
PA10-RFA60-2
PA10-RFA60-3
Level I
Level II
Level III
Level IV
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
Level 0 Figure 11. Cont. 0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA60-1
PA8-RFA60-2
PA8-RFA60-3 0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA60-1
PA9-RFA60-2
PA9-RFA60-3 10 (f)
(h)
Deflection (mm)
Level I
Level II
Level III
Level IV
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
Level 0 (e)
(g)
Deflection (mm)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA10-RFA60-1
PA10-RFA60-2
PA10-RFA60-3 (f) (e) Bending Strength (MPa) (h) (g) Table 6. Toughness performance level criteria. Table 6. Toughness performance level criteria. TPL Level
Residual loads (% of Design flexural strength)
at δ1 (=0.047 mm)
at L/600 (=0.5 mm)
at L/150 (=2.0 mm)
0
No fiber reinforced
I
13333 (100%)
2000 (15%)
667 (5%)
II
13333 (100%)
4000 (30%)
2000 (15%)
III
13333 (100%)
6667 (50%)
4000 (30%)
IV
13333 (100%)
10000 (15%)
6000 (45%) Residual loads (% of Design flexural strength) The determined TPL levels are presented in Table 7. 4.2. Toughness Performance Levels (a)
(b)
(c)
(d)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA50-1
PA7-RFA50-2
PA7-RFA50-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA50-1
PA8-RFA50-2
PA8-RFA50-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA50-1
PA9-RFA50-2
PA9-RFA50-3
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA60-1
PA7-RFA60-2
PA7-RFA60-3 0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA50-1
PA7-RFA50-2
PA7-RFA50-3 0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA8-RFA50-1
PA8-RFA50-2
PA8-RFA50-3 Deflection (mm) (b)
Deflection (mm)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA7-RFA60-1
PA7-RFA60-2
PA7-RFA60-3 (a)
Deflection (mm)
0
5
10
15
20
25
30
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Load (kN)
Bending Strength (MPa)
Deflection (mm)
PA9-RFA50-1
PA9-RFA50-2
PA9-RFA50-3 (b) (a) Bending Strength (MPa) Deflection (mm) Deflection (mm) (d) (c) 7648 Materials 2014, 7 Figure 11. Cont. (e)
(f)
(g)
(h)
Table 6. Toughness performance level criteria. 4.2. Toughness Performance Levels This study investigated at three points and took
the lowest level among all specimens for each specimen type. It is clearly shown in Table 7 that all
specimens of the RFA50 series satisfied Level III. However, for the RFA60 series, specimens with lower
PA fiber content exhibited Level II, and those with higher PA fiber contents exhibited Level III. Therefore, the RFA60 series requires greater PA fiber content compared to the RFA50 series. Moreover,
all the performance levels satisfied Level IV at δ1 and L/150 (=2.0 mm) although lower performance
levels were found at L/600 (=0.5 mm). As seen in Figure 11, the load-deflection curves rapidly decreased
after the first-peaks, but the loads increased as the deflection increased to satisfy Level IV. This finding
implies that the TPL analysis by Chen (1995) is not enough to explain the full toughness characteristics
of PAFRC that has a sudden drop after the first-peak in the load-deflection curve. Materials 2014, 7 7649 Table 7. Toughness performance levels. Table 7. Toughness performance levels. Specimen
at δ1
at L/600 (=0.5 mm)
at L/150 (=2.0 mm)
TPL
Level
SP1
SP2
SP3
Lvl. SP1
SP2
SP3
Lvl. SP1
SP2
SP3
Lvl. PA7-RFA50
IV
IV
IV
IV
III
III
III
III
IV
IV
IV
IV
III
PA8-RFA50
IV
IV
IV
IV
IV
IV
III
III
IV
IV
IV
IV
III
PA9-RFA50
IV
IV
IV
IV
III
III
III
III
IV
IV
IV
IV
III
PA7-RFA60
IV
IV
IV
IV
III
III
II
II
IV
IV
IV
IV
II
PA8-RFA60
IV
IV
IV
IV
III
III
II
II
IV
IV
IV
IV
II
PA9-RFA60
IV
IV
IV
IV
III
III
IV
III
IV
IV
IV
IV
III
PA10-RFA60
IV
IV
IV
IV
IV
III
IV
III
IV
IV
IV
IV
III 5. Conclusions This study described the development and processing of a macro-sized PA fiber to be used in concrete
reinforcement, and then investigated the influence of the PA fiber on flexural responses in accordance
with ASTM standards C1018-97 and C1609/C 1609M-05. The conclusions derived from this study are
as follows: The compressive strength and the first-peak were not notably influenced by the PA fiber contents
because PA fiber contents considered in this study did not deteriorate the compressive strength and
PA fibers were intended to resist tensile stresses. However, the PA fiber significantly improved
flexural responses and toughness after the deflection of L/600 as the PA fiber content was increased. Before L/600 deflection, PA fiber tensile strength was so low that allowed the first crack of concrete
specimens, but after L/600 deflection, PA fibers started to exert high tensile strength and increased
ductility and toughness of concrete. The PA fibers produced two peak bending strengths. The first-peaks apparently occurred near
0.005 mm of deflection and decreased up to 0.5 mm (L/600) of deflection, which was the
approximately lowest bending strength after the first peak. Then the bending strength increased up
to second peak until the deflections reached between 1.0 and 1.5 mm after the lowest peaks. The
load-deflection curve decreased moderately after the second-peak compared to the decreasing slope
after the first-peak. Toughness performance level (TPL) results exhibited Level III for all specimens in the RFA50 series
based on the design flexural strength of 4.0 MPa. However, for the RFA60 series, specimens with
less than 8 kg of PA fiber content were determined to be Level II, and specimens with 9 and 10 kg
of PA fiber content were determined to be Level III. Therefore, the RFA60 series requires greater
PA fiber content compared to the RFA50 series. However, due to the PA fiber characteristic,
load-deflection curves exhibited that PAFRC satisfy Level IV at δ1 and L/150 (=2.0 mm) except at
L/600 (=0.5 mm). Therefore, the TPL analysis is not enough to explain the full toughness
characteristics of PAFRC that has a drop after the first-peak in the load-deflection curve. Author Contributions Joong Kyu Jeon planned and performed experimental tests and WooSeok Kim analyzed the
experimental data. Chan Ki Jeon and Jin Cheol Kim reviewed and guided this study and provided
valuable comments. Materials 2014, 7 7650 Materials 2014, 7 by the Ministry on Land, Infrastructure, and Transport, and by Basic Science Research Program through
the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (NRF-2012R1A1A1044378). by the Ministry on Land, Infrastructure, and Transport, and by Basic Science Research Program through
the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (NRF-2012R1A1A1044378). Conflicts of Interest The authors declare no conflict of interest. Acknowledgments This research was supported by a grant from a Construction Technology Research Project
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distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/). © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).
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English
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Impacts of leaf age and heat stress duration on photosynthetic gas exchange and foliar nonstructural carbohydrates in <i>Coffea arabica</i>
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Ecology and evolution
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O R I G I N A L R E S E A R C H O R I G I N A L R E S E A R C H Danielle E. Marias1 | Frederick C. Meinzer2 | Christopher Still1 1Department of Forest Ecosystems and
Society, Oregon State University, Corvallis,
OR, USA Impacts of leaf age and heat stress duration on photosynthetic
gas exchange and foliar nonstructural carbohydrates in Coffea
arabica Danielle E. Marias1 | Frederick C. Meinzer2 | Christopher Still1 Received: 19 September 2016 | Revised: 28 October 2016 | Accepted: 27 November 2016
DOI: 10 1002/ece3 2681 Received: 19 September 2016 | Revised: 28 October 2016 | Accepted: 27 November 2016
DOI: 10 1002/ece3 2681 Received: 19 September 2016 | Revised: 28 October 2016 | Accepted: 27 November 2016 Received: 19 September 2016 DOI: 10.1002/ece3.2681 Abstract Abstract
Given future climate predictions of increased temperature, and frequency and inten-
sity of heat waves in the tropics, suitable habitat to grow ecologically, economically,
and socially valuable Coffea arabica is severely threatened. We investigated how leaf
age and heat stress duration impact recovery from heat stress in C. arabica. Treated
plants were heated in a growth chamber at 49°C for 45 or 90 min. Physiological recov-
ery was monitored in situ using gas exchange, chlorophyll fluorescence (the ratio of
variable to maximum fluorescence, FV/FM), and leaf nonstructural carbohydrate (NSC)
on mature and expanding leaves before and 2, 15, 25, and 50 days after treatment. Regardless of leaf age, the 90-min treatment resulted in greater FV/FM reduction
2 days after treatment and slower recovery than the 45-min treatment. In both treat-
ments, photosynthesis of expanding leaves recovered more slowly than in mature
leaves. Stomatal conductance (gs) decreased in expanding leaves but did not change in
mature leaves. These responses led to reduced intrinsic water-use efficiency with in-
creasing heat stress duration in both age classes. Based on a leaf energy balance
model, aftereffects of heat stress would be exacerbated by increases in leaf tempera-
ture at low gs under full sunlight where C. arabica is often grown, but also under partial
sunlight. Starch and total NSC content of the 45-min group significantly decreased
2 days after treatment and then accumulated 15 and 25 days after treatment coincid-
ing with recovery of photosynthesis and FV/FM. In contrast, sucrose of the 90-min
group accumulated at day 2 suggesting that phloem transport was inhibited. Both
treatment group responses contrasted with control plant total NSC and starch, which
declined with time associated with subsequent flower and fruit production. No treated
plants produced flowers or fruits, suggesting that short duration heat stress can lead
to crop failure. 2USDA Forest Service, Pacific Northwest
Research Station, Corvallis, OR, USA Correspondence
Danielle E. Marias, Department of Forest
Ecosystems and Society, Oregon State
University, Corvallis, OR, USA. Email: Danielle.Marias@oregonstate.edu Funding information
Division of Integrative Organismal Systems,
Grant/Award Number: 11-46746; National
Science Foundation Graduate Research
Fellowship Program. Ecology and Evolution 2017; 7: 1297–1310
| 1297
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© 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 1 | INTRODUCTION 1 relations and stomatal conductance (Wahid et al., 2007). At the whole
plant level, heat stress impacts leaf area, leaf abscission, visible foliar
damage (Cunningham & Read, 2006), budburst, growth, mortality,
and reproduction (Teskey et al., 2015). In response to high tempera-
ture stress, plants use assimilated carbon/photosynthate to produce
compounds used for repair, defense, and physiological recovery such
as primary and secondary metabolites, antioxidants, osmolytes, and
phytohormones (Bita & Gerats, 2013). However, we have limited
knowledge of how plant carbon allocation is altered in response to
heat stress. Climate models predict an increasing frequency and intensity of heat
waves and high temperature events throughout the 21st century
(Cramer et al., 2001; Diffenbaugh & Scherer, 2011; IPCC, 2014) that
are expected to influence plant species’ distributions, productivity,
and carbon balance, although the physiological impacts remain un-
clear. Heat waves are increasing under both drier and wetter condi-
tions (Hao, AghaKouchak, & Phillips, 2013). In contrast to drought,
much less is known about physiological responses to heat stress
(Barnabás, Jäger, & Fehér, 2008; Ruan, Jin, Yang, Li, & Boyer, 2010). This, along with the need to isolate the effects of heat and drought
stress to understand the interactions between the two on plant phys-
iological responses (Sevanto & Dickman, 2015), makes studies on the
physiological impacts of heat stress essential. Tropical species are
particularly vulnerable to heat stress because of the higher radiation
load, the increase in heat wave intensity and frequency expected in
the tropics (Corlett, 2011), and the narrower distribution of tempera-
tures typically experienced compared to extratropical species (Battisti
& Naylor, 2009). Nonstructural carbohydrates (NSCs) traditionally include starch
and free sugars (sucrose, glucose, and fructose) and are involved in
growth, storage, reproduction, metabolism, and repair (Kozlowski,
1992). Although NSCs play a role in plant responses to environmental
stress (Dietze et al., 2014) and it is understood that high temperature
shifts carbon metabolism enzymes, starch accumulation, and sucrose
synthesis (Bita & Gerats, 2013; Ruan et al., 2010), the role of NSCs
in the response to and recovery from heat stress is poorly under-
stood due to conflicting results (Génard et al., 2008; Sala, Woodruff,
& Meinzer, 2012). 1 | INTRODUCTION For example, NSCs may increase in response to
heat stress because NSCs are used for repair and damage preven-
tion (Couée, Sulmon, Gouesbet, & Amrani, 2006; Roitsch & González,
2004; Sevanto & Dickman, 2015; Sugio, Dreos, Aparicio, & Maule,
2009) and therefore are associated with heat stress tolerance (Liu &
Huang, 2000; Niinemets, 2010). In contrast, leaf NSCs have also been
shown to decrease in response to heat stress due to reduced carbon
gain and assimilation (i.e., decreased supply) by inhibited photosynthe-
sis and stomatal conductance (Zhao, Hartmann, Trumbore, Ziegler, &
Zhang, 2013) and/or increased utilization (i.e., increased demand) by
increased respiration and metabolic maintenance (Duan et al., 2013). The tropics support important agricultural crops such as coffee. Global coffee consumption continues to increase; over 9 billion kg
of coffee was consumed worldwide in 2014 (ICO, 2016). Coffea ara-
bica L. leads the world coffee trade and provides ~65% of commercial
production (ICO, 2016). Coffea arabica is a shade-adapted evergreen
species that originated in Ethiopia and is now grown in 80 countries
on four continents in the intertropical zone between 20 and 25°N in
Hawaii and 24°S in Brazil (DaMatta & Ramalho, 2006). Coffea arabica is
highly sensitive to fluctuations in temperature (Camargo, 2010; Silva,
DaMatta, Ducatti, Regazzi, & Barros, 2004) with an optimal mean an-
nual temperature range of 18–24°C (Camargo, 1985; Teketay, 1999). An increase in the frequency and intensity of heat waves in the trop-
ics would severely threaten suitable habitat to grow C. arabica (Bunn,
Läderach, Rivera, & Kirschke, 2014; de Camargo, 2010; Craparo, Van
Asten, Läderach, Jassogne, & Grab, 2015; DaMatta & Ramalho, 2006;
Davis, Gole, Baena, & Moat, 2012). Therefore, it is crucial to quantify
its physiological responses to and ability to recover from heat stress
(Martins, Tomaz, Lidon, DaMatta, & Ramalho, 2014; Martins et al.,
2016; Rodrigues et al., 2016). These divergent observations of NSC responses to heat stress
may be due to variation in the severity of the heat stress, which in-
fluences the extent of damage and the capacity to recover. Based on
the findings of Bauweraerts et al. (2013, 2014), episodic heat wave
events produced more stress than a constant increase in tempera-
ture, emphasizing the importance of considering duration and inten-
sity of heat stress when predicting plant responses and the capacity
to recover. Funding informationi K E Y W O R D S
chlorophyll fluorescence, heat stress, nonstructural carbohydrates, photosynthesis, stomatal
conductance | 1297
www.ecolevol.org www.ecolevol.org 1298 MARIAS et al. 2.2 | Heat stress duration treatments Plants were exposed to 49°C in a growth chamber (Model I-35LVL,
Percival, Boone, IA) that accommodated two plants at a time and was
equipped with cool white lighting (PAR = 25 μmol·m−2·s−1). The treat-
ment temperature of 49°C was selected based on the temperature
at which a 50% reduction in initial chlorophyll fluorescence occurred
(49.0 ± 0.5°C) on C. arabica leaf disks (Marias et al., 2016). Preliminary
experiments at other temperatures also showed that 49°C induced
enough heat stress to be damaging without completely scorching/
killing leaves, allowing us to monitor recovery. Plants were watered
to drainage directly before treatment to avoid drought effects and
to buffer changes in soil temperature during treatment. Fine-wire
thermocouples measured air, leaf, and soil (~10 cm depth) tempera-
tures during treatment exposure (Figure S2). Pots were completely
wrapped with reflective bubble wrap to isolate the soil and roots
from heat exposure. This prevented soil temperatures from exceed-
ing 30°C (Figure S2), which is realistic for soil temperatures in summer
(Zheng, Hunt, & Running, 1993). Two plants were heated for 45 min,
and two different plants were heated for 90 min on the same day in
two experimental rounds: one on 21 July 2014 and one on 19 August
2014 (N = 2 per round). Control (0 min) plants were not exposed to
a temperature treatment (N = 4 per round). Rounds were combined
(N = 4 for treated plants, N = 8 for controls) because environmental
conditions (Table S1) and physiological measurements did not differ
between rounds. The growth chamber did not have the capability to
adjust light levels. Although light can influence chlorophyll fluores-
cence (Buchner, Karadar, Bauer, & Neuner, 2013; Ludlow, 1987), we
assumed that the low light levels for the 45-min or 90-min treatment
duration did not substantially influence results. Photosynthesis, chlo-
rophyll fluorescence, and foliar NSC content were monitored in con-
trol and treated plants prior to treatment (day 0) and 2, 15, 25, and
50 days after treatment. Three leaves per leaf age class per individual
plant were marked for resampling. When leaf drop occurred, an intact
leaf was selected and marked for resampling for the remainder of the
experiment. The goal of this study was to investigate how leaf age and heat
stress duration influence NSC dynamics and physiological responses
to and recovery from heat stress in C. arabica. 2.2 | Heat stress duration treatments We exposed treated
plants to a simulated sudden heat wave at 49°C in a growth cham-
ber for two different heat stress durations (45, 90 min) and monitored
physiological responses and recovery in expanding and mature leaves
for 50 days after treatment using gas exchange, chlorophyll fluores-
cence, and leaf NSCs. Because C. arabica is grown in both sun and
shade (DaMatta, Ronchi, Maestri, & Barros, 2007) and to put this
study in the context of a combined heat and drought scenario, we
used a leaf energy balance model to investigate the effects of reduced
stomatal conductance on leaf temperature in partial and full sun con-
ditions. We hypothesized that 1) mature leaves would exhibit less
physiological damage and/or faster recovery than expanding leaves,
and 2) the 90-min heat stress duration would result in greater physi-
ological damage and/or slower recovery than the 45-min heat stress
duration. whether NSC levels regulate C. arabica photosynthesis (Batista et al.,
2011; DaMatta et al., 2016; Franck, Vaast, Génard, & Dauzat, 2006;
Ronchi et al., 2006; Vaast, Angrand, Franck, Dauzat, & Génard, 2005). However, to our knowledge, no study has investigated the impacts
of heat stress-induced reductions in photosynthesis as a cause rather
than a consequence of NSC dynamics in C. arabica. controlled glasshouse conditions, temperature, relative humidity, and
PAR did not substantially differ between rounds (Table S1, Figure
S1). Plants were kept well-watered and fertilized once every 2 weeks
(Miracle-Gro All-Purpose Liquid Plant Food, 12% N, 4% P2O5, 8%
K2O). Coffea arabica is a tropical evergreen species that continually pro-
duces new flushes of leaves year-round. Leaf age class (expanding,
mature) was determined by visually dividing a mid-canopy plagiotro-
pic branch into thirds where leaf age sequence increased from the
outermost leaves to the base of the branch (e.g., Wright, Leishman,
Read, & Westoby, 2006). Leaves in the outer third were the youngest
and still expanding (expanding), and leaves in the inner third were the
oldest, fully expanded, and mature (mature). Mean photosynthesis val-
ues were 6.3 and 7.0 μmol·m−2·s−1 for expanding and mature leaves,
respectively, consistent with previously reported values for C. arabica
(DaMatta et al., 2007). Previous work has shown that thermotolerance of tropical spe-
cies measured with chlorophyll fluorescence increases with leaf age,
an evolutionary adaptation to protect older and longer-lived foliage
from irreversible damage (Yamada, Hidaka, & Fukamachi, 1996; Zhang,
Poorter, Hao, & Cao, 2012). This pattern was also observed on de-
tached C. arabica leaf disks (Marias, Meinzer, & Still, 2016). Because
NSCs have been linked to heat stress responses and heat tolerance
(Liu & Huang, 2000), NSCs may influence the ability of plants to avoid
permanent damage, to tolerate heat stress, and/or to recover from
heat stress. Therefore, leaf age-related differences in thermotolerance
and the ability to recover from heat stress may be related to NSC dy-
namics (Filewod & Thomas, 2014; Teskey et al., 2015). However, this
has not been investigated in C. arabica plants in situ. 1 | INTRODUCTION Heat stress severity is a function of intensity (exposure
temperature) and duration of exposure (Colombo & Timmer, 1992). Bauweraerts et al. (2014) found that growth of Quercus seedlings in-
creased in a + 6°C treatment but decreased in a + 12°C treatment. Therefore, it is expected that NSC dynamics would also be influ-
enced by heat stress severity, although this remains unknown. Given
that even short heat events can have substantial impacts on carbon
gain (Filewod & Thomas, 2014), the predicted fluctuations in the du-
ration of summer heat waves (Della-Marta, Haylock, Luterbacher, &
Wanner, 2007), and the paucity of studies manipulating heat stress
duration, research on the effects of heat stress duration on physiolog-
ical recovery and NSC dynamics is needed. NSC dynamics in C. arabica
are tightly linked to sink demand from vegetative and reproductive
growth (Chaves, Martins, Batista, Celin, & DaMatta, 2012; Génard
et al., 2008; Ramalho et al., 2013), and many studies have investigated Examining the impacts of heat stress on plant carbon utilization is
critical for understanding plant responses to changes in climate and
potential feedbacks between vegetation and climate. Heat stress af-
fects plant physiology from the cellular to whole plant scales, induc-
ing shifts in the allocation of assimilated carbon/photosynthate to
repair and recovery processes. At the cellular and organelle levels, high
temperatures can damage photosystem II (PSII) photochemistry and
electron transport; reduce thylakoid membrane fluidity, RUBISCO ac-
tivity, and cell membrane stability; and induce heat-shock protein ex-
pression and the production of reactive oxygen species (ROS) (Teskey
et al., 2015; Wahid, Gelani, Ashraf, & Foolad, 2007). At the leaf level,
high temperature stress reduces photosynthesis, increases photore-
spiration and dark (mitochondrial) respiration, and influences water MARIAS et al. 1299 | 2.1 | Plant material Coffea arabica L. (Eritrean Mokka) plants about 6–9 months old, ob-
tained from the Hawaii Agriculture Research Center in January 2014,
were grown in a peat–perlite–pumice growing mix (Sunshine LA4P) in
9.6-L pots in a glasshouse in Corvallis, Oregon. Supplemental metal
halide and high-pressure sodium lighting (400 watts) was used to
maintain a 12-hour photoperiod during fall and winter months. The
first round of experiments began 21 July 2014, and the second round
began 19 August 2014. Plants were ~1 m tall. During the sampling
rounds, average daytime temperature was 23.5°C, average nighttime
temperature 18.1°C, average daytime relative humidity 64%, aver-
age nighttime relative humidity 79%, and average daily maximum
photosynthetically active radiation (PAR) 325 μmol·m−2·s−1. Due to 1300 MARIAS et al. samples, and NSC was extracted from the solutions by heating them
in steam for 1.5 hr. The concentration of free glucose + fructose was
determined photometrically on a 96-well microplate photometer
(Multiskan FC, Thermo Scientific, Waltham, MA, USA) after enzymatic
conversion of glucose + fructose to gluconate-6-phosphate. Samples
were hydrolyzed by enzymatic treatment: invertase for sucrose and
amyloglucosidase for total NSC. Photometric analysis was based on
absorbance of samples at 340 nm in solution with reference to the ab-
sorbance of a glucose reference solution. Total NSC was calculated as
the sum of starch, sucrose, and glucose + fructose. NSC values (mg/g
dry weight) are presented in figures as % dry weight. 2.7 | Statistical analysis A three-way factorial linear mixed-effects model was developed with
leaf age, treatment, and day as main fixed effects. Nested random ef-
fects in the model were plant and leaf within plant. Response vari-
ables were photosynthesis, stomatal conductance, iWUE, FV/FM, total
NSC, starch, sucrose, and glucose + fructose. To choose a correla-
tion structure that would account for the repeated measurements of
leaves within plants through time, four models that allowed for dif-
ferent residual correlation structures were fit and selected based on
the minimum Bayesian information criterion (BIC) value. Assumptions
of constant variance and normality were checked using residual and
quantile–quantile plots. Log-transformations were necessary to meet
assumptions for starch and glucose + fructose. For ease of interpre-
tation, we present back-transformed data in results and figures. All
interactive and main effects of factors on the response were tested 2.3 | Photosynthesis and stomatal conductance
measurements Photosynthesis and stomatal conductance were measured in the
morning during active gas exchange (before afternoon stomatal clo-
sure occurred) between 0700 and 1,000 hr (dawn was ~0500 hr) on
1–3 marked leaves per leaf age class per individual plant using a port-
able photosynthesis system (LI-6400, Li-Cor, Lincoln, NE, USA). The
ratio of photosynthesis to stomatal conductance (A/gs), an estimate
of intrinsic water-use efficiency (iWUE, Jones, 2009), was calculated. In the cuvette, PAR was set to 500 μmol·m−2·s−1, leaf temperature
was set to 25°C, [CO2] sample was set to 400 μmol/mol (to represent
ambient atmospheric [CO2]), and flow rate was set to 500 μmol/s. Day 0 photosynthesis values were estimated from photosynthesis-
intercellular CO2 (Ci) curves. To compare intrinsic photosynthesis at
the ambient atmospheric [CO2] of 400 μmol/mol, photosynthesis was
estimated at the average Ci value of the control (0 min) group aver-
aged over all sampling days, which was 246 μmol/mol for expanding
leaves and 254 μmol/mol for mature leaves. 2.4 | Chlorophyll fluorescence measurements Chlorophyll fluorescence was measured on 1–3 marked leaves per leaf
age class per individual plant at ambient temperature with a portable
pulse–amplitude modulated chlorophyll fluorometer (Mini-PAM, Heinz
Walz Gmbh, Germany) at predawn to ensure leaves were dark-adapted. Chlorophyll fluorescence was measured as the ratio of variable to maxi-
mum fluorescence (FV/FM) in the convention of Maxwell and Johnson
(2000). FV/FM measures the maximum quantum efficiency of PSII pho-
tochemistry (Genty, Briantais, & Baker, 1989) and is calculated as: Chlorophyll fluorescence was measured as the ratio of variable to maxi-
mum fluorescence (FV/FM) in the convention of Maxwell and Johnson
(2000). FV/FM measures the maximum quantum efficiency of PSII pho-
tochemistry (Genty, Briantais, & Baker, 1989) and is calculated as: (1)
FV
FM
= FM −FO
FM
=1−FO
FM (1) A measuring light (red light-emitting diode, 650 nm, 0.15 μmol·
m−2·s−1 PAR) with a pulse–width of 3 μs was turned on at a pulse mod-
ulation frequency of 0.6 kHz to induce the minimal level of fluorescence
(FO). FV/FM was then determined by applying a 0.8 s saturating pulse of
white light (18,000 μmol photons·m−2·s−1 PAR), which transiently closed
all PSII reaction centers (preventing any photochemical processes from
occurring), minimized heat dissipation (since leaves were dark-adapted),
and induced maximum and variable fluorescence. 2.6 A simple leaf energy balance model created by Kevin Tu (http://land-
flux.org/Tools.php) was used to estimate the effect of shifts in sto-
matal conductance (gs) on leaf temperature (Tleaf). The modified leaf
energy balance equation from Sridhar and Elliott (2002), Monteith and
Unsworth (2007), and Jones (2013) is described in detail in Supporting
Information and Table S2. To investigate the effect of changes in gs
at representative heat wave temperatures potentially experienced
by C. arabica in the tropics, air temperatures (Tair) of 35°C, 40°C,
and 45°C were used in the model. The range of gs values used was
0–0.15 mol·m−2·s−1, similar to that observed in this study as well as
previously reported values for field grown C. arabica (Meinzer et al.,
1990; Gutierrez & Meinzer, 1994; Silva et al., 2004). Environmental
and leaf parameters were set to represent conditions experienced
by C. arabica in the tropics: Short-wave radiation (SWR) was 600 and
1,000 W/m2 to simulate partial and full sun conditions, respectively,
wind speed was 2.0 m/s, relative humidity was 65%, leaf angle was
20° from horizontal, absorptance to SWR was 0.50, emissivity was
0.96, and leaf length in the direction of wind was 100 mm. Incident
photosynthetically active radiation (PAR) for each SWR level at a
wavelength of 550 nm (average wavelength for the 400–700 nm PAR
range) was ~1,380 and ~2,300 μmol·m−2·s−1, respectively. PAR is as-
sumed ~50% of SWR (Britton & Dodd, 1976). Like photosynthesis, FV/FM also declined 2 days after treat-
ment, yet the amount of the reduction in FV/FM 2 days after treat-
ment was significantly greater in the 90-min group than the 45-min
group in both leaf age classes (Figure 1b,d). Regardless of leaf age
class, FV/FM of the 45-min group recovered to control values by day
15, while the 90-min group did not fully recover by day 50, similar
to the slower recovery of the 90-min group compared to the 45-min
group as measured with photosynthesis. The slope of FV/FM recovery was determined for each plant by fit-
ting a line of best fit (linear regression) from the FV/FM value 2 days
after treatment (i.e., the minimum FV/FM value or maximum damage
after treatment) through the day at which FV/FM recovered to day 0
values. The slope of recovery was log-transformed, and its relationship
with the FV/FM 2 days after treatment was described by a logarithmic
nonlinear regression equation (f = y0 + a*ln(abs(x)). A two-way facto-
rial linear mixed-effects model was developed with leaf age and treat-
ment as main fixed effects for slope of recovery and minimum FV/FM
as response variables. Procedures to select a correlation structure and
check assumptions were the same as stated above.ii The log-transformed slope of recovery (ln(slope of recovery)
back to control values significantly decreased with increasing dam-
age assessed with FV/FM 2 days after treatment (p = .0007, R2 = .57,
Figure 2). Mean ln(slope of recovery) did not differ among leaf age
classes (p = .86) but significantly differed between treatments where
the 90-min treatment exhibited a significantly lower ln(slope of re-
covery) than the 45-min treatment (p = .002). Similarly, the mean
damage assessed with FV/FM 2 days after treatment was signifi-
cantly greater in the 90-min group compared to the 45-min group
(p = .006). Pearson product–moment correlation was used to quantify the
strength of the relationship between photosynthesis and each NSC
component (total NSC, starch, sucrose, glucose + fructose) within
each treatment group (control (0 min), 45, 90 min) for all days sam-
pled. Statistical analyses were conducted in SigmaPlot 13.0 (Systat
Software, San Jose, CA, USA) and R version 3.2.3 (2015-12-10, The R
Foundation for Statistical Computing). 3 | RESULTS Although mean post-treatment stomatal conductance (gs) of ex-
panding leaves exhibited a decline similar to that of photosynthesis (A,
Figure 1), mean post-treatment gs of mature leaves did not significantly
differ among treatments (p > .05, Figure 3a,c). This influenced mean
post-treatment intrinsic water-use efficiency (A/gs) where expanding
leaves of the 90-min group had a significantly lower mean A/gs than
the control and 45-min groups, and mature leaves of the 90-min group
had significantly lower mean A/gs than the controls (Figure 3b,d). Both the 45- and 90-min treatments induced significant shifts in gas
exchange, FV/FM, and NSC content and dynamics. It was also observed
that after the experiment, the controls (0 min) produced flowers and
fruits, whereas the 45- and 90-min treatment groups did not. 3.1 The leaf energy balance model results showed that Tleaf-Tair
increased with decreasing gs in both partial (600 W/m2) and full Interactions among the main effects (age, treatment, day) on pho-
tosynthesis and FV/FM are summarized in Table 1. Photosynthesis TABLE 1 Marginal F-tests for photosynthesis and FV/FM using marginal F-tests (also known as type III tests) that account for
unbalanced sample sizes. Post hoc comparisons were made using a
95% confidence interval and p < .05. Due to sufficient degrees of free-
dom, we did not make multiple comparisons corrections. If no signifi-
cant differences between leaf age classes existed, NSC components of
expanding and mature leaves were combined by averaging over leaf
age to simplify data visualization. significantly declined 2 days after treatment in both the 45- and
90-min groups for both expanding and mature leaves but slower
recovery to control values in the 90-min group compared to the 45-
min group (Figure 1a,c). Photosynthesis of mature leaves in the 45-
min group recovered to control values by day 15 while that of the
90-min group recovered by day 25. In contrast, expanding leaves took
longer to recover to control values than mature leaves regardless of
treatment with photosynthesis of the 45-min group recovering by
day 25 and that of the 90-min group recovering by day 50. Within-
day and within-treatment differences between leaf age classes were
variable. Like photosynthesis, FV/FM also declined 2 days after treat-
ment, yet the amount of the reduction in FV/FM 2 days after treat-
ment was significantly greater in the 90-min group than the 45-min
group in both leaf age classes (Figure 1b,d). Regardless of leaf age
class, FV/FM of the 45-min group recovered to control values by day
15, while the 90-min group did not fully recover by day 50, similar
to the slower recovery of the 90-min group compared to the 45-min
group as measured with photosynthesis. significantly declined 2 days after treatment in both the 45- and
90-min groups for both expanding and mature leaves but slower
recovery to control values in the 90-min group compared to the 45-
min group (Figure 1a,c). Photosynthesis of mature leaves in the 45-
min group recovered to control values by day 15 while that of the
90-min group recovered by day 25. In contrast, expanding leaves took
longer to recover to control values than mature leaves regardless of
treatment with photosynthesis of the 45-min group recovering by
day 25 and that of the 90-min group recovering by day 50. Within-
day and within-treatment differences between leaf age classes were
variable. ues indicate p < .05. NumDF and denDF are the degrees of freedom in the numerator and denominator, respectively. N = 4. Bolded p-values indicate p < .05. NumDF and denDF are the degrees of freedom in the numerator and denomina 2.5 | Nonstructural carbohydrate (NSC) analysis One leaf per age class per individual plant was collected early morning
(directly after FV/FM measurements were made) on each sampling date,
immediately put on ice in a cooler, and transported to the nearby labo-
ratory where samples were microwaved for 90 s to stop all enzymatic
activity and oven-dried at 75°C. Samples were stored in a freezer before
being ground to a fine powder. Leaf samples were analyzed for con-
tent of total NSC, starch, sucrose, and glucose + fructose as described
by Woodruff and Meinzer (2011). Water was added to the powdered MARIAS et al. 1301 TABLE 1 Marginal F-tests for photosynthesis and FV/FM Mean po
of expanding leaves of control plants was 0.0796
treated plants was 0.0451 mol·m−2·s−1 (Figure 3b). mated that this observed heat stress-induced redu
noted by vertical lines in Figure 4a,b) would yield at
of 41.6°C in partial sun and Tleaf of 44.5°C in full sun
Tleaf of 46.6°C in partial sun and Tleaf of 49.5°C in fu
of 45°C, Tleaf of 51.7°C in partial sun and Tleaf of 54
(Figure 4a,b). 3.2 | Nonstructural carbohydrates (NSC
NSC dynamics differed significantly across all t
groups (controls (0 min), 45, 90 min; Figures 5 and
among the main effects (age, treatment, day) on to
sucrose, and glucose + fructose are summarized
to lack of significant leaf age-related differences
FIGURE 2 FV/FM values reached 2 days after treatment (i.e.,
maximum damage after treatment) significantly related to the
log-transformed slope of FV/FM recovery across leaf age class and
treatment (R2 = .57, p = .0007). Nonlinear regression equation:
f = y0 + a*ln(abs(x). Each data point represents an individual plant. N = 4 (1,000 W/m2) sun conditions (Figure 4a,b). Tleaf was consistently
greater in full sun (Figure 4b) than partial sun (Figure 4a). Even at
higher values of gs (e.g., 0.15 mol·m−2·s−1), Tleaf in full sun at Tair of
35–45°C (Figure 4b) was 42.0–51.2°C. Mean post-treatment gs
of expanding leaves of control plants was 0.0796 mol·m−2·s−1 and
treated plants was 0.0451 mol·m−2·s−1 (Figure 3b). The model esti-
mated that this observed heat stress-induced reduction in gs (de-
noted by vertical lines in Figure 4a,b) would yield at Tair of 35°C, Tleaf
of 41.6°C in partial sun and Tleaf of 44.5°C in full sun; at Tair of 40°C,
Tleaf of 46.6°C in partial sun and Tleaf of 49.5°C in full sun; and at Tair
of 45°C, Tleaf of 51.7°C in partial sun and Tleaf of 54.5°C in full sun
(Figure 4a,b). TABLE 1 Marginal F-tests for photosynthesis and FV/FM Photosynthesis (μmol·m−2·s−1)
FV/FM
numDF
denDF
F-value
p-Value
numDF
denDF
F-value
p-Value
Intercept
1
93
366.41
<.0001
1
104
883.02
<.0001
Age
1
13
1.6028
.2277
1
13
6.846
.0213
Treatment
2
13
21.357
.0001
2
13
26.312
<.0001
Day
4
93
20.077
<.0001
4
104
94.940
<.0001
Age × Treatment
2
13
3.4666
.0621
2
13
2.8695
.0929
Age × Day
4
93
3.8058
.0065
4
104
1.2742
.2849
Treatment × Day
8
93
8.2791
<.0001
8
104
38.442
<.0001
Age × Treatment × Day
8
93
3.5207
.0013
8
104
0.6461
.7373
N = 4. Bolded p-values indicate p < .05. NumDF and denDF are the degrees of freedom in the numerator and denominator, respectively. 1302 |
MARIAS et al. FIGURE 1 Time courses of photosynthesis and FV/FM of expanding (a, b) and mature (c, d) leaves from the same plants (N = 4) of the 0-min
(controls), 45-min, and 90-min groups at 0, 2, 15, 25, and 50 days after treatment. Different uppercase letters represent significant differences
among treatments within each day and leaf age. Different lowercase letters represent significant differences among days within treatment and
leaf age. Asterisks represent significant differences between leaf age within treatment and day. Error bars represent ± SE MARIAS et al. MARIAS et al. 1302 FIGURE 1 Time courses of photosynthesis and FV/FM of expanding (a, b) and mature (c, d) leaves from the same plants (N = 4) of the 0-min
(controls), 45-min, and 90-min groups at 0, 2, 15, 25, and 50 days after treatment. Different uppercase letters represent significant differences
among treatments within each day and leaf age. Different lowercase letters represent significant differences among days within treatment and
leaf age. Asterisks represent significant differences between leaf age within treatment and day. Error bars represent ± SE FIGURE 2 FV/FM values reached 2 days after treatment (i.e.,
maximum damage after treatment) significantly related to the
log-transformed slope of FV/FM recovery across leaf age class and
treatment (R2 = .57, p = .0007). Nonlinear regression equation:
f = y0 + a*ln(abs(x). Each data point represents an individual plant. N = 4 (1,000 W/m2) sun conditions (Figure 4a,b). Tleaf w
greater in full sun (Figure 4b) than partial sun (Fig
higher values of gs (e.g., 0.15 mol·m−2·s−1), Tleaf in f
35–45°C (Figure 4b) was 42.0–51.2°C. 4.1 | Implications for combined heat and drought
scenarios The sudden heat stress disrupted coordination between A and gs,
leading to declining iWUE (A/gs) with increasing heat stress duration in
both expanding and mature leaves (Figure 3). Under a combined heat
and drought scenario expected throughout the 21st century (IPCC,
2014), sudden heat-induced reductions in the ratio of carbon gain to
water loss could accelerate the point at which drought, even in the
absence of heat, would further restrict carbon gain (Chaves et al.,
2002). Persistent reductions in gs and therefore evaporative cooling
(Ball, Cowan, & Farquhar, 1988; Monteith, 1981; Schymanski, Or, &
Zwieniecki, 2013) of expanding leaves on heat-stressed plants un-
dergoing drought would place them at additional risk of further heat
damage in closely spaced heat waves. The leaf energy balance model
indicated that the extent to which Tleaf would increase in response to
reduced gs would be greatest at higher Tair (e.g., 45°C) compared to
lower Tair (e.g., 35°C) and in full compared to partial sun (Figure 4a,b). Tleaf was especially sensitive to changes in gs at relatively low gs, which
would be expected during drought. For example, at Tair of 45°C and gs
of ~0.04 mol·m−2·s−1 as observed in expanding leaves of the treatment
groups (Figure 3a), Tleaf would be ~51.7°C in partial sun (Figure 4a)
and ~54.5°C in full sun (Figure 4b). These temperatures would cause
significant damage, if not death, because the temperature causing a
90% reduction in initial FV/FM estimated from chlorophyll fluores-
cence thermotolerance curves was 50.1°C in expanding leaves and
54.1°C in mature leaves (data not shown, see Marias et al., 2016). A factor not considered in the leaf energy balance model was sto-
matal sensitivity to VPD. Photosynthesis and gs in C. arabica decline
with increasing VPD (Oren et al., 1999) that typically ranges from
1.0 to 3.5 kPa throughout the day (Ronquim, Prado, Novaes, Fahl, &
Ronquim, 2006). This VPD-related decline in gs would further increase Sucrose of the control and 45-min groups did not significantly
change with day (Figure 5c). In contrast, sucrose of the 90-min group
was significantly greater than that of controls 2 days after treatment
before significantly declining and becoming significantly less than day
0 values at day 50 (Figure 5c). 4 | DISCUSSION 4 4.1 | Implications for combined heat and drought
scenarios 3.2 | Nonstructural carbohydrates (NSCs) FIGURE 2 FV/FM values reached 2 days after treatment (i.e.,
maximum damage after treatment) significantly related to the
log-transformed slope of FV/FM recovery across leaf age class and
treatment (R2 = .57, p = .0007). Nonlinear regression equation:
f = y0 + a*ln(abs(x). Each data point represents an individual plant. N = 4 NSC dynamics differed significantly across all three treatment
groups (controls (0 min), 45, 90 min; Figures 5 and 6). Interactions
among the main effects (age, treatment, day) on total NSC, starch,
sucrose, and glucose + fructose are summarized in Table 2. Due
to lack of significant leaf age-related differences within day and | 1303
MARIAS et al. FIGURE 3 Mean post-treatment
stomatal conductance (gs) and intrinsic
water-use efficiency (A/gs) for 0-min
(controls), 45-min, and 90-min groups. Different uppercase letters represent
significant differences among treatments
within leaf age class. Error bars
represent ± SE MARIAS et al. treatment, total NSC, starch, and sucrose of expanding and mature
leaves were combined by averaging over leaf age to simplify data
visualization (Figure 5). Total NSC and starch in the controls stead-
ily declined with time and were significantly less than day 0 values
by day 50 (Figure 5a,b). In the 45-min group, total NSC and starch
significantly declined 2 days after treatment and were significantly
less than that of any other day. At days 15 and 25, total NSC and
starch of the 45-min group significantly increased and became
significantly greater than that of the control and 90-min groups
(Figure 5a,b). In contrast to the control and 45-min groups, total
NSC and starch of the 90-min group did not significantly change
with day (Figure 5a,b). 4 | DISCUSSION 4.1 | Implications for combined heat and drought
scenarios Although sucrose of the 45-min group
did not significantly change with day, it was significantly greater than
that of controls and the 90-min group at days 15 and 25 and that of
the 90-min group at day 50. In contrast to total NSC, starch, and sucrose, glucose + fructose
was significantly affected by leaf age (F = 52.792, p < .0001, Table 2)
but did not significantly differ among treatments nor with day in ei-
ther age class (Figure 6a,b). Glucose + fructose of controls was greater
in expanding leaves than mature leaves at days 0, 15, 25, and in the
45-min group at day 2. There were no age differences in the 90-min
group. Pearson product–moment correlation indicated significant pos-
itive associations between photosynthesis and total NSC (r = .409,
p = .00145, Table 3) and between photosynthesis and starch in the
controls (r = .362, p = .00530, Table 3), and between photosynthesis
and starch in the 45-min group (r = .332, p = .0445, Table 3). In con-
trast, photosynthesis was not significantly related to any NSC compo-
nent in the 90-min group (p > .05, Table 3). 1304 |
MARIAS et al. FIGURE 4 Modeled relationships between stomatal conductance (gs) and leaf temperature (Tleaf) – air temperature (Tair) at Tair = 35°C, 40°C,
and 45°C at short-wave radiation (SWR) of 600 W/m2 (a) and 1000 W/m2 (b) to represent partial and full sun conditions, respectively. Vertical
lines indicate mean post-treatment gs values of controls (0.0796 mol·m−2·s−1) and of the 45-min and 90-min groups (0.0451 mol·m−2·s−1 for
expanding leaves). Incident photosynthetically active radiation (PAR) for each SWR level at a wavelength of 550 nm (average wavelength for the
400–700 nm PAR range) was ~1380 and ~2300 μmol·m−2·s−1, respectively. PAR is assumed ~50% of SWR (Britton & Dodd, 1976) MARIAS et al. MARIAS et al. 1304 FIGURE 4 Modeled relationships between stomatal conductance (gs) and leaf temperature (Tleaf) – air temperature (Tair) at Tair = 35°C, 40°C,
and 45°C at short-wave radiation (SWR) of 600 W/m2 (a) and 1000 W/m2 (b) to represent partial and full sun conditions, respectively. Vertical
lines indicate mean post-treatment gs values of controls (0.0796 mol·m−2·s−1) and of the 45-min and 90-min groups (0.0451 mol·m−2·s−1 for
expanding leaves). 4.2 | Greater sensitivity to heat stress in
expanding leaves Consistent with our first hypothesis, mature leaves showed faster re-
covery of photosynthesis than expanding leaves (Figure 1). However,
the extent of damage as measured with photosynthesis at day 2 did
not differ between leaf age classes nor treatment, suggesting that leaf
age may influence the capacity to recover more than the extent of
damage incurred, although both characteristics influence the ability
to withstand heat stress (Escandón et al., 2016). Consistent with our
second hypothesis, the 90-min duration of heat stress resulted in both
greater physiological damage and slower recovery than the 45-min
group as measured with FV/FM and slower recovery as measured with
photosynthesis (Figure 1). This was due to the apparent slower rate
of FV/FM recovery and greater damage at day 2 induced by the 90-
min treatment than the 45-min treatment (Figures 1 and 2). Taken to-
gether, these results showed that leaf age affected the recovery rate
of photosynthesis but not that of FV/FM, suggesting that PSII pho-
tochemistry may be conserved across different leaf ages in low light
(Ishida, Uemura, Koike, Matsumoto, & Hoe, 1999) and that longer-
lived foliage may be protected by the greater capacity of older leaves
to photosynthetically recover (Yamada et al., 1996). The declines in
FV/FM indicate that FO increased and/or FV decreased, both of which Interestingly, total NSC, starch, and sucrose content were not sig-
nificantly different between leaf age classes (Figure 5) suggesting that
these components were highly regulated. In contrast, glucose + fruc-
tose content was significantly greater in expanding leaves than mature
leaves (Figure 6), which may occur because free sugars are needed as
building blocks for structural carbohydrates (e.g., cellulose, lignin) in
cell walls of expanding leaves, whereas sugars in mature leaves have
already been allocated to structural carbohydrates required for struc-
tural support (Cavatte et al., 2012). 4.1 | Implications for combined heat and drought
scenarios Incident photosynthetically active radiation (PAR) for each SWR level at a wavelength of 550 nm (average wavelength for the
400–700 nm PAR range) was ~1380 and ~2300 μmol·m−2·s−1, respectively. PAR is assumed ~50% of SWR (Britton & Dodd, 1976) Tleaf and therefore the risk of damaging heat stress. In contrast to ex-
panding leaves, mature leaves maintained relatively high gs and rela-
tively high intercellular [CO2] across all treatments, which may have
contributed to the faster recovery of A than FV/FM in mature leaves
(Figure 1). However, plants experiencing drought would not be able to
maintain relatively high gs without risking hydraulic failure and death
(Adams et al., 2009). Therefore, recovery from heat stress would likely
be inhibited during drought. indicate stress-induced changes to photochemistry including PSII in-
activation, photodamage, heat dissipation, photoinhibition or dam-
age to the oxygen evolving complex and water splitting system that
disrupts electron donation to PSII reaction centers (Demmig, Winter,
Krüger, & Czygan, 1987; Havaux, 1993; Maxwell & Johnson, 2000;
Schreiber & Berry, 1977; Weis & Berry, 1987; Yamada et al., 1996;
Yamashita & Butler, 1968). In addition to their slower recovery, gs of expanding leaves declined
in the 45- and 90-min treatments while that of mature leaves did not
change with treatment. Besides reducing water loss, this response
may reflect the impact of damaged membrane integrity on the ability
to maintain gs (Bita & Gerats, 2013). As discussed above, reduced gs
would exacerbate the effects of heat stress on expanding leaves by
further increasing their temperature and putting them at greater risk
of additional heat-induced damage. 4.3 | Impact of heat-induced inhibition of flowering
on NSC dynamics In contrast to controls, none of the heat-treated plants produced
flowers or fruits. This is not surprising because coffee reproduc-
tion is highly sensitive to heat (Bita & Gerats, 2013; Camargo, 1985;
Camargo, 2010). Our results showed that even a short, sudden heat 1305 MARIAS et al. FIGURE 5 Time courses of mean total nonstructural
carbohydrates (total NSC), starch, and sucrose of expanding
and mature leaves from the same plants (N = 4) of the 0-min
(controls), 45-min, and 90-min groups at 0, 2, 15, 25, and 50 days
after treatment. Different uppercase letters represent significant
differences between treatments within each day. Different lowercase
letters represent significant differences between days within each
treatment. Error bars represent ± SE a sudden heat stress event simulated in this study (Bauweraerts et al.,
2013, 2014; Stone & Nicolas, 1995; Zou, 2009). However, dramatic
aseasonal fluctuations in temperature are expected to become more
frequent in the future (Filewod & Thomas, 2014). Further, elevated
ambient [CO2] may partially mitigate the impacts of high temperature
stress on C. arabica (Martins et al., 2016; Rodrigues et al., 2016) and
C. arabica may be able to maintain adequate mineral balance under
combined high temperature and elevated [CO2] situations (Martins
et al., 2014). a sudden heat stress event simulated in this study (Bauweraerts et al.,
2013, 2014; Stone & Nicolas, 1995; Zou, 2009). However, dramatic
aseasonal fluctuations in temperature are expected to become more
frequent in the future (Filewod & Thomas, 2014). Further, elevated
ambient [CO2] may partially mitigate the impacts of high temperature
stress on C. arabica (Martins et al., 2016; Rodrigues et al., 2016) and
C. arabica may be able to maintain adequate mineral balance under
combined high temperature and elevated [CO2] situations (Martins
et al., 2014). Because NSCs are utilized for reproduction (e.g., flowers, fruits) in
C. arabica (Cannell, 1976), the decline in total NSC and starch of con-
trols (Figure 5a,b) was likely due to the subsequent formation of flow-
ers and fruits (Chaves et al., 2012). Costa, Zambolim, and Rodrigues
(2006) also observed reduced starch in high fruit-bearing plants and
no decrease in soluble sugars, consistent with the patterns of starch,
sucrose, and glucose + fructose in the flower-bearing control plants
in this study. Since fruit production is the strongest carbohydrate sink in C. 4.3 | Impact of heat-induced inhibition of flowering
on NSC dynamics ar-
abica (Cannell, 1976) and competes with vegetative growth (Amaral,
Matta, & Rena, 2001; DaMatta et al., 2007, 2008; Vaast et al., 2005),
the lack of fruiting in the heat treatments should theoretically result
in more NSC available for growth, storage, metabolic maintenance,
and repair (Chapin, Schulze, & Mooney, 1990; Dietze et al., 2014;
Kozlowski, 1992). This helps elucidate the patterns in total NSC and
starch of the 45-min group (Figure 5a,b). The sharp but transient re-
duction in total NSC and starch at day 2 coincided with the treatment-
induced reductions in photosynthesis and FV/FM, indicating significant
heat stress-induced damage and reductions in carbohydrate produc-
tion (Wahid et al., 2007). The decline in starch with no significant shifts
in sucrose and glucose + fructose at day 2 may indicate the allocation
of NSC to metabolic maintenance and repair in response to the 45-min
treatment (Bita & Gerats, 2013). This may also be due to increased res-
piration (Way & Yamori, 2014), as well as the remobilization of starch
from source leaves to roots (Blessing, Werner, Siegwolf, & Buchmann,
2015), although we did not measure NSC in roots. By days 15 and 25,
total NSC, starch, and sucrose of the 45-min group had significantly
accumulated compared to controls, coinciding with the full recovery
of photosynthesis and FV/FM. Due to the lack of demand for repro-
duction and repair by day 15 and later, carbon supply was greater than
carbon demand (Génard et al., 2008) so starch was stored resulting in
an increase in total NSC. By day 50, starch and total NSC levels of the
45-min group declined to day 0 values suggesting that the stored NSC
may have been utilized for renewed vegetative growth and metabolic
maintenance. FIGURE 5 Time courses of mean total nonstructural
carbohydrates (total NSC), starch, and sucrose of expanding
and mature leaves from the same plants (N = 4) of the 0-min
(controls), 45-min, and 90-min groups at 0, 2, 15, 25, and 50 days
after treatment. Different uppercase letters represent significant
differences between treatments within each day. Different lowercase
letters represent significant differences between days within each FIGURE 5 Time courses of mean total nonstructural
carbohydrates (total NSC), starch, and sucrose of expanding
and mature leaves from the same plants (N = 4) of the 0-min
(controls), 45-min, and 90-min groups at 0, 2, 15, 25, and 50 days
after treatment. 4.3 | Impact of heat-induced inhibition of flowering
on NSC dynamics FIGURE 6 Time courses of glucose + fructose of expanding (a) and mature (b) leaves from the same plants (N = 4) of the 0-min (controls),
45-min, and 90-min groups at 0, 2, 15, 25, and 50 days after treatment. Different uppercase letters represent significant differences among
treatments within each day. Different lowercase letters represent significant differences among days within each treatment. Asterisks represent
significant differences between leaf age classes. Error bars represent ± SE reproduction. In contrast to the other NSC constituents that did not
change with time in the 90-min treatment, the transient increase in
sucrose content at day 2 may reflect an initial repair or defense re-
sponse to the substantial damage caused by the 90-min treatment
because sucrose has been linked to defense against ROS (Bita &
Gerats, 2013), antioxidant production (Couée et al., 2006), osmotic
adjustment (Cavatte et al., 2012), and stress response signaling (El
Sayed, Rafudeen, & Golldack, 2014; Secchi & Zwieniecki, 2011, 2016;
Sugio et al., 2009; Wang & Ruan, 2016). Also, soluble sugars such
as sucrose are among the primary metabolites and osmolytes known
to accumulate in response to heat stress (Wahid et al., 2007) and
are necessary for protection from elevated temperature and main-
taining water balance and membrane stability (Bita & Gerats, 2013;
Farooq et al., 2008). Sucrose is translocated from source leaves to
sink organs through the phloem, and its transient increase in plants
subjected to 90 min of heat stress may also be associated with a dis-
ruption or inhibition of phloem transport (Blessing et al., 2015; Sala
et al., 2012; Woodruff, 2014). The subsequent decline in sucrose
after day 2 suggests that the demand for sucrose for repair, renewed
growth, and ongoing metabolism was greater than the supply from
photosynthesis, which was still inhibited on day 50. repair. Escandón et al. (2016) also found that soluble sugars seemed
more closely associated with plant responses to increasing number of
days exposed to heat stress, although they did not measure starch. Interestingly, glucose + fructose was not significantly affected by day
or treatment in the 45- and 90-min groups (Figure 6), consistent with
Lafta and Lorenzen (1995) that found no effect of temperature on
sugar levels in potato and attributed this to coordinated control of
sugar metabolism in response to high temperature stress. Downregulation of photosynthesis has been associated with
NSC accumulation in C. 4.3 | Impact of heat-induced inhibition of flowering
on NSC dynamics Different uppercase letters represent significant
differences between treatments within each day. Different lowercase
letters represent significant differences between days within each
treatment. Error bars represent ± SE FIGURE 5 Time courses of mean total nonstructural
carbohydrates (total NSC), starch, and sucrose of expanding
and mature leaves from the same plants (N = 4) of the 0-min
(controls), 45-min, and 90-min groups at 0, 2, 15, 25, and 50 days
after treatment. Different uppercase letters represent significant
differences between treatments within each day. Different lowercase
letters represent significant differences between days within each
treatment. Error bars represent ± SE FIGURE 5 Time courses of mean total nonstructural Despite the greater damage induced by the 90-min heat treat-
ment compared to the 45-min treatment (Figures 1 and 2), total NSC,
starch, and glucose + fructose of the 90-min group unexpectedly did
not change significantly throughout the 50 days of the experiment
(Figures 5 and 6). This may reflect a balance between NSC supply
and sink demand in the 90-min treatment. The supply of NSC was
likely low due to the ongoing inhibition of photosynthesis and incom-
plete recovery of photochemistry (FV/FM) by day 50 (Figure 1). The
sink demand in this treatment was also low due to the inhibition of stress event can inhibit reproduction and have negative consequences
for C. arabica productivity (Bunn et al., 2014; Davis et al., 2012). The
treatment temperature of 49°C for 45- and 90-min durations may
occur in the field because leaf temperatures can exceed air tempera-
ture by 15–20°C in sun grown C. arabica (Alvim & Kozlowski, 2013;
Butler, 1977). This was supported by the leaf energy balance model
(Figure 4). It is possible that C. arabica may acclimate and respond dif-
ferently to gradual increases in average air temperature as opposed to 1306 |
MARIAS et al. FIGURE 6 Time courses of glucose + fructose of expanding (a) and mature (b) leaves from the same plants (N = 4) of the 0-min (controls),
45-min, and 90-min groups at 0, 2, 15, 25, and 50 days after treatment. Different uppercase letters represent significant differences among
treatments within each day. Different lowercase letters represent significant differences among days within each treatment. Asterisks represent
significant differences between leaf age classes. Error bars represent ± SE MARIAS et al. 1306 MARIAS et al. 4.3 | Impact of heat-induced inhibition of flowering
on NSC dynamics arabica (e.g., starch in DaMatta, Maestri,
Mosquim, and Barros (1997), sucrose in Franck et al., 2006), while
other studies have found no link between NSC and photosyn-
thetic downregulation (Batista et al., 2011; DaMatta et al., 2016;
Silva et al., 2004). In contrast, we found a positive relationship be-
tween starch and photosynthesis in controls and the 45-min group
(Table 3). These different results may be a consequence of previous
studies of NSC and photosynthesis in C. arabica having examined
NSC as a cause of photosynthetic regulation, whereas in this study,
we examined changes in NSC dynamics as a consequence of heat
stress-induced inhibition of photosynthesis and other physiologi-
cal processes. The lack of a significant relationship between pho-
tosynthesis and starch in the 90-min group suggests a decoupling
of starch dynamics and photosynthesis under greater heat stress
duration, complicating predictions of plant carbon allocation under
increasing temperature stress. ACKNOWLEDGMENTS This work was supported by the NSF Graduate Research Fellowship
Program and NSF grant IOS 11-46746. We are grateful to the OSU
Greenhouse Operations staff for their assistance with the mainte-
nance of plant material. The investigation of
the impacts of heat stress duration and leaf age on NSC dynamics
and recovery are essential for understanding plant carbohydrate me-
tabolism and how C. arabica may respond to future climate change
scenarios. that induced more damage and slower recovery exhibited an NSC
pattern of repair and/or phloem transport inhibition as indicated by
sucrose dynamics. Expanding leaves were more sensitive to heat
stress, exhibited by slower photosynthetic recovery and lower sto-
matal conductance with increasing heat stress duration, a response
likely to exacerbate heat stress effects during combined heat and
drought scenarios. Reproduction and NSC dynamics are tightly linked
in C. arabica and the heat-induced inhibition of flowering significantly
impacted NSC allocation dynamics, making the timing of heat stress
at key developmental stages such as reproduction critical for inter-
preting and predicting responses to heat stress. The investigation of
the impacts of heat stress duration and leaf age on NSC dynamics
and recovery are essential for understanding plant carbohydrate me-
tabolism and how C. arabica may respond to future climate change
scenarios. CONFLICT OF INTEREST None declared. TABLE 3 Pearson correlation coefficients (R) and p-values
describing the relationship between mean expanding and mature leaf
photosynthesis and NSC component (total NSC, starch, sucrose,
glucose + fructose) within the 0-min (controls), 45-min, and 90-min
groups TABLE 2 Marginal F-tests for total NSC, starch, sucrose, and glucose + fructose
Total NSC
Starch
Sucrose
Glucose + Fructose
numDF
denDF
F-value
p-Value
numDF
denDF
F-value
p-Value
numDF
denDF
F-value
p-Value
numDF
denDF
F-value
p-Value
Intercept
1
79
850.78
<.0001
1
79
271.56
<.0001
1
79
620.693
<.0001
1
79
144.68
<.0001
Age
1
13
3.202
.0969
1
13
0.5649
.4657
1
13
4.833
.0466
1
13
52.792
<.0001
Treatment
2
13
4.280
.0373
2
13
0.123
.8856
2
13
11.66
.0013
2
13
1.974
.1784
Day
4
79
3.420
.0124
4
79
8.526
<.0001
4
79
4.385
.0030
4
79
2.481
.0505
Age × Treatment
2
13
0.442
.6523
2
13
1.015
.3899
2
13
0.66
.5333
2
13
0.511
.6113
Age × Day
4
79
1.846
.1282
4
79
1.276
.2867
4
79
1.010
.4072
4
79
2.497
.0493
Treatment × Day
8
79
5.682
<.0001
8
79
11.670
<.0001
8
79
3.222
.0032
8
79
0.702
.6893
Age × Treatment × Day
8
79
0.956
.4767
8
79
1.457
.1865
8
79
0.509
.8466
8
79
2.146
.0408i Total
NSC
Starch
Sucrose
Glucose +
Fructose
Photosynthesis
0 min
R
.409
.362
.210
.141
p-Value
.00145
.00530
.113
.291
45 min
R
.318
.332
.0100
.161
p-Value
.0551
.0445
.953
.342
90 min
R
−.0427
−.0177
−.0920
.0606
p-Value
.805
.919
.594
.726
Bold values are significant p < .05. Bold values are significant p < .05. that induced more damage and slower recovery exhibited an NSC
pattern of repair and/or phloem transport inhibition as indicated by
sucrose dynamics. Expanding leaves were more sensitive to heat
stress, exhibited by slower photosynthetic recovery and lower sto-
matal conductance with increasing heat stress duration, a response
likely to exacerbate heat stress effects during combined heat and
drought scenarios. Reproduction and NSC dynamics are tightly linked
in C. arabica and the heat-induced inhibition of flowering significantly
impacted NSC allocation dynamics, making the timing of heat stress
at key developmental stages such as reproduction critical for inter-
preting and predicting responses to heat stress. 4.4 | Heat stress duration impacts on NSC dynamics The observed effect of heat stress duration on PSII photochemistry
and photosynthesis was associated with significant impacts on NSC
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Long term outcomes of intra-carotid arterial transfusion of circulatory derived autologous CD34+ cells for acute ischemic stroke patients—A randomized, open-label, controlled phase II clinical trial
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Research Square (Research Square)
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cc-by
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Long term outcomes of intra-carotid arterial
transfusion of circulatory derived autologous CD34+
cells for acute ischemic stroke patients—A
randomized, open-label, controlled phase II clinical
trial Hung-Sheng Lin
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Pei-Hsun Sung
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Shu-Hua Huang
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Wei-Che Lin
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
John Y. Chiang
National Sun Yat-Sen University Hung-Sheng Lin
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Pei-Hsun Sung
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Shu-Hua Huang
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Wei-Che Lin
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
John Y. Chiang
National Sun Yat-Sen University Ming-Chun Ma
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Yi-Ling Chen
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Kuan-Hung Chen
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Fan-Yen Lee
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Sheung-Fat Ko
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Hon-Kan Yip Ming-Chun Ma
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Yi-Ling Chen
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Kuan-Hung Chen
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Fan-Yen Lee
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Sheung-Fat Ko
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung
Hon-Kan Yip Yi-Ling Chen
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung Research Article Page 1/33 Page 1/33 Keywords: endothelial progenitor cells, acute ischemic stroke, intracarotid transfusion of CD34 + cells,
angiogenesis, neurological outcomes Posted Date: March 18th, 2024 Background This phase II randomized control trial tested whether intracarotid arterial administration of autologous
CD34 + cells to the patients within 14 ± 7 days after acute ischemic stroke (IS) could be safe and further
improve short- and long-term outcomes. DOI: https://doi.org/10.21203/rs.3.rs-3974840/v1 DOI: https://doi.org/10.21203/rs.3.rs-3974840/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Additional Declarations: No competing interests reported. Page 2/33 Methods and Results Between January 2018 and March 2022, 28 consecutive patients were equally randomly allocated into
group 1 (CD34 + cells/3.0 x 107/patient) and group 2 (received optimal-medical therapy). The CD34 +
cells were transfused into the ipsilateral brain infarct zone of group 1 patients at catheterization room. The results demonstrated that safety and success of the procedure were 100% and no long-term
tumorigenesis was observed in group 1 patients. In group 1 patients, the circulating EPC
number/angiogenesis capacity were significantly higher at post than at prior to granulocyte-colony-
stimulating factor treatment (all p < 0.001). Time courses of blood samplings from right-internal jugular
vein of the group 1 displayed a significant increase in the levels of SDF-1α and EPCs in time points of
5/10/30 minutes than in that of 0 minute (all p < 0.005). The National Institute of Health Stroke Scale
was similar upon presentation, whereas it showed a great response by days 30/90 and Tc-99m brain
perfusion was significantly increased by 180-days after acute IS in group 1 than in group 2 (p = 0.046). The long-term (4.1 ± 1.3 years follow-up) combined end points (defined as death/recurrent stroke/or
severe disability) were notably higher in group 2 than in group 1 patients (p = 0.103). Conclusion Intracarotid transfusion of autologous CD34 + cells was safe and might provide some benefits for acute
IS patients. Introduction Stroke, a growing epidemic, remains 2nd cause of death and 3rd leading cause of combined death and
disability worldwide [1-4]. Although various etiologies cause stroke, atheroembolic stroke is still the most
common cause of ischemic stroke (IS) worldwide [5-9], and, after initial IS development, there will be
associated with a high risk of frequently recurrent IS [10-12]. Additionally, previous clinical studies have
clearly observed that patients with a recent transient ischemic attack (TIA) or IS with severe stenosis
(defined as 70% to 90% stenosis of the diameter of a major intracranial artery) are at particularly high risk
of recurrent stroke in the territory of the stenotic arterial supplied area (estimated to be approximately
23% at 1 year) despite of treatment with aspirin and standard management of vascular risk factors
according to current AHA guideline recommendation [8, 13, 14]. Surprisingly, while the epidemiology, Page 3/33 Page 3/33 etiologies, mechanisms, classification, and prognostic outcomes of IS have been keenly investigated for
several decades [15-18], a safe and efficacious management for etiologies, mechanisms, classification, and prognostic outcomes of IS have been keenly investigated for
several decades [15-18], a safe and efficacious management for etiologies, mechanisms, classification, and prognostic outcomes of IS have been keenly investigated for
several decades [15-18], a safe and efficacious management for
all of the IS patients, especially when recurrent IS is seriously taken into consideration, has not been
fully developed. Previously, stenting for intra-cranial arterial stenosis (SAMMPRIS Trial), an alternative
therapy vs. conventional medication, has been conducted [19]. Regrettably, the results are
disappointing [19]. The majority of these patients, therefore, are still left without any categorical
treatment. Accordingly, finding a safe and effective therapeutic option for patients with IS is
fundamentally important for neurologists and physicians. Interestingly, our previous studies [20, 21] have shown that acute IS stimulated endothelial progenitor
cells (EPCs) mobilization into the circulation. Additionally, our other previous studies [20, 21] have further
identified that an increase in circulating level of EPCs was strongly associated with favorable clinical
outcomes after IS. Furthermore, our phase I and II clinical trials previously demonstrated that intra-
coronary transfusion of CD34+ cells improved left ventricular function in patients with diffuse coronary
artery disease (CAD) and non-candidates for coronary artery intervention [22, 23]. Intriguingly, it is well
recognized that CAD and cerebrovascular disease (CVD), which comprise the majority of the same causal
etiologies of endothelial dysfunction/damage and arteriosclerosis, constitute arterial obstructive
syndromes. Introduction Thus, CAD and CVD are two sides of the same coin. Accordingly, we proposed that
autologous EPC transplantation could be an alternative therapeutic modality for patients after acute IS. In fact, we have conducted a phase I clinical trial of intra-carotid arterial transfusion of autologous
circulatory derived CD34+ cells for old IS patients [24] and the results demonstrated that CD34+ cell
therapy was safe and offered some benefit to old IS patients. Surprisingly, since we designed the
protocol of phase II clinical trial with CD34+ cell therapy for acute IS in 2017, no data has been reported
to address the efficacy of intracarotid arterial transfusion of autologous EPCs/CD34+ cells for those
acute IS patients worldwide. Accordingly, the above-mentioned issues [1-24] and the result of our phase I
clinical trial of intracarotid arterial administration of CD34+ cells for old IS patients [24] encouraged us to
further conduct: Impact of intra-carotid arterial transfusion of circulatory derived autologous CD34+ cells
for acute IS patients—A randomized, open-label, controlled phase II clinical trial. Calculation of sample size for specific objective Within the study designed period of the study, there was no published paper on the utilization of CD34 +
in the treatment of acute IS. Therefore, we referred to our previous report [21] on the therapeutic effect of
erythropoietin (EPO) to estimate the National Institute of Health Stroke Scale (NIHSS) of both the
experimental group and control group for comparison. The score difference was assumed to be 2.5, and
the SD was estimated to be 4.5. In the case of alpha = 0.05 and power = 80%, the number of samples in a
group should be at least 51 participants. If the dropout rate was 10%, 58 participants should be enrolled
in each group. Accordingly, there were 116 participants in both groups. Thus, for the primary objective of
the study, an estimated sample size of 58 study subjects in each group was based on the effective size
with an α = 0.05, a power of 80%, an improvement of 1.5 in NIHSS score in control vs. 4.0 in NIHSS score
with EPC therapy and the SD was 4.5. Randomization method A total of 116 sealed envelopes were prepared for randomization. Within the envelopes, 58 patients
assigned to receive CD34 + cell treatment (i.e., group 1, i.e., 3.0 x 107 of CD34 + cells/patient from
intracarotid arterial administration) were included. On the other hand, 58 patients served as the control
group (i.e., group 2), i.e., 1: 1 homogenous distribution. These 116 sealed envelopes were then put into a
box after thorough mixture. The sealed envelope was one by one and randomized drawn out for each
participant. Study design and patient population This phase II clinical trial, which was approved by the Taiwan Food and Drug Administration (TFDA),
Republic of China (IRB No: 1076607733) and the Institutional Review Board (IRB) on Human Research at
Chang Gung Memorial Hospital (IRB No: 201700116A0C602) in 2018, was performed at Kaohsiung
Chang Gung Memorial Hospital (CGMH), a tertiary referral center. This study was funded by a program
grant from the Ministry of Science and Technology of the Republic of China (MOST 107-2314-B-182A-
056-MY3). This was a randomized, open-label, controlled phase II clinical trial with clinical trials register
number: ISRCTN15677760 (Registration date: 23/04/2018) to test whether intra-carotid arterial Page 4/33 Page 4/33 transfusion of circulatory derived autologous CD34 + cells for acute IS patients was safe and could
effectively further improve neurological function and outcomes in patients after acute IS. By TFDA and CGMH’s IRB permission, this study was designed to consecutively enroll 116 patients, i.e.,
58 patients in each group, who had history of acute IS [i.e., by clinical recording and brain magnetic
resonance imaging (MRI), or brain computerized tomography (CT) scan findings] and were willing to
participate the study. The enrollment time duration was permitted for three years and could start started
from January 2018. Unfortunately, during the enrollment period, severe COVID-19 pandemic appeared as
a public health emergency that was bestowed a great challenge and severely affected the enrollment of
the study patients. Thus, within January 2018 and March 2022, i.e., at the end of the study period, we
could only enroll 28 patients [i.e., 14 in study group (group 1) and 14 in control group (group 2),
respectively] who were willing to participate in the study. Since then, the clinical trial was stopped due to
(1) the time was up for enrollment of the patients and (2) the financial support was ending. respectively] who were willing to participate in the study. Since then, the clinical trial was stopped due to
(1) the time was up for enrollment of the patients and (2) the financial support was ending. Exclusion criteria 1. Age less than 45 years old or older than 80 years 2. Had received tissue plasminogen activator (tPA) therapy. 2. Had received tissue plasminogen activator (tPA) therapy. 3. Brain stem and other non-middle cerebral artery related acute IS, hemorrhagic strokes, or brain MRI
showed acute IS with hemorrhagic transformation. 4. NIHSS improved by more than 4 points within 12-24h after acute IS 4. NIHSS improved by more than 4 points within 12-24h after acute IS 5. Infectious agents, such as HIV, Covid-19, AIDS or syphilis 6. Myocardial infarction occurred within 3 months 7. Patients with severe stenosis of extracranial carotid arteries (including common carotid artery and
internal carotid artery) 8. Subjects with New York Heart Association Functional Class 3–4 9. Malignant tumors, hematopoietic disorders, or other serious organ diseases with expected survival of
less than one year 10. Patients with chronic kidney disease of creatine clearance rate (CCr) < 20ml/min Those who had received other clinical trial. 12. Patient who was not suitable for the examinations and treatments that included in the present study,
such as MRI. 12. Patient who was not suitable for the examinations and treatments that included in the present study,
such as MRI. 13. Patients had other brain diseases including tumors, infectious diseases, and neurodegenerative
diseases. 14 Contraindications for G CSF therapy 13. Patients had other brain diseases including tumors, infectious diseases, and neurodegenerative
diseases. 14. Contraindications for G-CSF therapy 14. Contraindications for G-CSF therapy Inclusion criteria 1. Ag more than 45 years old and less than 80 years old
2. Acute IS (onset within 14 days ± 7 days, and NIHSS ≥ 8 to < 22 scores), no midline-shift or
hemorrhagic transformation. 1. Ag more than 45 years old and less than 80 years old 1. Ag more than 45 years old and less than 80 years old 1. Ag more than 45 years old and less than 80 years old
2. Acute IS (onset within 14 days ± 7 days, and NIHSS ≥ 8 to < 22 scores), no midline-shift or
hemorrhagic transformation. 2. Acute IS (onset within 14 days ± 7 days, and NIHSS ≥ 8 to < 22 scores), no midline-shift or
hemorrhagic transformation. Page 5/33 3. The patients had already received the most appropriate medical treatment, including antiplatelet
treatment, blood lipid-lowering drugs, and blood pressure control. 3. The patients had already received the most appropriate medical treatment, including antiplatelet
treatment, blood lipid-lowering drugs, and blood pressure control. 4. The subjects/legal guardian had read and understood the proposal and study purpose in detail and
signed the inform consent. 4. The subjects/legal guardian had read and understood the proposal and study purpose in detail and
signed the inform consent. Methodology for preparation of autologous CD34 + cells
and intra-carotid arterial transfusion The procedure and protocol for the preparation of circulatory CD34 + cells have been described in our
previous clinical trials [22, 23]. In detail, prior to isolation of peripheral blood-derived autologous CD34 +
cells, granulocyte-colony stimulating factor (G-CSF) (5 µg/kg/every 12h for 8 doses) was subcutaneously
administered to all participants in group 1 to augment the circulatory level of CD34 + cells (i.e., migration
from bone marrow to circulation) for subsequent isolation by leukapheresis. After administration of the
final G-CSF, the peripheral blood-derived mononuclear cells isolated during leukapheresis were enriched
for CD34 + cells by utilizing a commercially available device [COBE Spetra 6.1 (Terumo BCT, INC.)] at 8:00
a.m. via a double-lumen catheter inserted into the right femoral vein. After a time- interval of 4h, bountiful
circulatory derived autologous CD34 + cells were isolated (purified via fluorescence-activated cell sorting
for CD34 + cells) and ready for intra-carotid arterial injection. Page 6/33 Page 6/33 Page 6/33 Based on the International Society of Hematotherapy and Grafting Engineering (ISHAGE) Guidelines for
CD34 + cell determination by flow cytometry to quantitate CD34 + cell in circulation, hematological stem
cells were characterized by the presence of the surface markers CD34high/CD45dim/SSClow that were
utilized tog quantitate the number of isolated CD34 + cells. The formula for estimating the number of
peripheral blood derived CD34 + cells was as follows: Number of CD34 + cells = (Percentage of CD34 +
cells) x WBC count x 103 x peripheral-blood stem cell (PBSC) volume (mL). In the present study, the flow
cytometric analysis was performed according to the College of American Pathology current guideline with
a performance coefficient of variation (CV): <4.0% (3.4 ± 2.5) (by definition < 10.0% was considered
acceptable). After completely collecting the CD34 + cells, each patient was quickly transferred to the cardiac
catheterization room for receiving ipsilateral intra-carotid artery injection of CD34 + cells through the
guiding catheter. Carotid arterial approach for slow transfusion (i.e., for about 5.0 to 7.0 minutes) of
CD34 + cells has been described in detail in our previous report [24]. Additionally, puncture of right internal
femoral vein and engagement of Swan Ganz into the right internal jugular vein (RIJV) were conducted for
estimating the time courses of EPCs in RIJV. Laboratory analysis of circulatory and RIJV levels of EPCs
by flow cytometric analysis The methodologies have been described in our previous studies [22, 23]. In detail, EPCs population in
circulation and RIJV were estimated by flow cytometry using double staining as depicted in our recent
reports [22, 23] by a fluorescence-activated cell sorter (FACSCalibur™ system; Beckman Coulter Inc, Brea,
CA, USA). Each analysis included 300,000 cells per sample. The assays for circulatory and RIJV EPCs
populations in each blood sampling were conducted in duplicate and mean levels were obtained. The collection of the blood sample in group 1 patients was performed at 8:00 a.m. prior to G-CSF
administration and the other was collected following G-CSF treatment for flow cytometric analysis. Additionally, for assessing the serial changes of EPCs levels in RIJV, serial blood samplings were drawn
from the RIJV at time points of 0 minute prior to and at 5, 10, and 30 minutes after CD34 + cell
administration into carotid artery. Time courses of neurological functional analysis and Neuro-Psychological Testing prior to and at 6-
month after CD34 + cell therapy NIHSS, Modified Rakin Scale and Barthel index were performed by a senior neurologist who completely
blinded to the grouping of the patients and the treatment protocol. Additionally, a clinical psychologist
who also blinded to each patient’s status conducted a neuro-psychological battery of tests that focused
on the attention, executive, speech, and language, memory, and visuospatial functional integrity. Attention
functions were measured by the digit span score from the Wechsler Adult Intelligence scale-III (WAIS-III)
[25, 26] and by the attention and orientation score from the Cognitive Ability Screening Instrument (CASI)
[25, 26]. Executive functions were assessed using the digit symbol coding, similarity, arithmetic, picture
arrangement, and matrix reasoning scores from the WAIS-III, and the abstract thinking scores from the
CASI. Memory functions were measured utilizing the short- and long- term memory scores from the CASI
and the information scores from the WAIS-III. Speech and language ability were assessed using the
vocabulary and comprehension scores from the WAIS- III and the language score from the CASI. Visuospatial functions were assessed using the picture completion and block design scores from the
WAIS-III and the drawing score from the CASI. To collect the peripheral blood-derived mononuclear cells for culturing EPCs and angiogenesis
assessment by Matrigel assay The procedure and protocol of Matrigel assay for determining the angiogenesis have been described in
detail in our previous reports [22, 23]. Briefly, for evaluating the angiogenesis ability of EPCs, 10 mL of
peripheral blood was collected from each patient in group 1 prior to G-CSF therapy. Blood sample from
each patient of group 2 was also obtained at the same time point for serving as the control. After
completing G-CSF treatment, 10 mL of peripheral blood was again drawn from each patient in group 1. In
addition, from the plasma-contained isolation of the CD34 + cells that was ready for transfusion after G-
CSF treatment in group 1 patients, 10 mL was collected. All samples were cultured for 21 days for the Page 7/33 Page 7/33 purpose of assessing the angiogenesis capacity of EPCs. Flow cytometric analysis was performed for
delineating the cellular characteristics (i.e., EPC surface markers) with appropriate antibodies after day 21
of cell culturing. purpose of assessing the angiogenesis capacity of EPCs. Flow cytometric analysis was performed for
delineating the cellular characteristics (i.e., EPC surface markers) with appropriate antibodies after day 21
of cell culturing. Time courses of neurological functional analysis and Neuro-Psychological Testing prior to and at 6-
month after CD34 + cell therapy Definition of the p value < 0.2 as great response replaced
the significance In view of small sample size (i.e., n = 14 in each group) that could possibly distort the statistical
significance, accordingly, those of variables with p < 0.2 were defined as having great response to CD34 +
cell therapy. Standardized formality of the clinical follow-up by TFDA
request and long-term follow-up In addition to regular follow-up of each patient at our outpatient clinic, a case report form that recorded all
clinical information of the patient including the presence or absence of acute or sub-acute events was
designed and completed by a research nurse regularly after each visit and on readmission as well as
through telephone interviews on an irregular basis. The TFDA and IRB committees required that all the
participants had to be followed up for one year. Page 8/33 Page 8/33 After the requirement of one-year follow up by TFDA and IRB committees, all the patients remained
followed up regularly at outpatient departments until the end of this study period. After the requirement of one-year follow up by TFDA and IRB committees, all the patients remained
followed up regularly at outpatient departments until the end of this study period. Statistical analysis All values are expressed as the mean ± SD, number, or percentage, as appropriate. Continuous data were
analyzed using independent or paired t test whereas categorical data were analyzed by chi-test or Fisher’s
exact test when appropriate. The time course of variables for longitudinal follow up was conducted by
repeated measures of ANOVA. Mean uptake readings of Tc-99m ECD brain perfusion SPECT before and
after treatment were analyzed by Wilcoxon Sign-rank test for paired data. Statistical analysis was
performed using SPSS statistical software for Windows version 13 (SPSS for Windows, version 28; SPSS,
IL, USA.). A value of p < 0.2 was considered statistically significant. The baseline characteristics in two groups of patients
(Table 1) Page 9/33 Table 1 Table 1
The baseline characteristics of study and control groups
Variables
Study group (n =
14)
Control group (n = 14)
p-value§
Age (yrs.)
65.2 ± 8.2
64.5 ± 9.6
0.833
Sex (male) (%)
57.1% (8/14)
64.3% (9/14)
0.699
Hypertension (%)
78.6% (11/14)
85.7% (12/14)
1.0
Hyperlipidemia (%)
71.4% (10/14)
57.1% (8/14)
0.430
Diabetes mellitus (%)
35.7% (5/14)
28.6% (4/14)
1.0
Smoking (%)
14.3% (2/14)
35.7% (5/14)
0.385
Old stroke (%)
21.4% (3/14)
7.1% (1/14)
0.596
Old myocardial infarction (%)
0% (0)
7.0% (1/14)
1.0
History of coronary artery disease (%)*
35.7% (5/14)
14.3% (2/14)
0.385
Clinical presentation of heart failure (%)
0% (0)
0% (0)
-
Atrial fibrillation (%)
35.7% (5/14)
28.6% (4/14)
1.0
Body heigh (BH) (cm)
164.3 ± 7.8
163.0 ± 7.8
0.654
Body mass (BM) index
27.0 ± 4.4
26.3 ± 5.7
0.724
Creatinine (mg/dL)
0.81 ± 0.24
0.87 ± 0.61
0.713
creatinine clearance rate (CCr)
(mm/min)
89.3 ± 24.2
95.7 ± 44.5
0.679
Medications upon presentation
Aspirin (%)
85.7% (12/14)
92.9% (13/14)
1.0
Clopidogrel (%)
7.0% (1/14)
28.6% (4/14)
0.326
Statins (%)
35.7% (5/14)
57.1% (8/14)
0.256
ACEIs/ARBs = angiotensin converting enzyme inhibitors/angiotensin II type 1 receptor blockers; LCCA
= left common carotid artery; LICA = left internal carotid artery; RCCA = right common carotid artery;
RICA = right internal carotid artery
* Defined as history of coronary artery obstruction > 50.0%
† Defined as the ratio of peak systolic velocity of LICA to LCCA ACEIs/ARBs = angiotensin converting enzyme inhibitors/angiotensin II type 1 receptor blockers; LCCA
= left common carotid artery; LICA = left internal carotid artery; RCCA = right common carotid artery;
RICA = right internal carotid artery
* Defined as history of coronary artery obstruction > 50.0%
† Defined as the ratio of peak systolic velocity of LICA to LCCA
‡ Defined as the ratio of peak systolic velocity of RICA to RCCA
§ Indicated continuous data were analyzed using independent t test whereas categorical data were
analyzed by chi-test or Fisher’s exact test when appropriate. The baseline characteristics in two groups of patients
(Table 1) Page 10/33 Page 10/33 Variables
Study group (n =
14)
Control group (n = 14)
p-value§
New oral anticoagulant (%)
28.6% (4/14)
28.6% (4/14)
1.0
ACEI/ARB (%)
35.7% (5/14)
28.8% (4/14)
1.0
Calcium channel blocker (%)
21.4% (3/14)
71.4% (10/14)
0.008
Beta blocker (%)
14.3% (2/14)
28.8% (4/14)
0.648
Carotid doppler findings
Peak systolic velocity of LCCA
76.0 ± 28.8
79.9 ± 20.9
0.688
Peak systolic velocity of LICA
69.1 ± 32.2
63.9 ± 10.0
0.572
Left carotid index†
0.88 ± 0.47
0.85 ± 0.26
0.855
Peak systolic velocity of RCCA
76.3 ± 22.4
81.3 ± 30.0
0.622
Peak systolic velocity of RICA
74.7 ± 49.6
72.7 ± 39.4
0.907
Right carotid index‡
0.96 ± 0.44
0.90 ± 0.29
0.673
ACEIs/ARBs = angiotensin converting enzyme inhibitors/angiotensin II type 1 receptor blockers; LCCA
= left common carotid artery; LICA = left internal carotid artery; RCCA = right common carotid artery;
RICA = right internal carotid artery
* Defined as history of coronary artery obstruction > 50.0%
† Defined as the ratio of peak systolic velocity of LICA to LCCA
‡ Defined as the ratio of peak systolic velocity of RICA to RCCA
§ Indicated continuous data were analyzed using independent t test whereas categorical data were
analyzed by chi-test or Fisher’s exact test when appropriate. The age and gender did not differ between the two groups. Additionally, the incidences of cerebrovascular
risk factors, including the diabetes mellitus and hyperlipidemia, were similar between these two groups of
patients. However, the incidences of cerebrovascular risk factors, including the hypertension and
smoking, were significantly higher in group 2 (i.e., control group) than in group 1 (study group). The incidences of old stroke, atrial fibrillation, history of CAD and clinical presentation of heart failure did
not differ between group 1 and group 2, whereas the old myocardial infraction was significantly higher in
group 2 than in group 1 patients. Additionally, the body mass index, body weight, circulatory level of
creatinine and CCr were similar between two groups of the patients. The baseline characteristics in two groups of patients
(Table 1) Except for calcium-channel blocker agent use that was significantly higher in group 2 than in group 1
patients, the percentage uses of antiplatelet agents, statins, beta- blocker agents, ACEIs/ARBs and new
oral anti-coagulant agents did not differ between two groups of patients.fi Page 11/33 Circulatory hematologic findings (Table 2)
Table 2
Circulatory hematologic findings
Variables
Study group (n = 14)
Control group (n = 14)
p-value‡
Baseline prior to G-CSF treatment
Red blood cells (RBC) (x 106)
4.98 ± 0.60
4.49 ± 0.56
0.036
White blood cells (WBC) (x 103)
8.7 ± 2.2
8.9 ± 3.8
0.866
Hemoglobin (g/dL)
14.6 ± 1.4
13.3 ± 1.7
0.033
Platelet (x 103/µL)
237.6 ± 52.3
270.9 ± 134.9
0.398
Segment (%)
72.8 ± 9.4
69.9 ± 8.3
0.395
Lymphocyte (%)
18.9 ± 8.4
19.6 ± 6.6
0.804
Compared prior to & after G-CSF treated*
Baseline†
After G-CSF treated†
Red blood cells (x 106)
4.98 ± 0.60
4.58 ± 0.57
< 0.001
White blood cells (x 103)
8.7 ± 2.2
49.6 ± 12.1
0.005
Hemoglobin (g/dL)
14.6 ± 1.4
13.5 ± 1.2
< 0.001
Platelet (x 103/µL)
237.6 ± 52.3
228.4 ± 67.9
0.068
Segment (SEG) (%)
72.8 ± 9.4
81.8 ± 4.4
0.408
Lymphocyte (LYM) (%)
18.9 ± 8.4
7.0 ± 2.7
0.553
HPCs (x 103/µL)
—
92.8 ± 208.4
—
* Indicated granulocyte colony stimulating factor (G-CSF) was only administered to the study group
(i.e., group 1) by subcutaneous administration (5 µg/kg twice a day for 4 days). † Indicated the parameters of prior to and after G-CSF treatment in group 1 patients
HPC = hematopoietic cells Circulatory hematologic findings (Table 2) Prior to G-CSF treatment, the circulatory levels of red blood cells (RBCs) and hemoglobin were
significantly higher in group 1 than in group 2, whereas the white blood cells (WBCs) and platelet counts,
and percentage of segment and lymphocyte did not differ within these two groups of patients. On the other hand, as compared to parameters prior to G-CSF treatment, the WBC count was significantly
increased whereas the RBC count and hemoglobin were significantly lower after G-CSF treatment among Page 12/33 the group 1 patients. Additionally, hematopoietic progenitor cells (HPCs) were substantially higher in
group 1 after receiving G-CSF treatment. However, the platelet counts, percentages of segment and
lymphocyte did not differ before and after G-CSF treatment among the group 1 patients. the group 1 patients. Circulatory EPC levels between group 1 and group 2 patients and in group 1 after G-CSF treatment (Fig. 1) First, to elucidate the time points of EPCs expression, the flow cytometric analysis was utilized in the
present study, As we expected, prior to G-CSF treatment, the circulating levels of EPCs (i.e., CD34 + KDR +
CD45dim, CD34 + CD133 + CD45dim, CD31 + CD133 + CD45dim, CD34 + CD133 + KDR + and CD133+) and
those of hematopoietic progenitor cells (HPCs) (CD34+) did not differ between group 1 and group 2. However, these parameters were significantly higher among the group 1 patients after receiving G-CSF
treatment, and more significantly increased in the plasma contained isolated EPCs. Our findings implied
that G-CSF treatment promoted the mobilization of EPCs and HPCs from bone marrow to circulation. The baseline characteristics in two groups of patients
(Table 1) Additionally, hematopoietic progenitor cells (HPCs) were substantially higher in
group 1 after receiving G-CSF treatment. However, the platelet counts, percentages of segment and
lymphocyte did not differ before and after G-CSF treatment among the group 1 patients. Estimation of angiogenesis by Matrigel assay in both
groups of patients (Fig. 2) Next, we utilized Matrigel assay to assess the angiogenesis ability (i.e., the parameters included cluster
formation, tubular length, and network formation). The result showed that these parameters were similar
between groups 1 and 2 prior to G-CSF treatment. However, these angiogenesis parameters were
significantly augmented in group 1 patients after receiving G-CSF treatment as compared to those of both
groups of patients prior to G-CSF treatment, suggesting that G-CSF would enhance the angiogenesis
capacity of EPCs. One particularly interesting finding was that the parameters of angiogenesis were
highest in plasma contained isolated EPCs. Serial changes of RIJV levels of stromal cell-derived factor (SDF)-1α and EPCs/HSC in 14 study patients
(Fig. 3) To evaluate the SDF-1α level at different time intervals of cerebral circulation after CD34 + intra-carotid
arterial transfusion, we collected the blood samples from the RIJV and measured this soluble
angiogenesis factor at baseline (i.e., 0 minute) and at 5, 10, and 30 minutes after CD34 + cell transfusion. As we expected, the RIJV levels of SDF-1α at baseline (i.e., at 0 minute) and the time point of 30 minutes
were significantly increased and further significantly increased at the time intervals of 5 and 10 than in
circulating level prior to G-CSF treatment, suggesting the EPCs, such as CXCR4 + cells, would be trapped
in cerebral blood vessels and capillary networks. Additionally, the SDF-1α level was significantly higher in
circulation at the time interval just after completing G-CSF treatment and more significantly higher in
plasma contained concentrated isolated CD34 + cells than in the RIJV at the time intervals of 5, 10, and
30 minutes post-CD34 + cell treatment. To elucidate the shedding rate of EPCs from RIJV after CD34 + cell intra-carotid artery administration,
time courses of EPC and HPCs evaluation were conducted by flow cytometry. The time courses of EPCs
(i.e., CD34 + KDR + CD45dim, CD34 + CD133 + CD45dim, CD31 + CD133 + CD45dim, CD34 + CD133 + KDR + Page 13/33 Page 13/33 and CD133 + surface markers) and HPCs (CD34+) were revealed to be continuously excreted from RIJV to
circulation at time points of 5, 10, and 30 minutes after intra-carotid arterial transfusion of CD34 + cells. Estimation of angiogenesis by Matrigel assay in both
groups of patients (Fig. 2) Additionally, except for CD34 + cells and CD34 + CD133 + KDR + cells, these parameters were significantly
higher in these three-time intervals as compared to prior to G-CSF treatment and to the time interval of 0
minute prior to CD34 + cell administration. Furthermore, among these patients, the circulating levels of
EPCs and HSC were remarkably higher after G-CSF treatment and more notably higher in plasma
contained isolated CD34 + cells than those before G-CSF treatment. This finding once more suggests that
G-CSF treatment allowed the EPC and HSC mobilization from bone marrow to circulation. and CD133 + surface markers) and HPCs (CD34+) were revealed to be continuously excreted from RIJV to
circulation at time points of 5, 10, and 30 minutes after intra-carotid arterial transfusion of CD34 + cells. Additionally, except for CD34 + cells and CD34 + CD133 + KDR + cells, these parameters were significantly
higher in these three-time intervals as compared to prior to G-CSF treatment and to the time interval of 0
minute prior to CD34 + cell administration. Furthermore, among these patients, the circulating levels of
EPCs and HSC were remarkably higher after G-CSF treatment and more notably higher in plasma
contained isolated CD34 + cells than those before G-CSF treatment. This finding once more suggests that
G-CSF treatment allowed the EPC and HSC mobilization from bone marrow to circulation. Estimation of angiogenesis by Matrigel assay in both
groups of patients (Fig. 2) ¶ defined as recurrent stroke or death
** Indicated continuous data were analyzed using independent t test, and categorical data were
analyzed by chi-test or Fisher’s exact test when appropriate. †† Indicated the followed-up time was 4.1 ± 1.3. # Defined as bed rident and totally dependent on life care. At the end of one-year follow up, the mortality rate was 0% in group 1 and 7.1% in group 2. Additionally,
the recurrent stroke was 0% in group 1 and 7 1% in group 2 Thus combined end points (defined as death Variables
Study group (n =
14)
Control group (n =
14)
p-
value**
Recurrent stroke
0% (0)
7.1% (1/14)
1.0
Mortality
0% (0)
7.1% (1/14)
1.0
Long-term combined end point††
14.3% (2/14)
50.0% (7/14)
0.103
Recurrent stroke
7.1% (1/14)
14.3% (2/14)
1.0
Mortality
7.1% (1/14)
14.3% (2/14)
1.0
Severe disability#
0% (0)
14.3% (3/14)
0.222
Tumorigenesis at long-term follow up††
0% (0)
0% (0)
—
NIHSS = National Institutes of Health Stroke Scale; LAAC = left atrial appendage closure. * Defined as the time point of early recovery phase after acute ischemic stroke (IS)
† Defined as the time point of late recovery phase after acute IS
‡ Defined as the time point of chronic phase after acute IS
§ Due to poorer condition was transferred to long-term care center. ¶ defined as recurrent stroke or death
** Indicated continuous data were analyzed using independent t test, and categorical data were
analyzed by chi-test or Fisher’s exact test when appropriate. †† Indicated the followed-up time was 4.1 ± 1.3. # Defined as bed rident and totally dependent on life care. At the end of one-year follow up, the mortality rate was 0% in group 1 and 7.1% in group 2. Additionally,
the recurrent stroke was 0% in group 1 and 7.1% in group 2. Thus, combined end points (defined as death
or recurrent stroke) at one-year followed up were 0% in group 1 and 14.3% in group 2. The neurological functional parameters of NIHSS, modified Rankin scale and Barthel index were similar
upon presentation (Table 3). Additionally, the parameters of Barthel index and modified Rankin remained
similar by days 60, 90 and 180 and that of NIHSS by day 180 did not differ between group 1 and group 2
patients. Estimation of angiogenesis by Matrigel assay in both
groups of patients (Fig. 2) One-year and long-term neurological and clinical outcomes
(Table 3) Page 14/33 Table 3
Time courses of neurological and prognostic outcomes
Variables
Study group (n =
14)
Control group (n =
14)
p-
value**
Neurological disability upon presentation
NIHSS
10.9 ± 3.2
11.9 ± 3.2
0.391
Modified Rankin scale
4.43 ± 0.65
4.36 ± 0.84
0.803
Barthel index
12.9 ± 11.0
18.9 ± 26.5
0.440
Baseline CASI score
67.1 ± 17.1
67.7 ± 25.3
0.946
Baseline WIAS-III
78.2 ± 13.2
81.8 ± 16.4
0.585
Baseline MMSE
20.7 ± 5.7
20.8 ± 7.3
0.990
Neurological disability at day 30*
NIHSS
7.6 ± 2.5
9.8 ± 4.5
0.130
Modified Rankin scale
3.36 ± 0.75
3.54 ± 0.66
0.511
Barthel index
50.4 ± 25.2
39.6 ± 27.9
0.0.076
Neurological disability at day 90†
NIHSS
6.6 ± 2.2
8.9 ± 4.7
0.144
Modified Rankin scale
3.14 ± 0.95
3.23 ± 0.83
0.801
Barthel index
59.6 ± 25.2
55.0 ± 33.8
0.688
Neurological disability at day 180‡
NIHSS
6.1 ± 2.6
7.5 ± 4.0
0.332
Modified Rankin scale
3.0 ± 1.0
2.90 ± 0.83
0.815
Barthel index
60.0 ± 27.7
63.2 ± 29.3
0.788
Death 3 month later after acute IS
0% (0)
7.1% (1/14)
1.0
Withdrawn study 3 months after acute
IS§
0% (0)
14.3% (2/14)
0.481
LAAC procedure
21.4% (3/14)
14.3% (2/14)
1.0
At 6-month CASI score
67.0 ± 21.9
65.4 ± 23.6
0.890
At 6-month WIAS-III
80.5 ± 18.1
72.0 ± 8.7
0.707
At 6-month MMSE
20.5 ± 6.3
16.8 ± 6.8
0.270
1 ear combined end point¶
0% (0)
14 3% (2/14)
0 481 Table 3 Page 15/33 Variables
Study group (n =
14)
Control group (n =
14)
p-
value**
Recurrent stroke
0% (0)
7.1% (1/14)
1.0
Mortality
0% (0)
7.1% (1/14)
1.0
Long-term combined end point††
14.3% (2/14)
50.0% (7/14)
0.103
Recurrent stroke
7.1% (1/14)
14.3% (2/14)
1.0
Mortality
7.1% (1/14)
14.3% (2/14)
1.0
Severe disability#
0% (0)
14.3% (3/14)
0.222
Tumorigenesis at long-term follow up††
0% (0)
0% (0)
—
NIHSS = National Institutes of Health Stroke Scale; LAAC = left atrial appendage closure. * Defined as the time point of early recovery phase after acute ischemic stroke (IS)
† Defined as the time point of late recovery phase after acute IS
‡ Defined as the time point of chronic phase after acute IS
§ Due to poorer condition was transferred to long-term care center. Estimation of angiogenesis by Matrigel assay in both
groups of patients (Fig. 2) However, by days 30 and 90 after acute IS, the NIHSS exhibited a remarkably great response
(defined as p value < 0.2) in group 1 than in group 2. On the other hand, the values of CASI score, WIAS-III
and MMSE, three indices of neuro-psychological scorings, did not differ at the time point of day 180 after
acute IS among group 1 or group 2 patients. The long-term follow-up time was 4.1 ± 1.3 years. The important finding was that the combined
cumulative end point (i.e., defined as recurrent stroke, mortality and severe disability) of this current study
during the long-term follow-up was remarkably higher in group 2 than in group 1, implicating that the Page 16/33 favorably outcomes in group 1 might be due to result of CD34 + treatment. Additionally, no tumorigenesis
was observed during one-year and long-term follow-up in group 1 and group 2 patients (Table 3). Two month later after acute IS, 3 patients in group 1 and 2 patients in group 2 due to atrial fibrillation
induced embolic stroke received catheter-based left atrial appendage closure and uneventfully discharge
from hospital (Table 3). The value of mean uptake readings of Tc-99m ECD brain
perfusion SPECT (Table 4) The value of mean uptake readings of Tc-99m ECD brain
perfusion SPECT (Table 4) Page 17/33 Table 4
Comparison of median uptake readings of Tc-99m ECD brain perfusion SPECT study before (baseline)
and after (by 6-month) CD34 + cell therapy in both groups of patients
Variables
Median (Q1, Q3)
(baseline) in group
1
Median
(Q1, Q3)
(6 month)
in group 1
p-
value*
Median
(Q1, Q3)
(baseline)
in group 2
Median
(Q1, Q3)
(6
month) in
group 2
p-
value*
Basal ganglia (L)
321.5 (263.8,
411.8)
326.5
(278.2,
384.7)
0.507
346.5
(275.5,
361.5)
303.1
(278.2,
359.2)
0.477
Basal ganglia (R)
333.5 (277.2,
465.1)
376.3
(250.4,
485.4)
0.807
354.3
(264.6,
410.8)
402.8
(333.7,
458.0)
0.110
Central region (L)
311.7 (244.6,
392.2)
325.9
(299.3,
401.6)
0.753
307.3
(274.7,
328.6)
291.1
(270.3,
334.5)
0.374
Central region (R)
317.5 (258.8,
442.5)
351.7
(322.0,
446.7)
0.753
329.1
(271.9,
367.3)
367.5
(295.0,
415.9)
0.130
Cerebellum (L)
355.7 (288.8,
477.7)
392.7
(310.9,
546.6)
0.463
355.6
(302.5,
392.9)
423.6
(341.4,
544.5)
0.110
Cerebellum (R)
343.3 (258.3,
446.2)
360.7
(280.4,
479.4)
0.552
317.7
(286.4,
372.1)
391.6
(329.9,
446.9)
0.110
Cingulate &
paracingulate gyri
(L)
336.1 (270.1,
422.8)
353.2
(296.4,
437.2)
0.861
305.1
(267.3,
374.5)
355.2
(307.6,
410.5)
0.286
Cingulate &
paracingulate gyri
(R)
322.4 (248.6,
427.5)
343.6
(291.1,
417.5)
0.753
323.4
(275.3,
343.6)
353.4
(300.6,
415.3)
0.213
Frontal lobe (L)
321.9 (250.3,
396.6)
325.3
(296.5,
412.0)
0.972
326.0
(262.9,
362.1)
333.3
(294.6,
375.8)
0.182
Frontal lobe (R)
303.7 (248.8,
426.9)
333.5
(294.3,
428.1)
0.753
299.4
(227.1,
321.4)
342.4
(308.6,
412.5)
0.131
Mesial temporal
lobe (L)
293.4 (228.3,
388.3)
306.5
(233.0,
360.2)
0.753
305.1
(249.0,
351.8)
284.1
(254.7,
334.8)
0.286
* I di
t d th
t ti ti
l
l
i b Wil
Si
k t
t f
i
d d t Page 18/33
* Indicated the statistical analysis by Wilcoxon Sign-rank test for paired data. L = left; R = right; M = month; SD = standard deviation. The value of mean uptake readings of Tc-99m ECD brain
perfusion SPECT (Table 4) Group 1 = study group; group 2 = control group Variables
Median (Q1, Q3)
(baseline) in group
1
Median
(Q1, Q3)
(6 month)
in group 1
p-
value*
Median
(Q1, Q3)
(baseline)
in group 2
Median
(Q1, Q3)
(6
month) in
group 2
p-
value*
Mesial temporal
lobe (R)
299.7 (248.0,
395.9)
324.1
(286.5,
413.9)
0.807
357.8
(331.5,
391.8)
360.8
(289.0,
384.6)
0.131
Occipital lobe(L)
372.0 (307.9,
485.0)
410.7
(326.4,
486.1)
0.701
363.5
(304.8,
422.2)
379.5
(323.4,
476.3)
0.182
Occipital lobe(R)
362.6 (300.1,
504.0)
415.9
(372.8,
509.3)
0.6
320.1
(290.2,
348.9)
415.3
(328.2,
484.9)
0.110
Parietal lobe (L)
343.4 (274.0,
399.5)
346.0
(322.4,
418.2)
0.861
330.1
(281.4,
380.8)
311.2
(283.0,
385.2)
0.182
Parietal lobe (R)
333.9 (280.0,
466.9)
378.0
(330.1,
468.3)
0.701
332.4
(274.8,
369.6)
388.9
(301.3,
425.6)
0.110
Temporal lobe (L)
336.1 (272.0,
389.9)
344.9
(268.9,
412.5)
0.701
364.5
(287.2,
397.6)
315.9
(269.8,
372.2)
0.328
Temporal lobe (R)
333.8 (281.0,
477.6)
384.9
(341.7,
481.4)
0.701
378.6
(328.9,
468.7)
394.6
(324.1,
460.5)
0.110
* Indicated the statistical analysis by Wilcoxon Sign-rank test for paired data. L = left; R = right; M = month; SD = standard deviation. Group 1 = study group; group 2 = control group To evaluate whether the intracarotid arterial administration of circulatory derived autologous CD34 + cells
would increase cerebral blood perfusion, the Tc-99m ECD brain perfusion SPECT was conducted, and
median value was utilized for determining the change between the baseline and 6-month after acute IS
among group 1 and group 2, respectively. Surprisingly, 100% (18/18) increased Tc-99m brain perfusion
uptake in all 18 different regions of brain was identified in group 1 patients, whereas merely 72.2%
(13/18) increased Tc-99m brain perfusion uptake in the same different regions were observed in group 2
patients (i.e., 100% vs. 72%, p = 0.046 measured by the Fischer’s Exact Test for count data), implicating
that the angiogenesis of cerebral system would be significantly increased in group 1 than in group 2. Time courses of neurological functions for one year follow-
up (Fig. 4) Discussion This phase II clinical trial which investigated the impact of intracarotid arterial transfusion of peripheral
blood derived autologous CD34 + cells yielded several striking clinical information. First, the results
demonstrated that procedural success and safety were 100% and 0% of CD34 + cell treatment-related
complication was observed as well as no tumorigenesis was observed during long-term clinical follow-up
in the study patients (i.e., group 1). Second, as compared with control group (i.e., group 2) patients, NIHSS
was greatly improved at early (defined as at day 30) and late (defined as at day 90) recovery phases in
study patients after acute IS. Finally, combined end point by the long-term follow up (i.e., mean follow-up
time: 4.1 ± 1.3 years) was markedly reduced in group 1 than in group 2 patients. While the safety and efficacy of autologous EPCs/CD34 + cell therapy have been extensively investigated
in setting of ischemic heart disease [22, 23, 27–30], such a novel strategic management for patients after
IS regrettably has been infrequently reported [24, 31, 32], implicating that there still exists a lot of
scientific research space. We designed this phase II clinical trial was in this context of time and space. One important finding in the present study was that all of the study patients who received the CD34 +
cells therapy were safe, and no tumorigenesis was observed during 1-year and long-term follow up. Intriguingly, our previous phase I clinical trial of old IS patients treated by autologous CD34 + cell
intracarotid arterial transfusion [24] and other two previous clinical trials [31, 32], including one by directly
intracerebral implantation [31] and another by intra-cranial artery injection [30] of autologous derived
stem cells have consistently identified that cell therapy for IS patients was safe and tolerable. Accordingly, the findings in our current and previous phase I clinical trial [24] and two other previously
reported clinical trials [31, 32] support autologous EPC progenitor cells/CD34 + cell therapy since no more
safe and ethical issues should be concerned. Interestingly, two previously clinical trials [31, 32] have demonstrated that hematopoietic stem cells (i.e.,
including bone marrow-derived mononuclear cells or circulatory derived CD34 + cells) treatment
effectively improved neurological outcomes in patients after IS. Time courses of neurological functions for one year follow-
up (Fig. 4) Page 19/33 When looked at the longitudinal follow-up of the prognostic neurological function, we found that the net
change (i.e., ∆ change) of improvement of the neurological function (i.e., including the NIHSS and Barthel
index) at time points of days 30 and 90 after IS was numerically higher in group 1 than in group 2. Discussion However, when looked at their reports [31,
32] in detail, we found that the so called “efficacy” totally depended on only the clinical follow-up
parameters and some of the illustrating imaging studies, implicating that this is still unmet need when
those of the underlying mechanistic basis regarding how to effectively improve the clinical outcomes is
taken into consideration. In comparison with these previous studies [31, 32], our findings from this phase
II clinical trial provided several impactful and novel findings that should be shared with the readers. First,
the results of our cell culture study, i.e., the Matrigel assay, demonstrated that the angiogenesis capacity
was markedly upregulated after G-CSF treatment in group 1 patients, implicating this strategic
management not only augmented the number of the hematopoietic progenitor cells/CD34 + cells
mobilized into circulation from bone-marrow nidus, but also implied that G-CSF treatment might have Page 20/33 Page 20/33 capacity of renovation and rejuvenation of the hematopoietic progenitor cells/CD34 + cells. Second, when
serially collected the blood samplings and utilized the flow cytometric analysis, we found that, as
compared with the baseline, the shedding of EPCs from cerebral circulation to the RIJV was continuously
highly excreted at time points of 5, 10, and 30 minutes after intracarotid arterial transfusion of
autologous CD34 + cells, suggesting that majority of transfused CD34 + cells were still retained/trapped
in the cerebral circulation for a rather longer duration for possible angiogenesis and for secretory
angiogenesis factors rather than original concept that they would be completely washed out from the
brain to the systemic circulation within serval cardiac outputs (usually 70 cardiac outputs in 60 seconds
with a total blood volume of 5000 cc to be pushed out from heart to circulation). Third, ELISA conducted
of blood samplings from RIJV identified that as compared with baseline and the control group in
circulation, the level of SDF-1α, a ligand of EPCs/CXCR4 + cells, was remarkably increased at the time
intervals of 5, 10 and 30 minutes of blood samplings, implicating that this angiogenesis factor which
would play a crucial role on trapping the EPCs/CXCR4 + cells (i.e., theory of ligand and receptor biding
together) in cerebral tissues/microvasculature, resulting an explanation for why the expression of
shedding EPCs could be identified as persistently increased in RIJV (i.e., a reflex of cerebral circulation) in
compared with the circulation. Discussion Fourth, the number of different brain-region perfusion evaluated by Tc-
99m ECD brain perfusion SPECT study (Table 4) was remarkably increased in group 1 than in group 2,
implicating CD34 + cell therapy may restore some of blood perfusion in cerebral circulation and
microvasculature through angiogenesis/neovascularization. Finally, the cardinal finding in the present study was that the neurological functional parameters of
NIHSS, one indicator of neurological outcome, had notably significant (i.e., great response) of
improvement in group 1 than in group 2 in early and late recovery phases (Table 3). Additionally, when
carefully looked at the longitudinal follow-up of serial changes of NIHSS and Barthel index, we identified
that these two parameters, especially in NIHSS parameter, were more notably improved in group 1 than in
group 2 patients (referred to Fig. 4). Furthermore, of paramount finding was that the long-term combined
end point was remarkably lower in group 1 than in group 2, resulting in indicating that the prognostic
outcome was favorable in acute IS patient received CD34 + cells treatment. In this way, our findings
supported that early intracarotid arterial transfusion of autologous CD34 + cells might offer benefits for
patients after acute IS, especially when long-term outcome was taken into consideration. Conclusions In conclusion, the present study by utilizing the imaging studies, neurological functional and neuro-
psychological evaluations, and clinical follow-up as well as the basic research studies provided
convincing scientific data to prove that the intra-carotid arterial injection of autologous CD34 + cell
therapy was safe and might be effective for improving the favorable outcomes in short- and long-term
intervals in patients after IS. Abbreviations CAD
coronary artery disease
CCr
creatine clearance rate
EPC
endothelial progenitor cell
G-CSF
granulocyte-colony stimulating factor
HPCs
hematopoietic progenitor cells
HSC
hematopoietic stem cells
IS
ischemic stroke
NIHSS
National Institute of Health Stroke Scale
RIJV
right internal jugular vein
tPA
tissue plasminogen activator CAD
coronary artery disease
CCr
creatine clearance rate
EPC
endothelial progenitor cell
G-CSF
granulocyte-colony stimulating factor
HPCs
hematopoietic progenitor cells
HSC
hematopoietic stem cells
IS
ischemic stroke
NIHSS
National Institute of Health Stroke Scale
RIJV
right internal jugular vein
tPA
tissue plasminogen activator Declarations Availability of data and materials Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author
upon reasonable request. Study limitations Page 21/33 This study has limitations. First, as mentioned in the introduction section, the small sample size that did
not completely comply with the designed calculation of sample size was really due to the COVID-19
pandemic developed during the enrollment time intervals. Second, we could not completely rule out the
statistical significance was, therefore, greatly distorted by small sample size. Third, the NIHSS and
Barthel index became no more difference at the time point of 180-day follow-up between groups 1 and 2
that could be attributed to one patient died and another patient withdrawn from the study in group 2 in
the time interval of 3 month after acute IS as well as the small cohort study. The two patients have
relatively higher NIHSS and notably lower Barthel index. Thus, interpretation of our neurological
functional parameters should be cautioned at the time interval of 180 day after IS. Acknowledgement Page 22/33 Hung-Sheng Lin Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and
Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan. Institute for Translational
Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, 83301, Taiwan. Center
for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung,
83301, Taiwan. Pei-Hsun Sung, Yi-Ling Chen, Hon-Kan Yip Department of Nuclear Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University
College of Medicine, Kaohsiung, 83301, Taiwan. Department of Nuclear Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University
College of Medicine, Kaohsiung, 83301, Taiwan. Shu-Hua Huang Department of Diagnostic Radiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung
University College of Medicine, Kaohsiung, 83301, Taiwan. Department of Diagnostic Radiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung
University College of Medicine, Kaohsiung, 83301, Taiwan. Funding This study was supported by a program grant from the Ministry of Science and Technology of the
Republic of China (MOST 107-2314-B-182A-056-MY3). Page 22/33 Page 22/33 We would like to thank patients and their relatives, physicians, nurses, technicians and clinical research
staff of the Kaohsiung Chang Gung Memorial Hospital for their involvement in this study. Wei-Che Lin Department of Computer Science and Engineering, National Sun Yat-Sen University, Kaohsiung, 804201,
Taiwan. Department of Healthcare Administration and Medical Informatics, Kaohsiung Medical
University, Kaohsiung, 80708, Taiwan. Authors and Affiliations Department of Neurology, Cognition and Aging Center, Kaohsiung Chang Gung Memorial Hospital and
Chang Gung University College of Medicine Kaohsiung, 83301,Taiwan. Sheung-Fat Ko Department of Nursing, Asia University, Taichung 41354, Taiwan. 14 Department of Medical
Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan. Department of Nursing, Asia University, Taichung 41354, Taiwan. 14 Department of Medical
Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan. Hon-Kan Yip Consent for publication Not applicable. Ethics approval and consent to participate The study protocol was approved by ethics committee [Taiwan Food and Drug Administration
(1076607733) and Institutional Review Board of Chang Gung Medical Foundation (201700116A0C602)]. Informed consent was obtained with the patient or its relatives. Contributions Study conception or design: HSL, PHS, SHH, WCL, YLC, MCM, HKY. Data acquisition, analysis, or
interpretation: HSL, PHS, SHH, WCL, SFK, HKY. Drafted manuscript or critical revision of manuscript: HSL,
PHS, SHH, WCL, JYC, YLC, MCM, KHC, FYL, SFK, HKY. All authors read and approved the final manuscript. Corresponding authors Correspondence to Hon-Kan Yip. Fan-Yen Lee Department of Radiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College
of Medicine, Kaohsiung, 83301, Taiwan. John Y. Chiang Division of Hema-Oncology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital
and Chang Gung University College of Medicine Kaohsiung, 83301, Taiwan. Ming-Chun Ma Ming-Chun Ma Page 23/33 Page 23/33 Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University
College of Medicine, Kaohsiung, 83301, Taiwan Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University
College of Medicine, Kaohsiung, 83301, Taiwan Kuan-Hung Chen Kuan-Hung Chen Division of Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital
and Chang Gung University College of Medicine Kaohsiung, 83301, Taiwan. Division of Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital
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Book: Pathophysiology, Diagnosis, and Management: Elsevier Health Sciences, 2011. 7. Barnett, H. J., D. W. Taylor, M. Eliasziw, A. J. Fox, G. G. Ferguson, R. B. Haynes, R. N. Rankin, G. P. Clagett, V. C. Hachinski, D. L. Sackett, K. E. Thorpe, H. E. Meldrum, and J. D. Spence. "Benefit of
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Medical Management for Preventing Recurrent Stroke in Intracranial Stenosis Trial. "Aggressive
Medical Treatment with or without Stenting in High-Risk Patients with Intracranial Artery Stenosis
(Sammpris): The Final Results of a Randomised Trial." Lancet 383, no. 9914 (2014): 333 – 41. 20. Yip, H. K., L. T. Chang, W. N. Chang, C. H. Lu, C. W. Liou, M. Y. Lan, J. S. Liu, A. A. Youssef, and H. W. Chang. "Level and Value of Circulating Endothelial Progenitor Cells in Patients after Acute Ischemic
Stroke." Stroke 39, no. 1 (2008): 69–74. 21. Yip, H. K., T. H. Tsai, H. S. Lin, S. F. Chen, C. K. Sun, S. Leu, C. M. Yuen, T. Y. Tan, M. Y. Lan, C. W. Liou,
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(2020): 324. 29. Losordo, D. W., T. D. Henry, C. Davidson, J. Sup Lee, M. A. Costa, T. Bass, F. Mendelsohn, F. D. Fortuin,
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Therapy for Refractory Angina." Circ Res 109, no. 4 (2011): 428–36. 30. Quyyumi, A. A., A. Vasquez, D. J. Kereiakes, M. Klapholz, G. L. Schaer, A. Abdel-Latif, S. Frohwein, T. D. Henry, R. A. Schatz, N. Dib, C. Toma, C. J. Davidson, G. W. Barsness, D. M. Shavelle, M. Cohen, J. Poole, T. Moss, P. Hyde, A. M. Kanakaraj, V. Druker, A. Chung, C. Junge, R. A. Preti, R. L. Smith, D. J. Mazzo, A. Pecora, and D. W. Losordo. "Preserve-Ami: A Randomized, Double-Blind, Placebo-Controlled
Clinical Trial of Intracoronary Administration of Autologous Cd34 + Cells in Patients with Left
Ventricular Dysfunction Post Stemi." Circ Res 120, no. 2 (2017): 324 – 31. Page 27/33 Page 27/33 31. Chen, D. C., S. Z. Lin, J. R. Fan, C. H. Lin, W. Lee, C. C. Lin, Y. J. Liu, C. H. Tsai, J. C. Chen, D. Y. Cho, C. C. Lee, and W. C. Shyu. "Intracerebral Implantation of Autologous Peripheral Blood Stem Cells in
Stroke Patients: A Randomized Phase Ii Study." Cell Transplant 23, no. 12 (2014): 1599 – 612. 32. Bhatia, V., V. Gupta, D. Khurana, R. R. Sharma, and N. Khandelwal. "Randomized Assessment of the
Safety and Efficacy of Intra-Arterial Infusion of Autologous Stem Cells in Subacute Ischemic Stroke."
AJNR Am J Neuroradiol 39, no. 5 (2018): 899–904. Figure 1 Figure 1 Figures Page 28/33
Figures Circulatory EPC levels between group 1 and group 2 patients and in group 1 after G-CSF treatment A1 to A4) Illustrating the flow cytometric analysis for identification of CD34+KDR+CD45dim cells. A5)
Analytical result of number of CD34+KDR+CD45dim cells, * vs. other groups with different symbols (†, ‡),
p<0.0001. B1 to B4) Illustrating the flow cytometric analysis for identification of CD34+CD133+CD45dim
cells. B5) Analytical result of number of CD34+CD133+CD45dim cells, * vs. other groups with different
symbols (†, ‡), p<0.0001. C1 to C4) Illustrating the flow cytometric analysis for identification of
CD31+CD133+ CD45dim cells. C5) Analytical result of number of CD31+CD133+ CD45dim cells, * vs. other
groups with different symbols (†, ‡), p<0.0001. D1 to D4) Illustrating the flow cytometric analysis for
identification of CD34+CD133+KDR+ cells. D5) Analytical result of number of CD34+CD133+KDR+ cells,
* vs. other groups with different symbols (†, ‡), p<0.0001. E1 to E4) Illustrating the flow cytometric
analysis for identification of CD133+ cells. E5) Analytical result of number of CD133+ cells, * vs. other
groups with different symbols (†, ‡), p<0.0001. F1 to F4) Illustrating the flow cytometric analysis for identification of CD34+ cells. F5) Analytical result
of number of CD34+ cells, * vs. other groups with different symbols (†, ‡), p<0.0001. All statistical
analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test
(n=14 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). EPC = endothelial progenitor
cell; G-CSF = granulocyte-colony stimulating factor. G1 = control group. G2A (study group) = pre-G-CSF
treatment; G2B = post G-CSF treatment. G3 = plasma contained isolated CD34+ cells. Page 29/33 Figure 2
Estimation of angiogenesis by Matrigel assay with and without G-CSF treatment in both groups of
patients
A) Illustrating the example for how to identification of angiogenesis variables, including the tubular length
(blue line), cluster formation (red circle) and network formation (blue triangle). B to E) Illustrating the
Matrigel assay for identification of angiogenesis capacity in G1 (i.e., control group) (B), in G2 (study
group) at time point of pre-G-CSF treatment (C) and post G-CSF treatment (D) as well as at the condition
of plasma contained isolation of CD34+ cells (E), respectively. Pink arrows indicated tubular length. Red
arrows indicated cluster formation. Black dotted line circle indicated network formation. F) Analytical
result of number of tubule, * vs. other groups with different symbols (†, ‡), p<0.001. Circulatory EPC levels between group 1 and group 2 patients and in group 1 after G-CSF treatment G)Analytical result of Figure 2 Estimation of angiogenesis by Matrigel assay with and without G-CSF treatment in both groups of
patients
A) Illustrating the example for how to identification of angiogenesis variables, including the tubular length
(blue line), cluster formation (red circle) and network formation (blue triangle). B to E) Illustrating the
Matrigel assay for identification of angiogenesis capacity in G1 (i.e., control group) (B), in G2 (study
group) at time point of pre-G-CSF treatment (C) and post G-CSF treatment (D) as well as at the condition
of plasma contained isolation of CD34+ cells (E), respectively. Pink arrows indicated tubular length. Red
arrows indicated cluster formation. Black dotted line circle indicated network formation. F) Analytical
result of number of tubule, * vs. other groups with different symbols (†, ‡), p<0.001. G)Analytical result of Estimation of angiogenesis by Matrigel assay with and without G-CSF treatment in both groups of
patients A) Illustrating the example for how to identification of angiogenesis variables, including the tubular length
(blue line), cluster formation (red circle) and network formation (blue triangle). B to E) Illustrating the
Matrigel assay for identification of angiogenesis capacity in G1 (i.e., control group) (B), in G2 (study
group) at time point of pre-G-CSF treatment (C) and post G-CSF treatment (D) as well as at the condition
of plasma contained isolation of CD34+ cells (E), respectively. Pink arrows indicated tubular length. Red
arrows indicated cluster formation. Black dotted line circle indicated network formation. F) Analytical
result of number of tubule, * vs. other groups with different symbols (†, ‡), p<0.001. G)Analytical result of Page 30/33 Page 30/33 total tubular length, * vs. other groups with different symbols (†, ‡), p<0.001. H)Analytical result of mean
of tubular length, * vs. other groups with different symbols (†, ‡), p<0.0001. I)Analytical result of cluster
formation, * vs. other groups with different symbols (†, ‡), p<0.0001. J)Analytical result of network
formation, * vs. other groups with different symbols (†, ‡), p<0.0001. All statistical analyses were
performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=14 for each
group). Symbols (*, †, ‡) indicate significance (at 0.05 level). G-CSF = granulocyte-colony stimulating
factor. G1 = control group. G2A (study group) = pre-G-CSF treatment; G2B = post G-CSF treatment. G3 =
plasma contained isolated CD34+ cells. total tubular length, * vs. other groups with different symbols (†, ‡), p<0.001. Circulatory EPC levels between group 1 and group 2 patients and in group 1 after G-CSF treatment H)Analytical result of mean
of tubular length, * vs. other groups with different symbols (†, ‡), p<0.0001. I)Analytical result of cluster
formation, * vs. other groups with different symbols (†, ‡), p<0.0001. J)Analytical result of network
formation, * vs. other groups with different symbols (†, ‡), p<0.0001. All statistical analyses were
performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=14 for each
group). Symbols (*, †, ‡) indicate significance (at 0.05 level). G-CSF = granulocyte-colony stimulating
factor. G1 = control group. G2A (study group) = pre-G-CSF treatment; G2B = post G-CSF treatment. G3 =
plasma contained isolated CD34+ cells. total tubular length, * vs. other groups with different symbols (†, ‡), p<0.001. H)Analytical result of mean
of tubular length, * vs. other groups with different symbols (†, ‡), p<0.0001. I)Analytical result of cluster
formation, * vs. other groups with different symbols (†, ‡), p<0.0001. J)Analytical result of network
formation, * vs. other groups with different symbols (†, ‡), p<0.0001. All statistical analyses were
performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=14 for each
group). Symbols (*, †, ‡) indicate significance (at 0.05 level). G-CSF = granulocyte-colony stimulating
factor. G1 = control group. G2A (study group) = pre-G-CSF treatment; G2B = post G-CSF treatment. G3 =
plasma contained isolated CD34+ cells. Page 31/33 Figure 3
Serial changes of RIJV levels of stromal cell-derived factor (SDF)-1α and EPCs/HSC
A) Illustrating the ELISA finding for identification of the time courses of stromal cell-
(SDF) 1
i
1
ti
t
l ti
l
lt f SDF 1
*
th
ith diff Figure 3
Serial changes of RIJV levels of stromal cell-derived factor (SDF)-1α and EPCs/HSC in 14 study patients
A) Illustrating the ELISA finding for identification of the time courses of stromal cell-derived factor
(SDF)-1α in group 1 patients; analytical result of SDF-1α, * vs. other groups with different symbols (†, ‡, §,
¶), p<0.0001. B) Flow cytometric analysis of the baseline and time courses of CD34+KDR+CD45dim cells, Fi
3 Serial changes of RIJV levels of stromal cell-derived factor (SDF)-1α and EPCs/HSC in 14 study patients Page 32/33
changes of RIJV levels of stromal cell-derived factor (SDF)-1α and EPCs/HSC in 14 study patients
ustrating the ELISA finding for identification of the time courses of stromal cell-derived factor
)-1α in group 1 patients; analytical result of SDF-1α, * vs. Circulatory EPC levels between group 1 and group 2 patients and in group 1 after G-CSF treatment other groups with different symbols (†, ‡, §,
0.0001. B) Flow cytometric analysis of the baseline and time courses of CD34+KDR+CD45dim cells, Serial changes of RIJV levels of stromal cell-derived factor (SDF)-1α and EPCs/HSC in 14 study patients
A) Illustrating the ELISA finding for identification of the time courses of stromal cell-derived factor
(SDF)-1α in group 1 patients; analytical result of SDF-1α, * vs. other groups with different symbols (†, ‡, §, of stromal cell-derived factor (SDF)-1α and EPCs/HSC in 14 study patients A) Illustrating the ELISA finding for identification of the time courses of stromal cell-derived factor
(SDF)-1α in group 1 patients; analytical result of SDF-1α, * vs. other groups with different symbols (†, ‡, §,
¶), p<0.0001. B) Flow cytometric analysis of the baseline and time courses of CD34+KDR+CD45dim cells, Page 32/33 Page 32/33 analytical result of number of CD34+KDR+CD45dim cells, * vs. other groups with different symbols (†, ‡,
§), p<0.0001. C) Flow cytometric analysis of the baseline and time courses of CD34+CD133+CD45dim
cells, analytical result of number of CD34+CD133+CD45dim cells, * vs. other groups with different
symbols (†, ‡, §), p<0.0001. D) Flow cytometric analysis of the baseline and time courses of
CD34+CD133+KDR+ cells, analytical result of number of CD34+CD133+KDR+ cells, * vs. other groups
with different symbols (†, ‡), p<0.0001. E) Flow cytometric analysis of the baseline and time courses of
CD31+CD133+CD45dim cells, analytical result of number of CD31+CD133+CD45dim cells, * vs. other
groups with different symbols (†, ‡, §, ¶, α), p<0.0001. F) Flow cytometric analysis of the baseline and
time courses of CD34+ cells, analytical result of number of CD34+ cells, * vs. other groups with different
symbols (†, ‡, §), p<0.0001. G) Flow cytometric analysis of the baseline and time courses of CD133+
cells, analytical result of number of CD133+ cells, * vs. other groups with different symbols (†, ‡, §),
p<0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple
comparison post hoc test (n=14 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). G-
CSF = granulocyte-colony stimulating factor; RIJV = right internal jugular vein; EPCs = endothelial
progenitor cells; hematopoietic stem cells. Figure 4
Longitudinal evaluation of serial changes of the NIHS and Barthel index in each group of the patients
(A)Indicated the time courses of National Institute of Health Stroke Scale (NIHSS) in group 1 and group 2
respectively. Circulatory EPC levels between group 1 and group 2 patients and in group 1 after G-CSF treatment Analytical result by repeated measures of ANOVA, p<0.0001 in group 1 and p<0.001 in group
2. Figure 4 Longitudinal evaluation of serial changes of the NIHS and Barthel index in each group of the patients (A)Indicated the time courses of National Institute of Health Stroke Scale (NIHSS) in group 1 and group 2
respectively. Analytical result by repeated measures of ANOVA, p<0.0001 in group 1 and p<0.001 in group
2. (B) Indicated the time courses of Barthel index in group 1 and group 2, respectively. Analytical result by
repeated measures of ANOVA, p<0.0001 in group 1 and p<0.0001 in group 2. (B) Indicated the time courses of Barthel index in group 1 and group 2, respectively. Analytical result by
repeated measures of ANOVA, p<0.0001 in group 1 and p<0.0001 in group 2. Page 33/33
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Approach for Modelling Process Effects during Friction Stir Welding of Composite Extruded Profiles
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Advanced materials research
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Keywords: Finite element analysis, friction stir welding, composite aluminium extrusions, light
weight construction Keywords: Finite element analysis, friction stir welding, composite aluminium extrusions, light
weight construction Abstract. In the field of light weight frame structures the assurance of dimensional accuracy and the
prediction of structural properties especially during and after welding processes are of great
importance. The problem in this regard mostly arises from the used welding technique which is
characterised by complex interactions of various parameters. A simulative approach is useful in
order to predict the structural behaviour and to improve the geometrical quality of joined light
weight components after welding. As such, it contributes to reduce process adjustments in the early
stage of the product life cycle, and therefore helps to save time and costs. In this paper an approach
for modelling the innovative joining process of composite extruded profiles by friction stir welding
is presented. This article is an open access article under the terms and conditions of the Creative Commons Attribution (CC BY) license
(https://creativecommons.org/licenses/by/4.0) Approach for Modelling Process Effects during Friction Stir Welding of
Composite Extruded Profiles pproach for Modelling Process Effects during Friction Stir Weldin
Composite Extruded Profiles
Michael F. Zaeh1,a and Alexander Schober1,b
1Institute for Machine Tools and Industrial Management (iwb)
Technische Universität München, Boltzmannstr. 15, D-85748 Garching, Germany
amichael.zaeh@iwb.tum.de, balexander.schober@iwb.tum.de Michael F. Zaeh1,a and Alexander Schober1,b
1Institute for Machine Tools and Industrial Management (iwb)
Technische Universität München, Boltzmannstr. 15, D-85748 Garching, G
amichael.zaeh@iwb.tum.de, balexander.schober@iwb.tum.de Advanced Materials Research
ISSN: 1662-8985, Vol. 43, pp 105-110
doi:10.4028/www.scientific.net/AMR.43.105
© 2008 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Advanced Materials Research
ISSN: 1662-8985, Vol. 43, pp 105-110
doi:10.4028/www.scientific.net/AMR.43.105
© 2008 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Online: 2008-04-08 Introduction This paper discusses some results that were achieved in sub-project B4 within the Transregional
Collaborative Research Centre 10. The scientific objective of this project is the simulation of the
interactions between the considered thermal joining processes (friction stir welding and laser beam
welding) and the component structure [1]. In particular the joining of composite extruded profiles
will be investigated. In doing so, various new aspects have to be considered. For instance, the
different material parameters – in particular the coefficient of thermal expansion – of the matrix and
the reinforcing elements cause the generation of stresses during a heat treatment, particularly when a
welding process is performed. In this regard it is of interest to study low heat joining technologies
such as friction stir welding. The benefit of applying such an innovative process should be proved. Processes during which a great amount of heat is applied to the treated components will be
examined as well, in order to test the limits of their applicability. The particular joining technique
that will be studied in this regard is laser beam welding. The evaluation of the applicability of these
joining techniques for various profile types will be clarified in cooperation with other sub-projects
that investigate the process sensibility, particularly sub-project A11. It is intended to compare
experimental results to those of appropriate simulations for mutual benefit, meaning, on the one
hand, to gain information for optimising the process and, on the other hand, to validate the results
obtained from the simulation. Depending on the geometrical properties and the used materials of the
various profiles, methods will be derived to calculate the state of the regarded component during
and after the examined welding process. Flexible Manufacture of Lightweight Frame Structures, 2008 106 Presentation of the considered example The joining process of two composite extruded profiles by means of friction stir welding provides
an application example for the examination discussed within this paper. This example describes the
experiments that were conducted by sub-project A11, which studies the joining techniques of
friction stir welding and laser beam welding. In the course of that sub-project various experiments
were and will be conducted at the authors’ institute. As a starting point the setup of the above
mentioned experiment provides the basis for modelling. In detail this experiment involves a butt
joint of two extruded composite sheet metals – each made of an aluminium matrix reinforced with
six steel wires that are distributed equidistantly – with the dimensions 35 × 56 × 5 mm. These
extruded composite profiles are shown in Fig. 1. A detailed description of the experimental setup is
discussed in a following paragraph. A transient temperature field is calculated in a thermophysical
simulation of the welding process until cooling of the structure to the ambient temperature. Within a
thermomechanical simulation, the welding distortion and the residual stresses of the workpiece are
finally calculated considering the influence of the reinforcing elements in the structure and the
clamping conditions during friction stir welding. Setup of the simulation To conduct the above mentioned computations the finite element method is applied. Therefore the
complex physical effects that occur during a welding operation become computable due to a
discretisation in space and time – by using a finite element mesh and a partition in discrete time
steps respectively. The increasing available computing power and the advancement of commercial
software both contribute to increase the accuracy of the results of a simulation and to decrease the
amount of time to compute them. From another point of view, it is possible to examine structures
with more complex geometries or include more physical effects by coupling different computations. To compute the structural results, commercial software is used. There are two main computation
steps in this regard. Firstly the transient temperature field is computed. The definition of the
intensity of the heat source as an input parameter is determined iteratively. To validate the results of
the simulation various ways are possible. These include examinations of cross sections of the weld
seam, temperature measurements during the welding process, evaluations of experience based data
sets or a combination of the above. Secondly the effects of the thermomechanical and mechanical
loads during the joining process within the component structure are calculated. The result herefrom
is a global state of residual stresses, which combines the consequences of the various influences. In
the simulation it is important to take into consideration both the modelling of the clamping situation
and the stress formation that occurs on the interface between the different materials of matrix and
reinforcing elements. All simulations are conducted using commercial software. As a starting point all meshing tasks
are handled with HyperMesh (Altair) while all further modelling tasks and computations are
performed with SYSWELD (ESI Group). Further software tools will be used as well in the course
of the project. Therefore, it will be possible to compare results even between two or more numerical
simulations using different software tools. In addition numerous experiments will be conducted to
validate the numerical results. Advanced Materials Research Vol. 43 107 Approach for modelling In addition to conduction, heat is also
transferred from the sheets to the surroundings via convection and radiation, although its
contribution to the overall heat transfer is lower – taking into account the temperature range that is Flexible Manufacture of Lightweight Frame Structures, 2008 108 attained during the welding process up to a maximum of 500°C for aluminium alloys – than that of
heat conduction. Nevertheless, once the workpiece is unclamped, heat transfer will occur only
through convection and radiation. attained during the welding process up to a maximum of 500°C for aluminium alloys – than that of
heat conduction. Nevertheless, once the workpiece is unclamped, heat transfer will occur only
through convection and radiation. All experimental process parameters that are of importance for the simulation are going to be
adopted for the modelling in this case. These parameters are listed in Table 1 below. parameter
value [unit]
feed of the tool
200 [mm/min] ≈ 3.33 [mm/s]
length of the weld seam
56 [mm]
sheet thickness
5 [mm]
reinforcing element diameter
1 [mm]
initial temperature
20 [°C]
Table 1: Selected process parameters of the friction stir welding experiments Table 1: Selected process parameters of the friction stir welding experiments Table 1: Selected process parameters of the friction stir welding experiments Approach for modelling The basic setup of the described friction stir welding experiment − size of the sheets to be joined,
clamping conditions etc. − will be adopted for the modelling. Furthermore, measurement results
which were captured during the experiments are used to calibrate various influencing parameters in
the simulation. Fig. 1 shows the schematic clamping situation of the experiments. The two sheets that are to be
joined are indicated in the figure. Additionally a third sheet – marked as supporting sheet in the
figure – can be seen in the back. These three sheets are mounted onto the support surface by two
clamps. The supporting sheet is used to allow an easier experimental performance. In this sheet the
pin of the tool plunges in vertically at the beginning of the friction stir welding operation. After that
the tool moves in a horizontal motion along the joining line of the two sheets. By the use of a
supporting sheet a stable welding process can be achieved in the experiments. The supporting sheet
can either be considered or omitted in the simulation. To clarify this aspect, both variations will be
examined in the course of the project. For the investigations in this paper the latter case was
selected. support surface
sheets to be joined
supporting sheet
clamp
clamp
reinforcing elements
Fig. 1: Schematic clamping situation for the regarded friction stir welding experiments supporting sheet clamp clamp support surface Fig. 1: Schematic clamping situation for the regarded friction stir welding experiments The definition of the clamping situation plays an important roll in the simulation. The sheets are
fixed tightly so that no movement during the welding process is allowed. Through this setup the
heat transfer is specified. There are three different ways of heat transfer [2]. The majority of the heat
that is input into the structure by the welding process will be transferred from the sheets to the
support surface through heat conduction. The contact area between each clamp and the sheets is a
thin rectangle that is smaller than the area between the sheets and the support surface. Nevertheless
heat conduction will occur through these two areas as well. Modelling of the friction stir welding The first step in the modelling is the generation of an appropriate mesh of solid elements for the
composite structure. Usually the mesh is subdivided into two sections − a fine mesh and a coarse
mesh. The fine mesh is used to handle the high temperature gradients that appear in the elements
where the heat source is applied. Once such a mesh for a single sheet is generated, the mesh of the
second sheet is created by mirroring the whole mesh structure in respect to the weld line. If a weld
gap shall be considered, solid elements need to be implemented between the two sheets. The
handling of the material behaviour of these elements is described below. In addition to the solid
elements, groups of two-dimensional elements are defined in order to model the heat exchange. Finally, to describe the welding trajectory, groups of one-dimensional elements are defined. Two
nodes indicate where the trajectory starts and where it ends respectively. In the intended simulation
the trajectory is a straight line. j
y
g
Three different materials have to be specified for the simulation. The aluminium matrix consists
of EN AW-6060 and the reinforcing steel elements are made of S 355 J2. If a weld gap should be
considered, the elements defined between the two sheets need to be handled by setting Young’s
modulus to a value that corresponds to 0.7% of Young’s modulus of aluminium [3]. To define the
material properties, appropriate databases are used. These are included in the commercial software,
which was referred to above, and are complemented by experimental data provided by sub-project
A3. Furthermore, the heat input needs to be defined. As in the first phase of sub-project B4, a conical
heat source is one type of heat source that can be used [4]. Fig. 2 illustrates such a heat source and
indicates the parameters that define it. These parameters are the maximum source intensity Q0
[W/mm³] and four geometrical parameters that define the position relative to the workpiece and the
shape of the cone. Advanced Materials Research Vol. 43 109 workpiece surface
x
y
z
ri
zi
ze
Q0
re
Fig. 2: Conical heat source with its defining parameters workpiece surface Fig. Outlook Both friction stir welding and laser beam welding are complex processes each with its own specific
effects that need to be taken into consideration within a simulation. As an example for such a
specific effect the rotation of the friction stir welding tool was examined. As the rotating tool moves
along the workpiece it causes a material flow in the welding zone. This process phenomenon is very
complex to model and, therefore, it is difficult to be included in a simulation. As usual, various
simulations have to be conducted to quantify the different influences and to decide which effects are
important and should be considered in the simulation and which are of minor or no importance and
can therefore be neglected. In addition to that, some new aspects have to be taken into account in
future investigations. The fact that the composite extruded profiles are made of different materials
with different material properties leads to additional structural effects that have to be taken into
consideration. Taking into account these influencing factors will yield accurate results of the
examined welding processes. Acknowledgement This paper is based on investigations of the Transregional Collaborative Research Centre 10 which
is kindly supported by the German Research Foundation (DFG). Flexible Manufacture of Lightweight Frame Structures, 2008 Flexible Manufacture of Lightweight Frame Structures, 2008 110 The shape of the weld seam that can be observed in Fig. 3 is typical for friction stir welding. Both the tool shoulder diameter and the tool pin diameter are indicated in the figure. These
diameters are mainly responsible for the shape of the weld seam. For a good recreation of the real
welding process the parameters of the used heat source have to be chosen in a way that the shape of
the temperature field, which is calculated during the simulation, matches well with the shape of the
weld seam in the cross section. More heat sources of varying types can be combined simultaneously
to accomplish this intention. Modelling of the friction stir welding 2: Conical heat source with its defining parameters The five defining parameters are determined using cross sections which are provided by sub-
project A11 and temperature measurements that were generated by various friction stir welding
experiments at the authors’ institute [5]. In addition to the conical heat source other types of heat
sources are going to be tested in order to replicate the real process as close as possible. The
influence of a cross section for the parameter definition is discussed in the following. Such a cross
section is shown in Fig. 3. tool shoulder diameter = 10 mm
tool pin diameter = 2 mm
weld seam
Fig. 3: Cross section of a weld seam from a friction stir welding experiment tool shoulder diameter = 10 mm tool pin diameter = 2 mm weld seam Fig. 3: Cross section of a weld seam from a friction stir welding experiment [5] P. Gebhard, M. F. Zaeh: Empirical Model for the Tool Shoulder Temperature during Friction
Stir Welding. Sixth International Symposium on Friction Stir Welding 2006, Saint-Sauveur,
10th-13th October 2006. References [1] S. Roeren: Komplexitätsvariable Einflussgrößen für die bauteilbezogene Struktursimulation
thermischer Fertigungsprozesse. Diss. Technische Universität München. Herbert Utz Verlag,
München, 2007, ISBN 978-3-8316-0680-1, iwb Forschungsbericht Nr. 203. [2] D. Radaj: Heat Effects of Welding: Temperature Field, Residual Stress, Distortion. Springer-
Verlag Berlin Heidelberg 1992, ISBN 3-540-54820-3. [3] L. Papadakis, S. Roeren: Anwendungsnahe Modelle zur FEM-Berechnung des Verhaltens
gefügter Bauteile beim Laserstrahlschweißen. Tagungsband SYSWELD Forum 2005, S. 37-46,
Weimar, 1st September 2005. [3] L. Papadakis, S. Roeren: Anwendungsnahe Modelle zur FEM-Berechnung des Verhaltens
gefügter Bauteile beim Laserstrahlschweißen. Tagungsband SYSWELD Forum 2005, S. 37-46,
Weimar, 1st September 2005. [4] SYSWELD Reference Manual. ESI Group, 2000. [5] P. Gebhard, M. F. Zaeh: Empirical Model for the Tool Shoulder Temperature during Friction
Stir Welding. Sixth International Symposium on Friction Stir Welding 2006, Saint-Sauveur,
10th-13th October 2006.
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https://openalex.org/W4237410472
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https://scielo.conicyt.cl/pdf/rchdp/n29/0718-0233-rchdp-29-0337.pdf, http://www.scielo.cl/pdf/rchdp/n29/0718-0233-rchdp-29-0337.pdf
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Revista
Chilena
[diciembre
2017]
Diciembre
2017de Derecho Privado, Nº 29, pp. 337-345
Derecho
de familia
, sucesorio y regímenes matrimoniales
Susana Espada Mallorquín
Profesora de Derecho Civil
Universidad Adolfo Ibáñez
La denuncia falsa por violencia intrafamiliar como causa justificada de
desheredación del ar. 1208.1° del CC. Corte Suprema, 21 de septiembre
de 2017, rol 79128-2016.
En 2007 Ricardo A.B.A. contrajo matrimonio con Cristina J.S. y resultado
de esta unión nació su hija Josefa V.B.J. El 15 de octubre de 2010 se produce
la separación de hecho del matrimonio por la infidelidad de Cristina; eso sí,
el 29 de octubre de 2010, Cristina deduce acción de violencia intrafamiliar,
fundándola en que durante quince años Ricardo la violentaba de forma permanente con maltratos psicológicos tales como: insultos verbales, menoscabos
como mujer, amenazas de agresión y expulsión de la casa. Ricardo resultó
inocente en ese proceso, por cuanto los hechos comunicados no fueron probados, rechazándose la denuncia. En octubre de 2013, Ricardo deja la casa
familiar, quedando en ella Cristina y Josefa.
Posteriormente, en julio de 2014, Ricardo otorgó testamento abierto en una
notaría de Villarrica, instituyendo como única heredera de todos sus bienes a
su hija Josefa y en la cláusula sexta de dicho testamento procedió a desheredar a Cristina, fundando su actuar en la injuria grave que cometió en contra
de su honor, al haberlo denunciado como autor de violencia intrafamiliar,
querella desestimada por no haberse acreditado el daño causado producto
de la violencia. Así la cláusula sexta del testamento señalaba:
Comentarios de jurisprudencia
DERECHO DE FAMILIA, SUCESORIO
Y REGÍMENES MATRIMONIALES
337
“Declaro además que desheredo a mi cónyuge doña Cristina J. S.
cédula de identidad [...], por cuanto ha cometido injuria grave en
contra de mi honor al haberme denunciado como autor de violencia
intrafamiliar, según consta en la causa Rit 303 del año 2013 del Juzgado de Familia de Villarrica. En dicha causa, faltando a la verdad,
estipuló que en forma permanente y durante quince años fue víctima
de maltratos sicológicos de mi parte, consistentes en insultos verbales,
menoscabos como mujer, amenazas de agresión y expulsión de la
casa, hechos que fueron desestimados en dicha causa”.
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Susana Espada Mallorquín
338
RChDP Nº 29
Fallecido Ricardo, Cristina interpone demanda en juicio ordinario de
reforma del testamento contra su hija Josefa y solicita que se modifique el
referido testamento, específicamente en lo que respecta a la cláusula sexta del
mismo, con el objetivo de que se le reconozca su calidad de legitimaria y se
repare la lesión que está sufriendo en relación con la legítima rigorosa de la
herencia sobre la cual tiene derecho. La parte demandada contestó solicitando
su rechazo por cuanto la acción acometida por la ahora demandante en contra
del causante por violencia intrafamiliar significó para el testador un daño moral
devastador, que le afectó gravemente, constituyendo un caso de injuria atroz, ya
que se basó en hechos falsos y no probados, desestimándose por ello la acción
deducida. Por ello la causal de desheredamiento es legalmente procedente y,
por tanto, la acción de reforma de testamento debe ser desestimada.
El Juzgado de primera instancia rechaza la demanda, pues considera
justa la causa de desheredación por injuria grave al considerar probada la
falsedad de la acusación de violencia familiar realizada contra el testador.
Así en su considerando vigésimo noveno tuvo en vista el considerando sexto
de la sentencia dictada en la causa sobre violencia familiar que estableció:
“[...] el informe del Centro de la Mujer señala que no hay daño en
la denunciante, que tiene red de apoyo, respecto a la situación de
la hija señala que no está vulnerada, no hay maltrato que afecte la
integridad de la niña”.
De igual forma, dicho sentenciador de primera instancia tuvo por ciertas
las declaraciones de las testigos R.M. y N.V. (art. 384 N° 3 del CPC) que
coinciden en señalar que la denuncia por violencia intrafamiliar de su propia esposa afectó física y psicológicamente al testador, atendido, además, el
estado de salud que aquejaba a Ricardo. Ambos afirmaron el dolor inmenso
que sufrió el testador por la difamación plasmada por su cónyuge con quien
convivió quince años, después de tener una hija en conjunto pese a todas las
dificultades que implicaba y por tener que dejarla a ella en la casa familiar,
sin poder convivir junto a su hija.
La demandante interpone recurso de apelación y la Corte de Apelaciones de Temuco acoge la demanda de reforma del testamento, ya que en su
considerando duodécimo establece:
“Es un hecho de la causa y de los antecedentes tenidos a la vista, que
el requisito del art. 1209 CC no se da en la especie ya que, por un
lado, en la causa referida a la denuncia por violencia intrafamiliar,
el juez la desestima por falta de prueba y no hace calificación alguna
de la existencia de una denuncia temeraria o carente de fundamento,
situación que en estos propios autos tampoco ocurre ya que no existe
declaración del Tribunal de primera instancia en orden a dar por
establecida la existencia de la causal (como tampoco los intervinien-
Revista Fueyo 29 arreglada 080118.indd 338
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Derecho de familia, sucesorio y regímenes matrimoniales
Se recurre a la Corte Suprema de casación en el fondo argumentando
que se habrían infringido los arts. 1207, 1208 N°1 y 1209 del CC. La Corte
Suprema acoge el recurso de casación y dicta sentencia de reemplazo donde
se considera que la denuncia falsa de violencia intrafamiliar interpuesta por
su cónyuge es una justa causa de desheredación por constituir una injuria
grave hacia la persona, el honor y los bienes del testador.
Fundamento, excepcionalidad y requisitos de las causales
de desheredación
En la presente sentencia, la Corte Suprema parte destacando que ante la
existencia de asignatarios forzosos en nuestro ordenamiento, la acreditación
de la concurrencia de una causal de desheredación debe ser especialmente
rigurosa, ya que sirve para justificar la privación a los legitimarios de todo o
parte de su legítima (art. 1207 del CC).
En este sentido, podemos afirmar que la protección que el legislador otorga
a los asignatarios forzosos se justifica porque se presume que estos asignatarios
van a tener una conducta acorde con los cercanos lazos familiares que le unen
con el causante, basados en un respeto recíproco. Son los lazos familiares
unidos a un comportamiento de respeto mutuo lo que justifica la atribución
patrimonial1. Es por ello que el desheredamiento solo estará justificado en
casos excepcionalmente graves, como sanción a la mala conducta de los
legitimarios y, por ello, la Corte Suprema afirma que las causales de desheredación son “una media extrema para casos excepcionales” (cons. quinto).
Respecto a este punto quisiera hacer una precisión. Es cierto que la doctrina
chilena afirma que no hay más causas de desheredamiento que las contempladas
en el art. 1208 del CC, aun cuando la conducta que el heredero haya podido llevar
a cabo pueda ser considerada más grave de las allí señaladas e igualmente, se
sostiene que por ser causales excepcionales y limitativas deben interpretarse restrictivamente, no admitiendo interpretaciones amplias o aplicaciones analógicas2.
En mi opinión, el hecho de que se exija una interpretación restrictiva de
las causas taxativas no puede significar una exigencia tan estricta que impida
cualquier tipo de interpretación de dichas causales por parte del juez. Respecto
de la interpretación estricta y amplia de las disposiciones excepcionales, Karl
1
2
Comentarios de jurisprudencia
tes solicitan su declaración), sino que se limita a rechazar la acción
interpuesta incluso, declarando que la demandante tuvo motivos
plausible para litigar, criterio, que, por lo demás esta Corte comparte,
lo que sumado a los demás argumentos señalados precedentemente,
justifican la revocación de la sentencia...”.
339
Elorriaga de Bonis (2010), p. 487.
Op. cit., p. 489.
Revista Fueyo 29 arreglada 080118.indd 339
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Susana Espada Mallorquín
RChDP Nº 29
Comentarios de jurisprudencia
Larenz señala:
340
“Se tiene que evitar que mediante una interpretación demasiado
amplia de las disposiciones excepcionales o mediante su aplicación
analógica la intención del legislador se trueque finalmente en lo contrario a ella. Pero esto no significa que la disposición excepcional haya
de interpretarse tan estrictamente como sea posible o que la analogía
quede excluida en todo caso. Aquí es decisiva de nuevo la razón por
la que el legislador ha exceptuado precisamente esos casos”3.
Por ello, de acogerse esta argumentación a pesar de afirmar el carácter
extremo y excepcional de las causales de desheredación, considero que esto
no impide a los jueces interpretar las causales de desheredación adecuándose
al tiempo y a la realidad social actual en la que se aplican, sin que ello se
considerase una interpretación prohibida por su carácter extensivo4.
Hecha esta aclaración, en su considerando quinto la Corte señala los
requisitos que son precisos que concurran para que válidamente proceda el
desheredamiento:
“1°. Que se haga por una disposición testamentaria expresa (artículo
1209). Debe hacerse por medio de una disposición testamentaria, en
que el testador expresamente ordene que el legitimario sea privado
del todo o parte de su legítima. Se trata de una medida de rigor y
ella debe expresarse de un modo claro y terminante; en caso de duda
sobre la voluntad del testador, no existiría el desheredamiento.
2°. Que se funde en alguna de las causas indicadas por la ley (art. 1208).
3°. Que la causa legal en que se funda el testador sea expresada
específicamente en el testamento (art. 1209). No basta que se diga en
el testamento que se deshereda a tal o cual legitimario; es menester
que se exprese específicamente en el testamento la causal legal en
que se funda el desheredamiento. Deberá, pues, referirse el testador
expresamente a alguna o algunas de las cinco causales enumeradas en
el artículo 1208. Esta es una solemnidad especial que la ley quiere que
se cumpla, el desheredamiento hecho sin expresión de causa no vale, y,
4°. Que la causal de desheredamiento debe ser probada judicialmente en vida del testador, o por los interesados después de su
muerte (art. 1209)”.
Respecto de los mencionados requisitos, en la sentencia objeto de ese
comentario dos son los principales problemas que se plantean. Por un lado,
la necesidad de precisar que es una injuria grave en el sentido de la causal
3
4
Larenz (2001), pp. 352-353.
En este mismo sentido véase Betti (2009), pp. 105-108.
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Derecho de familia, sucesorio y regímenes matrimoniales
El concepto de injuria grave
del art. 1208.1° del CC
Como pone de manifiesto la Corte Suprema en el considerando octavo de
la presente sentencia:
“[...] nuestro Código Civil no ha dado una definición ni describe siquiera lo que debe entenderse por injuria grave o atroz, que permitan
determinar cuáles son los elementos que autorizan para calificar de
grave o atroz la ofensa o injuria hecha a una persona. La jurisprudencia ha señalado que la injuria consiste en un hecho ejecutado o dicho
proferido en deshonra o menosprecio de otro. El móvil de la injuria es
siempre el perjuicio, molestia o daño que se trata de infligir al injuriado,
a fin de satisfacer alguna venganza, odio o resentimiento de parte del
injuriante (RDJ. Tomo 13, sec. 1ª, p. 13). También ha sostenido que para
dar por establecido la existencia de un acto injurioso y, sobre todo,
de un acto que pueda consistir injuria grave, se necesita que medie
una intención dolosa de parte del que lo ejecuta, dirigido a perjudicar,
molestar o dañar al injuriado (RDJ. Tomo 64, sec. 1ª, p. 176)”.
Comentarios de jurisprudencia
establecida en el art. 1208.1° del CC y, por otro, que se determine cuándo se
considera probada en esos casos la causal mencionada, así como su mención
testamentaria y su posible revisión casacional.
341
En este sentido la doctrina también considera la que el término “injuria
grave” no puede tomarse con una significación penal (art. 416 del Código
Penal), sino con un significado más genérico que hace referencia a un daño
o perjuicio, como se define en el art. 44 del CC5. Según el citado precepto la
culpa grave en el ámbito civil equivale al dolo y este consiste en la intención
positiva de inferir injuria a la persona o propiedad de otro.
Luego, es posible interpretar que podrá ser desheredado justamente quien
haya realizado un comportamiento susceptible de causar un grave daño o
menoscabo intencional en contra de alguna de las personas que se mencionan
en el art. 1208.1º del CC, pues dicho comportamiento será susceptible de ser
calificado como injuria grave.
La falsedad de la acusación de violencia intrafamiliar
La determinación de si existe un daño y la gravedad del mismo es una cuestión de hecho que deberá ser apreciada por los tribunales del fondo. En este
5
Elorriaga de Bonis (2010), p. 492.
Revista Fueyo 29 arreglada 080118.indd 341
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Comentarios de jurisprudencia
Susana Espada Mallorquín
342
RChDP Nº 29
sentido y teniendo en cuenta los hechos relatados en el fallo, cuando existe
una falsa acusación de violencia intrafamiliar, la gravedad del daño de esa
falsa acusación derivará de que la única motivación de llevarla a cabo sea
causar –con una intención dolosa por parte de quien acusa– un daño, perjudicar y molestar al injuriado, lesionando su honra, a fin de satisfacer con ello
alguna venganza, odio o resentimiento.
En nuestro caso, en la contestación a la demanda se señalaba que el
único objetivo de la acusación de violencia intrafamiliar fue dañar al causante gravemente en aquello que para él es del máximo valor: su honor. Al
imputarle acciones de poca hombría, de vileza y acoso psicológico cobarde
y permanente, que no corresponden de un hombre hacia su mujer, fue la
imputación más dañina que para el honor de Ricardo pudieron realizarle.
Al respecto, considero que los tribunales deben tener claro que para
aplicar la concurrencia de esta causal de desheredación, lo esencial es cerciorarse de la falsedad de la acusación de violencia intrafamiliar y, por ello,
que su única finalidad sea esta intención dolosa de dañar mediante injurias
al causante. Por lo tanto, el constatar meramente el hecho de que no se logró
probar la violencia intrafamiliar no siempre va a implicar que la acusación de
quien la realiza sea falsa y que esto justifique una desheredación por injuria.
La ausencia de prueba de la violencia no es sinónimo de la falsedad de la
acusación. Eso sí de constatarse en los hechos que la demanda por violencia
se funda en hechos falsos y que la intención de su interposición es deshonrar,
afrentar, envilecer y desacreditar al demandado, será un comportamiento
susceptible de calificarse como injuria grave que justifique una desheredación
de aquel que lo realice.
Sin embargo, que lo relevante a los efectos de aceptar la denuncia falsa
como causal de desheredación sea la constatación por parte de los tribunales
de la falsedad de la misma no significa que se tenga que existir, como afirma la
sentencia recurrida de la Corte de Apelaciones de Temuco, “una declaración
objetiva del juez que afirme el carácter temerario y carente de fundamento de
la demanda de violencia intrafamiliar” (considerando primero de la sentencia
de la Corte Suprema). Si el juez de instancia a raíz de los hechos probados
logra constatar la falsedad de la denuncia de violencia, así como el daño a
la honra del testador (art. 1208.1° del CC), podría declarar la desheredación
como justa, a pesar de que no exista una declaración del juez que califique
la interposición de la demanda como temeraria y carente de fundamento.
En estos casos de acusación de violencia intrafamiliar, quizá podría resultar oportuno establecer como criterio la exigencia de que en el juicio o en
la resolución se constatase la condena en costas al desheredado (art. 144 del
CPC). Como sabemos, el tribunal puede eximir de dichas costas cuando se
hayan tenido motivos plausibles para litigar, de lo cual debe hacerse declaración expresa en la resolución. Cuando se condena en costas respecto de la
acusación de violencia intrafamiliar y el juez expresa que dicha condena se
basa en la ausencia de un motivo plausible para litigar, tenemos un elemento
Revista Fueyo 29 arreglada 080118.indd 342
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Derecho de familia, sucesorio y regímenes matrimoniales
La prueba de los hechos constitutivos
de injuria grave
Como pone de relevancia la sentencia de la Corte Suprema en su considerando noveno:
“Que las causas que autorizan el desheredamiento se apoyan en
hechos, que deben ser acreditados por los que tienen interés en que
la voluntad del testador sea cumplida, en este caso, el peso de la
prueba corresponde al demandado. Por lo demás, procesalmente,
quien afirma es quien debe probar el hecho, como norma general.
En suma, debe probarse que los hechos en que se apoyó el testador
eran ciertos”.
Comentarios de jurisprudencia
de peso para poder afirmar la falsedad de la acusación, sin necesidad de tener
que exigir que un juez declare la temeridad de la acción. El comportamiento
injurioso que justifica la desheredación del legitimario es la falsedad de la
acusación, que en parte se acredita por la ausencia de un motivo plausible
para acusarle ante los tribunales de hechos de extrema gravedad como son
los considerados en la violencia intrafamiliar.
Luego no toda ausencia de prueba de los hechos constitutivos de violencia intrafamiliar justifica la desheredación del acusador; pero tampoco solo
podrá causar injurias aquella acusación de violencia que haya sido temeraria
en sus términos. Eso sí, probada la falsedad de una acusación de violencia
intrafamiliar –lo que puede presumirse si existe condena en costas– es posible
desheredar al acusador, pues, sin duda, dicha acusación falsa provoca un
grave daño a la honra del causante.
343
En este sentido también la doctrina6 complementa esta afirmación señalando que procesalmente, para los fines del art. 1209 del CC, los hechos
constitutivos de la injuria grave los determinan soberanamente los jueces del
fondo, es decir, los de la instancia; pero la precisión de si esos hechos tienen
la gravedad suficiente requerida por ley para justificar una desheredación es
una cuestión de calificación que quedaría sujeta al control de casación.
Por lo tanto, una vez identificada la causal de desheredación es necesaria
la referencia al factum cuasal desheredatorio [“haber cometido injuria grave
contra el testador en su persona, honor o bienes (art. 1208.1°del CC)”], pero
también del factum probans respecto del factum probandum7 (expresando la
causa en el testamento y probado judicialmente en vida del testador o por
los interesados tras el fallecimiento, exart. 1209 del CC).
6
7
Domínguez y Domínguez (2011), p. 1093.
Martinez Gallego (2006), p. 1620.
Revista Fueyo 29 arreglada 080118.indd 343
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Comentarios de jurisprudencia
Susana Espada Mallorquín
344
RChDP Nº 29
En este caso, el testador en la cláusula sexta de su testamento hacía
referencia expresa a los hechos de los que falsamente se le acusaba8, por lo
tanto, esos son los hechos que deben quedar probados en la instancia y, en
casación, la Corte podrá entrar a calificar si los mismos son suficientemente
graves para constituir una causa justa de desheredación. Así, en la sentencia
que se comenta, en casación se estimó en los considerandos decimotercero
y decimocuarto:
“[...] el acto ejecutado en desdoro del testador, consistente en la
denuncia indebida y sin fundamento por violencia familiar de su
cónyuge, unido a la infidelidad de la misma, ha debido ser hecha
con la intención de deshonrar, afrentar, envilecer, desacreditar, hacer
odiosa, despreciable o sospechosa la conducta de éste, elementos
suficientes para considerarse injuriado. [...] Que, a la luz de lo expuesto en las consideraciones precedentes, no cabe más que concluir
que se encuentra configurada la causa de injuria grave consagrada
en el artículo 1208 N° 1 del Código Civil, la cual, además, ha sido
probada durante el proceso y, al no establecerlo así los jueces de la
instancia, han infringido lo dispuesto en los artículos 1208 causal 1a
y 1209 inciso primero del Código Civil, por cuanto la causal ha sido
válidamente probada en el juicio”.
En este sentido, para calificar la gravedad de la injuria, la Corte Suprema
tuvo en cuenta, por un lado, la sentencia dictada en la causa sobre violencia
intrafamiliar que afirmaba la inexistencia de daño en la denunciante y en su
hija; y, por otro, las declaraciones de testigos –que el sentenciador de primer
grado tuvo por ciertas– y que coincidían en señalar que la denuncia por violencia intrafamiliar de la esposa afectó física y psicológicamente al testador,
el dolor y aflicción padecidos por este, teniendo en cuenta su delicado estado
de salud y el dolor inmenso que le supuso la difamación de su cónyuge con
quien convivió quince años, después de tener una hija en común a pesar de
la dificultades y de ahora tener que dejarla a ella en la casa familiar junto a
su hija.
Bibliografía citada
Betti, Emilio (2009). La interpretación jurídica. Santiago: Legal Publishing.
Domínguez Águila, Ramón y Ramón Domínguez Benavente (2011). Derecho
sucesorio. Santiago: Editorial Jurídica de Chile. Tomo i.
8
En este sentido se puede señalar que en nuestro Código Civil no está prevista la posibilidad
de que sea el propio causante el que inicie un juicio en vida destinado específicamente a
establecer la veracidad y certeza de los hechos que invoca para desheredarlo.
Revista Fueyo 29 arreglada 080118.indd 344
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Diciembre 2017
Derecho de familia, sucesorio y regímenes matrimoniales
Comentarios de jurisprudencia
Elorriaga de Bonis, Fabián (2010). Derecho sucesorio. Santiago: Abeledo Perrot
Legal Publishing.
Larenz, Karl (2001). Metodología de la Ciencia del Derecho. Barcelona: Ariel Derecho.
Martínez Gallego, Eva. (2006). “La desheredación”. Actualidad Civil. N° 16,
Tomo ii. Madrid.
345
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Fast Augmented Reality Authoring: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations
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RICCARDO PALMARINI1, IÑIGO FERNÁNDEZ DEL AMO1, DEDY ARIANSYAH1,
SAMIR KHAN1, (Member, IEEE), JOHN AHMET ERKOYUNCU
1,
AND RAJKUMAR ROY
2 1Cranfield Manufacturing, School of Aerospace, Transport and Manufacturing, Cranfield University, MK43 0AL Cranfield, U.K. 2School of Mathematics, Computer Science and Engineering, City, University of London, EC1V 0HB London, U.K. Corresponding author: John Ahmet Erkoyuncu (j.a.erkoyuncu@cranfield.ac.uk) is work was supported in part by the High-Speed Sustainable Manufacturing Institute (HSSMI), and in part by the Engineering a
ysical Sciences Research Council (EPSRC) by the Project ‘‘Digi-TOP’’ under Grant EP/R032718/1. This work involved human subjects or animals in its research. Approval of all ethical and experimental procedures and protocols was
granted by Cranfield University. ABSTRACT Augmented Reality (AR) has shown great potential for improving human performance in
Maintenance, Repair, and Overhaul (MRO) operations. Whilst most studies are currently being carried out at
an academic level, the research is still in its infancy due to limitations in three main aspects: limited hardware
capabilities, the robustness of object recognition, and content-related issues. This article focuses on the last
point, by proposing a new geometry-based method for creating a step-by-step AR procedure for maintenance
activities. The Fast Augmented Reality Authoring (FARA) method assumes that AR can recognise and track
all the objects in a maintenance environment when CAD models are available, to knowledge transfer to a
non-expert maintainer. The novelty here lies in the fact that FARA is a human-centric method for authoring
animation-based procedures with minimal programming skills and the manual effort required. FARA has
been demonstrated, as a software unit, in an AR system composed of commercially available solutions
and tested with over 30 participants. The results show an average time saving of 34.7% (min 24.7%; max
55.3%) and an error reduction of 68.6% when compared to the utilisation of traditional hard-copy manuals. Comparisons are also drawn from performances of similar AR applications to illustrate the benefits of
procedures created utilising FARA. INDEX TERMS Augmented reality, authoring, content, digital engineering, maintenance. his work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Received 19 December 2022, accepted 30 December 2022, date of publication 24 January 2023, date of current version 27 January 2023. Digital Object Identifier 10.1109/ACCESS.2023.3235871 VOLUME 11, 2023
This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons I. INTRODUCTION range of fields: medical applications, marketing, entertain-
ment, education, maintenance, and manufacturing [3], [4]. This article focuses on Maintenance, Repair, and overhaul
(MRO) operations. MRO operations impact the lifecycle cost
of industrial equipment [5]. The increasing complexity and
automation of industrial machinery require new technolo-
gies for ensuring reliability and productivity through MRO
operations. In the aviation field, MRO operations costs can
reach 80% of the entire aircraft lifecycle [6]. MRO operations
strongly rely on maintenance technicians’ expertise [7]. The
latter can affect both the errors and completion time involved AUGMENTED Reality (AR) is an innovative technology that
aims to enhance the human perception of reality by providing
digitally created content in the real context [1]. Another
definition has been provided by Azuma [2] who stated that
an AR system should have three characteristics: Combine real
and virtual, 2) Interactive real-time, and 3) Registered in 3-D. AR applications have been developed and tested in a wide The associate editor coordinating the review of this manuscript and
approving it for publication was Yu Liu
. 8407 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations and expensive [15]. The contents are now implemented in
AR as ‘‘standalone’’ programs by programmers [16]. Com-
prehensive reviews of AR authoring for maintenance appli-
cations have been done [10]. The remaining paragraphs
highlight some of the existing efforts for content creation and
identify the crucial gap of AR authoring for industrial imple-
mentation to be addressed in this work. The main knowledge
gap is that current authoring environments require com-
prehensive programming and graphical expertise. The
most common tools for authoring AR content comprise plug-
ins, software development kits (SDK), and graphical pro-
gramming languages. Among these, it is worth mentioning
Unity, Unreal, Panda3D, ArToolkit, Vuforia, and Max/MPS. in the MRO operation thus influencing the MRO cost. AR can
help reduce errors and completion time by allowing easy
access to MRO information which today belongs mostly
to the expert maintainers’ memory [8]. Even though the
advantages (time savings and error reductions) of AR in
maintenance have been proven by academics, the technology
still lacks the robustness and flexibility to become of com-
mon use. I. INTRODUCTION Among the main research topics, it is possible to
find [9], [10], [11]: • Tracking and recognition of performances • Hardware (head-mounted displays) capabilitie • Contents-related issues The last one comprises difficulties in creating and manag-
ing content for AR applications. The traditional process of
creating content (a.k.a. authoring) for AR requires different
professionals: programmer, animator, CAD modeller, and
AR developer. More innovative authoring solutions which
provide a friendly user interface and content adaptation have
also been proposed ([12], [13], [14]). However, they still
require a lot of human effort and have limited flexibility. This
article contributes to this gap by implementing an AR within
a maintenance application. For authoring ‘‘AR step-by-step’’
procedures to guide a non-expert technician in carrying out a
maintenance task ‘‘Step-by-step AR instructions’’ or ‘‘proce-
dures’’ is a common terminology which refers to the action
of gradually providing a set of information at each step of
an MRO operation. The data considered in this project is
visual (3D animations). The method developed and validated
has been named FARA: Fast Augmented Reality Authoring. The novelty of this work lies in the approach that FARA
is a humancentric method for authoring animation-based
procedures with minimal programming skills and manual
effort required when learning to use the software features
for creating step-by-step AR instructions. It takes inspi-
ration from Fast Programming Robots that enable users to
teach robots by demonstration. Similarly, FARA allows users
to teach an AR system how to overlay the virtual content into
a real environment without programming. y,
,
,
,
,
Nowadays, only a few have attempted to ease and de-skill
the authoring process. Shim et al proposed an interactive
features-based AR authoring tool [17]. This allows users to
rotate, move, enlarge merge and occlude virtual objects the
virtual objects visualized over a 2D printed marker. The men-
tioned transformations are done through marker interaction
and gesture interaction. Similarly, Yang et al proposed an
authoring tool that takes advantage of a mobile device to inter-
act with the virtual objects visualized through an HMD [18]. Both approaches do not require any programming skills and
are not time-consuming for the content creator. Still, the
solution does not allow the creation of animation, which is
powerful in the maintenance environment [6], [19].1 Csurka
et al proposed SUGAR, as an easy-to-use AR editor that does
not require programming skills [20]. 1It must be noted that step-by-step animations might not always be
required. I. INTRODUCTION However, part of the
creation of the content must be done through the SUGAR
editor. The latter requires the content creator to input the
picture of the working environments and manually create or
import the virtual objects and animation that users want to
over-impose on the real environment at each step of the main-
tenance procedure. Even though the advantages compared
with the traditional authoring methods have been proven,
it is the authors’ opinion that most methods are still time-
consuming. Zhu et al proposed an on-site authoring tool that
allows maintenance technicians to change or create informa-
tion instances related to maintenance procedures [21]. This
means that only text information can be created and edited. Other authoring solutions have been used or proposed in
the literature [22], [23], [24], [25], [26], [27]. Using these
authoring solutions requires some knowledge and a manual
process in understanding how to use the software features
such as creating and positioning the 3D animation on top of
the real component. This article is structured as follows. The research back-
ground and motivation are provided in Sections I and II. This
is followed by a description of FARA: how it works and its
structure and the detailed methodology for FARA’s valida-
tion. This includes the quantitative test design (Section IV-A),
the case study utilised (Section IV-B), and FARA’s implemen-
tation in an AR system for testing purposes (Section IV-C). Analysis and results are reported in Section IV-C. Finally, the
discussion of the results and the conclusions are presented in
Sections V and VI. Taking advantage of the valuable contributions of previous
studies, the aim of simplifying the authoring process of AR
procedures should remain and will likely help to implement
AR in the industry. The availability of AR authoring tools that
can be operated by non-programmers and non-AR experts is
essential for the success of AR technology in both the main-
tenance field and other areas [15]. The approach proposed A. HOW FARAIS WORKS: A PRACTICAL EXAMPLE The FARAIS shown in this example has been developed using
commercial hardware and the open-source limited versions
of Vuforia and Unity 3D software. These will be described
in Section IV-C. The procedure selected is the assembly of
a mock-up designed and utilised for testing purposes. It will
be described in Section IV-B. First, consider the maintenance
environment shown in Fig 1. It includes the maintainer (Nr.1),
the product to be maintained (Nr.2) and FARAIS (Nr.3). There are two scenarios: 1) involving an expert technician,
and 2) involving a non-expert technician. in this article is an authoring technique that automates the
creation and positioning of 3D animation content overlaid on
the physical components. III. FAST AUGMENTED REALITY AUTHORING Fast Augmented Reality Authoring (FARA) aims to over-
come the contents-related issues previously described and
therefore ease the implementation of AR in the industry. Its name implies ‘‘fast programming’’ when implemented
in an existing AR system for maintenance. In this context,
‘‘fast programming’’ indicates ‘‘fast AR contents-creation for
maintenance procedures’’. From now on, the authors will
refer to any AR system for maintenance implemented with
FARA as FARAIS: FARA Implemented System. FARAIS is
a tool for easy knowledge transfer from experts to non-expert
technicians within procedural operations (e.g., dis/assembly,
repair, inspections). It will allow the expert (user confident
with the maintenance procedures) to ‘‘record’’ the MROs
and the non-expert to access the MROs in a ‘‘step-by-step’’
format. Ideally, a FARAIS would be suitable for any oper-
ation involving humans, e.g., both preventive and corrective
maintenance. FARA is based on two assumptions. The first
one is that current object recognition and tracking issues [10]
will be solved by providing reliable and real-time tools that
can identify objects independently from the light condition
and background noise. The second hypothesis is that CAD
models are available for the components involved in any
maintenance procedure. II. RESEARCH BACKGROUND AND MOTIVATION II. RESEARCH BACKGROUND AND MOTIVATION This research focuses on the creation of content for AR
(authoring) for maintenance applications which are known as
one of the main problems that prevent AR to become main-
stream. The simple and easy creation of AR content is not
currently available. The authoring process is time-consuming 8408 VOLUME 11, 2023 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 1. Maintenance environment simulation for testing purposes. The
environment includes the technician (Nr 1), the product to be maintained
(Nr 2) and FARAIS (Nr 3). FIGURE 2. 2D graphical representation of ‘‘transform’’. The transformed
vector of object 2 is (1x, 1y, 1α). FIGURE 1. Maintenance environment simulation for testing purposes. The
environment includes the technician (Nr 1), the product to be maintained
(Nr 2) and FARAIS (Nr 3). FIGURE 2. 2D graphical representation of ‘‘transform’’. The transformed
vector of object 2 is (1x, 1y, 1α). FIGURE 2. 2D graphical representation of ‘‘transform’’. The transformed
vector of object 2 is (1x, 1y, 1α). FIGURE 1. Maintenance environment simulation for testing purposes. The
environment includes the technician (Nr 1), the product to be maintained
(Nr 2) and FARAIS (Nr 3). B. FARA METHOD Once the
procedure is completed, the expert technician can quit the
‘‘record mode’’ through the UI. FARAIS will automatically
build the AR step-by-step ‘‘Procedure 1’’. The ‘‘How’’ is
explained in Section IV. The maintainer’s effort in creating
the AR step-by-step procedure is low as the only duty is to
press the record button and perform the maintenance proce-
dure as usual. It is worth mentioning that video recording is
performed by FARAIS. 1) SCENARIO 1 – EXPERT TECHNICIAN In this scenario, FARAIS ‘‘captures’’ the expert technician’s
knowledge. The expert has to carry out a maintenance pro-
cedure that they are confident with, on the product shown
in Fig 1 (Nr.2). He accesses FARAIS through the hardware
provided. Here, it is a ‘‘head-mounted video-see-through dis-
play’’. Before starting the procedure, he will select ‘‘record
mode’’ to ‘‘capture’’ the procedure and select a name: e.g.,
‘‘Procedure 1’’. Once started the procedure, FARAIS will
simultaneously perform three actions: • Recognise and track the real objects in the Field of View
(FOV) of the technician • Store the transforms of the real objects in the table
dedicated to ‘‘Procedure 1’’ • Overlay the virtual objects over the real ones utilising
the ‘‘basic overlay rule’’ available on a database (DB) The first action comprises tracking the position and orien-
tations of the objects. The second one means that the positions The first action comprises tracking the position and orien-
tations of the objects. The second one means that the positions VOLUME 11, 2023 8409 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 3. AR step-by-step procedure animation example. The latter consists of animating the virtual objects on the
real ones suggesting the positions and orientations that the
real objects must reach at each step. An example is reported
in Fig 3. First, FARAIS recognises the objects and overlays the
corresponding virtual objects utilising the basic overlay rule
available on the DB (Fig 3a). Then, the virtual object animates
detaching from the real object and moving to the target posi-
tion and orientation as suggested by the selected procedure
(Fig 3b, 3c, and 3d). It is worth noting that FARAIS also takes
into account user interaction. For instance, if the technician
moved the real object in the opposite direction, the animation
would start from the position of the real object and make a
new trajectory to get to the final position. It will be done
by getting first closer to the recorded trajectory, and then
continuing with the recorded animation. When the technician
puts the real object in the target position, the virtual object
will become green (Fig 3e). FARAIS will then move to the
next step of the procedure showing the next animation. The
screen shows the message ‘‘Procedure Completed’’ when the
procedure is completed. B. FARA METHOD FIGURE 3. AR step-by-step procedure animation example. FARA is a method that, integrated with an AR system, forms
what in this article has been named FARAIS. On one hand,
the AR system can recognise the environment by perform-
ing object recognition and tracking and overlaying virtual
objects on the real environment following pre-programmed
rules (e.g., overlying the virtual object over the real one by
overlapping the corners). On the other hand, it provides the
maintainer with the ability to produce the virtual overlay rule
by collecting the data from the MRO. This formalizes the AR
step-by-step procedures. The FARA method is schematized
in Fig 4. The figure is divided into three main squared areas:
FARAIS, AR system, and FARA. This division is meant to
show that the union of an AR System and the FARA method
becomes FARAIS. In simple words, FARAIS consists of an
AR system (hardware and software) which utilises FARA to
record and display AR step-by-step procedures. The inputs
(arrows in) required by FARA are the AR system outputs
(arrows out) reported at the top of Fig 4: and orientations are stored as ‘‘transform’’. A transform is
a vector which consists of the linear and angular distances
between an object and an anchor object. The third one refers
to the capability of an AR system to overlay a virtual object
on a real one following a predetermined rule. The basic rules
of alignment and scale, as well as the rendering information,
are called, in this article, the basic overlay rule. Once the
procedure is completed, the expert technician can quit the
‘‘record mode’’ through the UI. FARAIS will automatically
build the AR step-by-step ‘‘Procedure 1’’. The ‘‘How’’ is
explained in Section IV. The maintainer’s effort in creating
the AR step-by-step procedure is low as the only duty is to
press the record button and perform the maintenance proce-
dure as usual. It is worth mentioning that video recording is
performed by FARAIS. and orientations are stored as ‘‘transform’’. A transform is
a vector which consists of the linear and angular distances
between an object and an anchor object. The third one refers
to the capability of an AR system to overlay a virtual object
on a real one following a predetermined rule. The basic rules
of alignment and scale, as well as the rendering information,
are called, in this article, the basic overlay rule. 2) SCENARIO 2 – NON-EXPERT TECHNICIAN • The object recognition and tracking data In this scenario, FARAIS suggests a ‘‘step-by-step’’ AR pro-
cedure to a non-expert technician. The non-expert operator
must do a maintenance procedure that he has not done before
on the product. He/she accesses FARAIS through the hard-
ware provided. In this case, it is a head-mounted video-see-
through display. Before starting the procedure, he will select
‘‘play mode’’ and input the procedure name he wants to
perform (e.g., ‘‘Procedure 1’’). At this point FARAIS will: • Virtual object overlay basic rule • The User Kind (UK) • The Procedure Number (PN) The first input consists of the geometrical transforms in space
related to the objects in the environment. Usually, an AR
system can recognise an object and track it by estimating
its pose: the relative position and orientation of an object in
space concerning the camera. These can be translated into
a transform relative to an anchor object in the scene. The
second input consists of the basic information for overlaying
the virtual object over the real object. The third input indicates
the experience level of the operator utilising the AR system. For this study FARA only considers two levels of users: • Recognise and track the real objects in the Field-Of-
View (FOV) of the technician • Overlay the virtual objects over the real ones following
the basic overlay rule • Find ‘‘Procedure 1’’ in its DB • Show the step-by-step AR ‘‘Procedure 1’’ 8410 VOLUME 11, 2023 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 4. FARA method. The arrows in and out represent the inputs and outputs of each process. The dotted lines
refer to the pre-defined input that do not depend on any decisional choices. The firm lines are driven by decisional
choices among alternative options. FIGURE 4. FARA method. The arrows in and out represent the inputs and outputs of each process. The dotted lines
refer to the pre-defined input that do not depend on any decisional choices. The firm lines are driven by decisional
choices among alternative options. 1) PROCESSING DATAi Data processing applied by FARA in process 2∗shown in Fig TABLE 1. INclination questionnaire. TABLE 1. INclination questionnaire. clarifying that these processes are automated by selecting the
threshold of time and distance required for both the smooth-
ing and the step identification. The step information is then
stored and, together with the tracking data, represents the AR
step-by-step procedure. 2) SHOW AR PROCEDURE OVERLAY RULE This process aims to create and show the step-by-step AR
procedure created to the non-expert operator. The process for
its creation takes three inputs: • Live stream of object recognition and tracking data • Transforms table was corrected and updated with the
steps created in the process ‘‘2’’ • Virtual object basic overlay rule The first one is provided in the same format as the one
stored: transforms of the objects involved in the maintenance
procedure. This will be compared every second with the
first transform of the AR step-by-step procedure built-in
process ‘‘2’’ Fig 4. If they differ, the AR step-by-step overlay
rule will be created by gradually positioning the virtual object
on the target position. An example of the animation produced
is shown in Fig. 3. When the real object transform reaches
the correct transform, the correct transform becomes the next
row of the table, and another animation will be shown. When
all the transforms in the table related to the first step are
completed, process ‘‘3’’ will move to the next step until the
procedure is completed. IV. TEST DESIGN AND METHODOLOGY 1) PROCESSING DATAi Experienced (E) and Non-Experienced (NE). The last one is
an ‘‘id’’ used for identifying the maintenance procedure that
is going to be carried out by the maintainer. It is relevant to
note that potentially, all of them can be identified without
input from the operator. Having all these 4 inputs, FARA
will first check the procedure ‘‘id’’ and then the user expe-
rience level. Only in two cases, FARA will proceed. More
specifically, if the procedure ‘‘id’’ is not already available on
the DB and the user is experienced, FARA will go through
the processes ‘‘1∗’’, and ‘‘2∗’’ in Fig 4. This is the scenario
described in the practical example in Section III-A1. On the
other hand, if the procedure is already available on DB and
the user is non-expert, FARA will go through the process
‘‘3∗’’. This is the scenario described in Section III-A2. In the
other two possible combinations, ‘‘new procedure/non-expert
user’’ and ‘‘available procedure/expert user’’, FARA will
not go through any process. While process 1∗is commonly
utilised for software development, processes 2∗, and 3∗,
in Fig. 4, have been designed for FARA. This process modifies the data acquired by the AR system
and temporarily stored at process 1∗in Fig 4. The ‘‘raw’’
data is acquired in real-time. Whilst progressively filling
the rows of a 2D table as time advances, each row needs
object recognition and tracking information related to one of
the objects in the environment at each time, ti. FARA is a
geometrical-based method. Hence, the information utilised is
(x, y, z) positions and (α, β, γ ) rotations. As stated before,
these together build the transformed vector (x, y, z, α, β, γ ). An example of the data collected for one object within one
MRO is reported in Fig 5a. For simplicity, rotations are not
shown. The data acquired is then smoothed (Fig 5b). The
data acquisition will have different errors due to the object
recognition and tracking system. These have to be deleted or
modified to store the correct information. In this example,
due to the dimensions (distances and time) of the case, the
author used exponential smoothing, applying a threshold of
40mm and 2 seconds. It means that any transformation in 8411 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 5. A. THE QUANTITATIVE TEST This section describes the test carried out for quantifying the
time/error improvements within a maintenance procedure due
to the utilisation of FARAIS versus a hard-copy manual. The
quantitative test methodology is described in Fig 7. Starting
from the top, the participant is asked to answer a short Likert
scale inclination questionnaire in Table 1. q
A higher average score for the first three or the last
three questions corresponds to a more AR or maintenance-
oriented profile. Based on the results, the participant will
be assigned to one between the Contents Creator (CC) and
Contents Tester (CT) groups. The first is for maintenance-
oriented profiles and the second is for AR-oriented ones. In the first case, the CC is then asked to carry out different
MRO operations using a hard-copy manual. He is given an
initial time to read the manual and become confident with
the objects and then start the procedure. The observer will
measure the time and errors by filling out a pre-designed form
that lists the errors inserted in the mockup. These will provide
the ‘‘dataset1’’ regarding the hard-copy manual supported
maintenance procedure. At this point, the CC will be allowed
any amount of time until he becomes an expert in performing
the maintenance procedure. No data is collected in this phase. The CC can now utilise FARAIS described in Section III-A1
and create an AR procedure. Each CC had to create four tasks
in which each task was taken and mixed up with each task
from different CCs. In total, there were four tasks from four
different CCs to be used by each CT. If identified as CT, the
participant is firstly inducted about the AR application and
how it works and then is asked to perform the same MRO
operation carried out by CCs. During the latter, the observer
will collect the time and error data which will provide the
FARA-support maintenance procedure ‘‘dataset2’’. In both
datasets, the time has been recorded in seconds and the
number of errors has been stored. It is worth mentioning that
the MRO operations and the product to be maintained have
been designed ad-hoc. Explicit errors have been inserted in
the mock-up design for ensuring the objectivity of the data
collection. ‘‘dataset1’’ and ‘‘dataset2’’ will then be compared
to quantitatively extract eventual improvements in terms of
time-saving and number of errors. IV. TEST DESIGN AND METHODOLOGY FARA has been described in Section II. Its advantages
in terms of time-saving and low-effort requirements for
creating AR step-by-step procedures have been described
(Section III-A1). It is essential to validate if FARAIS’ step-
by-step procedures created with the method described in
Section III-B are as valuable as the contents created using
traditional methods. If so, it would be clear that FARA could
provide a step forward to ease AR implementation in Industry
and partially solve content-related issues by providing an
intuitive tool for creating AR content. The approach taken by
the authors to validate FARA’s method consists of two steps: FIGURE 5. Data processing applied by FARA in process 2∗shown in Fig 4. space smaller than 40 mm that lasted for less than 2 seconds
has been deleted since it is not considered a movement, but
a tracking error. The threshold has been selected arbitrarily
based on the author’s experience in this case. The process
can potentially be automated. Once the table is corrected,
process 2 will divide the transforms into groups to identify
the procedure steps. For splitting the steps, FARA considers
that each step is completed when the transforms of the objects
in the environment do not change for a predefined amount of
time. In this case, the minimum amount of time considered
is 2 seconds. For instance, in Fig 5c, it is possible to see that
‘‘Step 3’’ has been identified between the non-variation of
(x, y, z) after ‘‘Step2’’ until the re-stabilization of (x, y, z) that
follows the variation of y from 220mm to 10mm. It is worth • Quantification of the average time and errors improve-
ments of a maintenance procedure carried out by using a
FARAIS versus the same maintenance procedure carried
out by using a hard-copy manuali • Comparison of the results of the quantification with the
average time and error improvements of a maintenance
procedure carried out using a traditional AR system 8412 VOLUME 11, 2023 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations study is the dis/assembly maintenance of a hydraulic valve In
the selected case study, the authors designed and 3D-printed
a ‘‘maze’’ which has similar translations and rotations of its
component. The mock-up CAD model is shown in Fig 6c
and the 3D printed version is shown in Fig 6d. A. THE QUANTITATIVE TEST It has been done through
the inferential statistical analysis reported in Section V-A. The quantified results will then be compared with the ones
found in the literature related to traditional AR systems for
supporting maintenance. Results are reported qualitatively in
Section V-B. FIGURE 6. Mock-up designed for testing purposes. FIGURE 6. Mock-up designed for testing purposes. IV. TEST DESIGN AND METHODOLOGY The assembly
consists of three components: the basement, the board, and
the top. The basement has a planar maze that has to be com-
pleted by sliding (right, left, up, and down) the bottom part
of the board. Three errors have been inserted in the planar to
ease the collection of the data during the test. Once completed
the assembly of the board into the base, the top component
will be assembled into the board. It has a cylindrical maze
that has to be completed by rotating (CW or ACW) and
sliding (up and down) the top component on the top side of
the board component. Also in this, the authors inserted three
dis/assembly errors for testing purposes. These consist of the
misplacement of the assembly components concerning each
other in three different phases of the assembly. For easing
Vuforia’s object recognition, the surfaces of the objects have
been enriched with colour. versus the same maintenance procedure carried out
using a hard-copy manuali While the latter can be found in literature, the first one has
been calculated utilising the test described in the following
Section IV-A. C. FARA IMPLEMENTATION This section describes how the FARA method has been imple-
mented as a software unit in an AR system. This has been
done for validating the FARA method within this study, but
its implementation can be different in terms of Hardware and
Software for other research or industrial purposes. B. THE MAINTENANCE CASE STUDY FOR
TESTING PURPOSES This section describes the hardware of the AR system
where FARA has been implemented and become a FARAIS. These are shown in Fig 1. FARA requires hardware with In this section, the case study utilised for testing purposes is
described. The real case maintenance scenario chosen in this 8413 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 7. Schematic representation of the validation test. input/output capabilities. FARAIS has to collect data from the
proposed environment and transfer the processed information
to the operator through the output device. In this specific
case, an RGB camera and touchscreen are used as the input
device and a display as the output device; both are installed
on most of the commercial mobiles available, i.e., the Sam-
sung Galaxy S8. Moreover, the display has been fitted on
a headset developed by XVENO. It has to be mentioned
that, even though the software has been designed for these
specific input/output characteristics, FARA’s method could
be utilised with different sensors and devices. For instance,
rather than capturing the current environment with an RGB
camera, it could use a depth camera and infrared cameras. The input device could consist of a laptop or a head-mounted
display (HMD). As an example, Microsoft Hololens or Epson
Moverio HMDs could be used to meet these characteristics. As an input device, it is not unexpected that depth sensors
will be soon utilised for more efficient object recognition and
tracking. In the same way, see-through displays will soon
be preferred to the video see-through display utilised in this
example. Finally, a virtual server has been set up utilising
XAMPP. V. ANALYSIS AND RESULTS To understand whether there is a statistically significant dif-
ference between the means of timely completion of the main-
tenance tasks performed using FARAIS support vs hard-copy
manual, the author decided to carry out the one-way ANOVA
test. The two different supports, as described in section IV-A
are 1) Hard-copy manual instructions, and 2) FARAIS. The
maintenance procedures tested are: This section reports the analysis and test results described in
Section IV. Firstly, the results of the quantification test are
reported in Section V-A. Then the results of the comparison
with the current AR system’s performances in maintenance
are shown. 2) SOFTWARE The AR system software and FARA software unit have
been developed together as a tablet/mobile-based application
which mainly carries out three duties/units: FIGURE 7. Schematic representation of the validation test. • Hardware control complete and included the execution of an average of 3 ran-
domly chosen tasks per participant. The data collected has
been stored in compliance with Cranfield’s research ethics
policy. The following subsections report the statistical anal-
ysis and the results for the dependent variables ‘‘completion
time’’ and ‘‘the number of errors’’ affected by the utilisation
of the FARAIS support vs. hard-copy manual support. • Data processing and storage unit • Provide a responsive user interface (UI) It has been developed utilising Unity3d as a game engine and
Vuforia SDK and Android SDK. Moreover, a local virtual
server with an SQL DB has been set up to, not only provide
storage for the information collected but also process them
offline easing the workload of the mobile device. A. QUANTITATIVE TEST • Task 1: Assembly of the Board into the Basement This section reports the results of the quantification test which
aims to quantify the improvements due to the utilisation of
FARAIS vs hard-copy manuals as support for carrying out
MRO operations. A total of 30 participants (18m/12f) took
part in the study. These include students, staff, and industrial
personnel. The average age was 28.8 (20, 36, SD = 4.28). The
participant’s background has been assessed mainly through
the inclination questionnaire. None of the participants was
working in maintenance. Because of the first inclination ques-
tionnaire, half of them have been asked to perform the test as
CC and half as CT. Each test took from 35 to 65 minutes to • Task 2: Assembly of the Top on the Board • Task 3: Disassembly of the Top from the Board • Task 4: Disassembly of the Board from the Basement • Overall Task: The complete assembly and disassembly
of the product. (Task1 + Task2 + Task3 + Task4) The number of tasks and their characteristics has been
chosen based on the case study described in Section IV-B
and the authors’ experience. To apply ANOVA to a sam-
ple, the normality and homoscedasticity of the latter must
be validated. Even though it is generally correct to make 8414 VOLUME 11, 2023 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations TABLE 2. Homoscedasticity and normality test results for the completion
time dataset collected in the test. Both are validated. TABLE 2. Homoscedasticity and normality test results for the completion
time dataset collected in the test. Both are validated. TABLE 3. Homoscedasticity and normality test results for the number of
errors dataset collected in the test. Normality and homoscedasticity are
verified only for the overall task. TABLE 3. Homoscedasticity and normality test results for the number of
errors dataset collected in the test. Normality and homoscedasticity are
verified only for the overall task. FIGURE 8. Overall task completion time with hard-copy manual and
FARAIS support. TABLE 4. Quantitative test results summary. TABLE 4. Quantitative test results summary. completion time described in the previous section. In per-
forming the normality tests of the error datasets, it has been
found that the data collected regarding the errors performed in
the single tasks are not normally distributed because these did
not pass the normality test. B. QUANTITATIVE COMPARISON OF RESULTS This section reports the comparison between AR systems
reported in the literature and FARAIS in which the valida-
tion results are presented in Section V-A. The quantitative
performance results of FARAIS for time and error reductions
are summarized in Table 4. The results have been collected
by applying the empirical test methodology described in
Section IV and reported in detail in Section IV-C. These
can be qualitatively compared with the ones found in the
literature. The latter reports the results of the literature studies
which compared the utilisation of AR systems for supporting
maintenance (designed and tested within their projects) vs the
utilisation of hard-copy manual supports. A. QUANTITATIVE TEST As shown in Table 3, the p-values
calculated through the Shapiro-Wilk test for the errors related
to Task1, Task2, Task 3, and Task 4, are smaller than 0.05. Thus, applying the ANOVA test to single-task datasets has not
been possible. On a positive note, the overall task error dataset
resulted to be both normally distributed and homogeneous in
terms of variance. FIGURE 8. Overall task completion time with hard-copy manual and
FARAIS support. the ‘‘assume of normality’’ for relatively big-sized samples,
in this study case it is required to validate the normality. Since the sample is smaller than 50, the Shapiro-Wilk test
is carried out and each task sample results are normal since
all the p values are greater than 0.05 as shown in Table 2. The homoscedasticity of the sample has been validated by
applying the Levene test. Also, in this case, the p values
resulted are greater than 0.05 hence the samples have the
same variance. Due to this above, the ANOVA test has been performed
only for the overall task errors. The analysis of variance
showed that the effect of the support method on the overall
task number of errors is significant, F (1,28) = 30.919,
p ≤0.05 (95% confidence). Utilising FARAIS decreased the
number of errors of the overall task by 68.6% (1.53 vs 4.87)
compared to the hard-copy manual support. No sample has been removed from the dataset after these
tests. The analysis of variance showed that the effect of
the support method on the overall task completion time
is significant, F (1,28) = 32.013, p ≤0.05 (95% confi-
dence). Utilising FARAIS improves the completion time of
the overall task by 34.7% (501s vs 768s) compared to the
hard-copy manual support. Similarly, each task separately
showed improvements in time completion. More specifically:
Task1 - F (1,28)=39.793, p ≤0.05 - 55.3% (79s vs 177s). 2) NUMBER OF ERRORS The approach utilised for analyzing the number of errors
collected is the same as the one utilised for analysing the 8415 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 9. Task 1 completion time with hard-copy manual and FARAIS
support. It has to be mentioned that not all the studies which propose
an AR application for maintenance report in detail the time
and error reductions (AR vs. hard-copy manual) as well as
the insight of the methodology and testing material utilised
for collecting the data and analysing it. For instance, some
authors clearly explain the methodology utilised for vali-
dating a large-screen AR application [28]. In the study, the
independent variables have been the instruction mode (paper
vs. AR) and the task. Measures have been carried out on
completion time and errors. Still, the reader does not have
access to the complete paper instructions or AR step-by-
step procedure. What kind of information has been provided
to participants to perform the maintenance task with the
paper instructions? Was there a lot to read which could have
affected the completion time? Were the instructions clear
or this could lead to misinterpretations? How were the AR
instructions displayed? Similarly, others have outlined a vali-
dation methodology with four instruction modes utilised [29]:
printed manual, LCD instructions, HMD AR and spatially
registered AR. Still, not having access to the actual instruc-
tions makes it difficult to understand whether their structure
and contents could have affected the test results. Therefore,
it would not be accurate to quantitatively compare the liter-
ature outcomes with this study’s results which might have
used different user-AR interfaces and printed manual levels of
detail. For this reason, the authors of this study decided to do
a qualitative comparison of the results against others similarly
published research in the literature. More specifically, the
qualitative comparison is made with the eight studies which
the authors considered to have more similarities in terms of
field of application and validation procedure. FIGURE 9. Task 1 completion time with hard-copy manual and FARAIS
support. FIGURE 10. Task 2 completion time with hard-copy manual and FARAIS
support. TABLE 5. Maintenance performance improvements in terms of time and
errors reductions found in the literature. TABLE 5. Maintenance performance improvements in terms of time and
errors reductions found in the literature. FIGURE 10. 2) NUMBER OF ERRORS This method was tested by having main-
tainers perform the task in front of an AR system (therefore
among the other things capable of recognizing the objects)
to create a step-by-step procedure which can be understood
by untrained users. Ideally, if all the maintainers would use
a FARAIS, lots of step-by-step procedures could be created
without additional programming.i FIGURE 12. Task 4 completion time with hard-copy manual and FARAIS
support. 2) NUMBER OF ERRORS It must be mentioned that one limitation
of FARA and in general of AR systems will remain the
recognition of similar and symmetrical objects which have a
different internal composition (e.g., two spheres with differ-
ent weights but the same radius). Furthermore, registration
also poses an important issue in AR, particularly in handling
occlusion between real and virtual objects. In an AR envi-
ronment, improper occlusion can hinder process efficiency
rather than enhance it. The utilisation of multiple cameras
could be a potential workaround for this issue. The second
hypothesis can be considered generally true for the industrial
environment. FARA has been described as a method and its
implementation in an AR system has been named FARAIS. method but an algorithm (FARA method) that automates the
authoring process from the tracking data. The setup is pro-
gressively being reduced as the advancement in AR technolo-
gies are advancing. It must be mentioned that one limitation
of FARA and in general of AR systems will remain the
recognition of similar and symmetrical objects which have a
different internal composition (e.g., two spheres with differ-
ent weights but the same radius). Furthermore, registration
also poses an important issue in AR, particularly in handling
occlusion between real and virtual objects. In an AR envi-
ronment, improper occlusion can hinder process efficiency
rather than enhance it. The utilisation of multiple cameras
could be a potential workaround for this issue. The second
hypothesis can be considered generally true for the industrial
environment. FARA has been described as a method and its
implementation in an AR system has been named FARAIS. The intent of having two acronyms (FARA and FARAIS) is
to emphasize the difference between the method (FARA) and
its actual practical utilisation once implemented in an already
existing AR system (FARAIS: FARA Implemented System). Even if different FARAIS can be proposed (Section IV-C1),
the authors believe that this would not negatively affect the
result of the test reported in this article. The FARAIS pro-
posed in this project has been developed to comply with the
minimum requirements in terms of performance and user
interface because this project aimed to validate FARA and
not the AR system. 2) NUMBER OF ERRORS Task 2 completion time with hard-copy manual and FARAIS
support. similarities with the one proposed in this study since both
concern mechanical components with axial and rotating
movements involved. Ong et al [4] and Sanna et al [32],
had reported time reductions from 36%. The tasks involved
in their studies were notebook disassembly and inspection
operation. These hardly relate to the mockup utilised in this
work and are shown in Section IV-B. FARAIS error reduc-
tion calculated is around 70% (Table 4). The average time
reduction in Table 5 is about 72%. Not all the studies which
observed and reported the time reductions also observed or
reported the errors reductions. The systematic observation
of the errors occurring in a maintenance operation requires
a methodology which lists in detail how and when an error
should be detected. In this study the author designed in
the mock-up design, three dis/assembly errors explained in
Section IV-B. By qualitatively comparing the performance in Table 5,
it is possible to understand that the performance of FARAIS
is close to other reported AR systems in the literature. The
time reduction calculated for FARAIS is close to the average
time reduction shown in previous studies and listed in Table 5. More specifically in this study, the average time reduction
is around 36% (Table 4). The average time reduction of
referenced studies (Table 5) is 35.5%. It is interesting to
note that Fiorentino et al observed a time reduction close
to the one observed in this study [28]. It might be because
the assembly task utilised for testing purposes presents 8416 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 11. Task 3 completion time with hard-copy manual and FARAIS
support. FIGURE 13. Overall task number of errors with hard-copy manual and
FARAIS support. FIGURE 11. Task 3 completion time with hard-copy manual and FARAIS
support. FIGURE 13. Overall task number of errors with hard-copy manual and
FARAIS support. FIGURE 13. Overall task number of errors with hard-copy manual and
FARAIS support. FIGURE 11. Task 3 completion time with hard-copy manual and FARAIS
support. FIGURE 12. Task 4 completion time with hard-copy manual and FARAIS
support. method but an algorithm (FARA method) that automates the
authoring process from the tracking data. The setup is pro-
gressively being reduced as the advancement in AR technolo-
gies are advancing. VI. DISCUSSION The authors’ intent in developing FARA is to provide a
method for allowing technicians to create AR-based main-
tenance step-by-step procedures while performing the task
and with minimum effort. FARA is developed based on two
assumptions: 1) To have robust and reliable object tracking
and recognition; and 2) When CAD models are available. The
authors believe that both these assumptions will be validated
in the near future. The current research effort is working on
object recognition and tracking solutions which, through the
utilisation of new sensors and technology (depth cameras,
point cloud, etc.) will overcome current lighting, occlusions,
and background noise issues. As AR technology gets more
matured over time so does the tracking system and the use
of 3D models for visualization purposes. Therefore, there
will be no additional hardware for implementing the FARA To validate FARA, the authors quantified its per-
formance and compared it with the literature findings. 8417 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations FIGURE 14. Schematic representation of the FARA method implemented with Intelligent Learning. FIGURE 14. Schematic representation of the FARA method implemented with Intelligent Learning. The average error reduction (FARAIS vs hard-copy manual
support) is 68.6%. It has not been possible to calculate the
error reductions of the single tasks because the hypothesis of
normality and homoscedasticity were not verified. It has to
be considered, for future studies, the need of implementing
more variance in terms of errors collected. This could be
achieved by testing longer maintenance tasks or artificially
creating more tricks for the study participants. It must be
mentioned that the case study utilised for validation is limited
to an assembly and disassembly procedure and does not
validate FARP in other maintenance operations. However,
the application of the FARP method for repair, inspections,
and overhaul operations seems feasible and needs further
investigation. The FARA method can be improved to detect
assembly/disassembly errors to inform the AR user regarding
the correct procedure (e.g., misalignments). In addition, this The methodology for quantifying the time and error reduc-
tion has been explained. It is worth mentioning that
similar methodologies have been utilised by other stud-
ies [19], [28], [29]. For these reasons, even though for rel-
atively small samples a non-parametric approach such as
the Friedman test would be recommended, the authors still
preferred to assume normality and homoscedasticity based
on previous results. VI. DISCUSSION The average time reduction in perform-
ing the test’s maintenance tasks in Section V-A1 (FARAIS
vs hard-copy manual support) is 35.8%. The biggest time
reduction has been observed in Task 1, followed by Task 3,
Task 2, and Task 4. The authors believe this is because
the participants who had to perform Task 1 with the hard-
copy manual needed to take confidence in the manual itself. On the other side, the participants performing Task 1 with
FARAIS intuitively followed the instructions on the screen. 8418 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations sufficient to run the application and the transformations in
space of the objects in the FOV will be recorded and utilised
to automatically build the AR maintenance procedure. This
can then be accessed by a non- expert user to carry out the
same operation. Future studies should aim for more testing
that represents the actual industrial case study to test the
usability of the FARA method. The objective of the study
should expand validation and take more advantage of the
information recorded. The validation also needs to consider
both the learning time and the time required for creating an
AR step-by-step procedure. The first consists of the time a
maintainer needs to gain confidence with FARA and create
AR procedures. It should be compared with the time required
for learning how to use other authoring tools available. The
time required for creating AR procedures, on the other side,
should be quantified by collecting the time required when
utilising FARA and when utilising other authoring tools but
all applied to the same case study. Figure 14 shows the
planned expansion of the method. Comparing this with the
current schematic representation of FARA, it is possible to
see one new process: Machine Learning (ML) for improving
AR step-by-step procedures. Furthermore, AR should not be
limited to animation overlay only. The approach needs to be
formalized into a software package which can easily integrate
with existing AR authoring tools to enrich the informative-
ness of AR procedures. For example, automated arrows to
show the direction and rotation, text boxes for enriching each
step with descriptions, pdf links for opening manuals, and
vocal indications to support the operation. The idea should be
to utilise the data collected during any maintenance procedure
for automatically creating and/or enhancing the AR step-by-
step procedures. Data supporting this study are openly available from (CORD)
at DOI: 10.17862/cranfield.rd.21858771. Data supporting this study are openly available from (CORD)
at DOI: 10.17862/cranfield.rd.21858771. Data supporting this study are openly available from (CORD)
at DOI: 10.17862/cranfield.rd.21858771. VI. DISCUSSION By the application of intelligent learning,
FARA could potentially, classify the MRO operation steps
and propose a different solution to a similar maintenance
problem, without the need for expert training. Finally, future
works should remove the assumptions made at the outset
of this study and more validation should be performed on
various maintenance scenarios. method is currently more suited to maintenance applications
because the mechanical part is more likely to have its CAD
model available. The methodology for comparing FARA’s performance
results is not as strict as the quantifying one. This is due
to that not all the studies about AR application in mainte-
nance have as results a quantified time saving and error rate
number. Based on the author’s experience, the results of the
comparison test can still be considered satisfying. It must
be said that, for more effective validation of FARA as an
authoring method, it should have been directly compared with
the authoring methods developed by other research centres
and applied to the same case study. Unfortunately, this pro-
cess would have required, not only access to the conceptual
authoring methods but also access to the actual tool that
different research centres have utilised for the validation. This
includes software and hardware. This approach for validating
FARA seemed impractical and not suitable at this stage of the
study. Therefore, to have a comparative evaluation of the AR
system across different studies with traditional methods, there
is a necessity in creating a standard for the components to be
assembled (e.g., a mockup) and the assembly manuals for a
particular task that will be compared against an AR solution. The authors have included materials in this article that are
used to test this approach such as paper-based manuals that
describes the task being tested and 3D models. In this way,
it is possible to have a fair comparison by replicating the
task and comparing the efficacy of FARA implementation
with other types of authoring systems. Nevertheless, since
FARA implementation enables ‘‘task recording’’ for building
step-by-step instruction, it could be hypothesized that there
is much more time saving gained in comparison to any other
platforms that require time spent in programming or develop-
ing AR step-by-step instruction. VII. CONCLUSION FARA is a geometry-based method for authoring AR content
for maintenance. It is based on two assumptions: 1) machines
can recognise the objects in a working environment, and
2) CAD models are available for all the real objects involved
in the maintenance procedure. FARAIS is intrinsically not
time-consuming and not tedious since it does not require
the maintainer to do anything, but only perform the main-
tenance task. At the same time, it can provide a similar
amount of time and error reductions as other AR systems
for maintenance. For these reasons, the authors consider
FARA as a step forward in the development of authoring
solutions for AR (for maintenance but not only). It does
not require any programming skills to be operated. FARA
has been presented and its validation has been reported in
this article. The proposed method can be applied to any
AR system and has to be implemented as a software unit. To the best of the authors’ knowledge (literature and expe-
riences) it is the only method that allows users to create
AR step-by-step procedures with minimum effort. It will be RICCARDO PALMARINI RICCARDO PALMARINI received the M.Sc. degree in aircraft vehicle design from Cranfield
University and the Ph.D. degree from Cranfield
University, in 2019. He is currently working as
an Instrument and Automation Engineer at Tech-
nipFMC. His Ph.D. project focused on developing
an augmented reality (AR) based on visualization
system to support with the delivery of maintenance
and manufacturing. [13] G. Klinker, O. Creighton, A. H. Dutoit, R. Kobylinski, C. Vilsmeier, and
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pp. 657–679, 2012. [15] C. Knopfle, J. Weidenhausen, L. Chauvigne, and I. Stock, ‘‘Template based
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Authoring tool for mixed reality contents,’’ in Proc. IEEE Int. Symp. Mixed
Augmented Reality, Oct. 2010, pp. 219–220. IÑIGO FERNÁNDEZ DEL AMO received the
degree in industrial engineering from the Poly-
technic University of Madrid (UPM), in 2016, the
degree in manufacturing consultancy from Cran-
field University, in 2016, and the Ph.D. degree
from Cranfield University. He received the schol-
arship from Babcock International for his Ph.D. degree. His primary interest is to study innovative
software methods to capture maintenance experts’
knowledge and reduce the cost of AR’s industrial
implementation. Besides his Ph.D. research, he has co-supervised various
M.Sc. thesis and group projects that involve the implementation of AR and
digital twins in the industrial workplace using SW technologies. His overall
aim is to enable the transformation of operative roles into a source of valuable
information for through-life engineering services using innovative visualisa-
tion and data-structuring technologies. His research interests include the use
of augmented reality (AR) and semantic web (SW) for knowledge capture in
maintenance diagnosis. He won the 2017 Cranfield Vice-Chancellor’s Prize
for being the most outstanding M.Sc. student. [17] J. Shim, M. Kong, Y. Yang, J. Seo, and T.-D. Han, ‘‘Interactive features
based augmented reality authoring tool,’’ in Proc. IEEE Int. Conf. Consum. Electron. (ICCE), Jan. 2014, pp. 47–50. [18] Y. Yang, J. Shim, S. Chae, and T.-D. REFERENCES [1] P. Milgram and F. Kishino, ‘‘A taxonomy of mixed reality visual displays,’’
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Jan. 2017. 8419 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations [6] F. Lamberti, F. Manuri, A. Sanna, G. Paravati, P. Pezzolla, and
P. Montuschi, ‘‘Challenges, opportunities, and future trends of emerg-
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pp. 95–102. RICCARDO PALMARINI Han, ‘‘Interactive augmented reality
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system to assist the maintenance operators,’’ Int. J. Interact. Des. Manuf. (IJIDeM), vol. 8, no. 4, pp. 293–304, Nov. 2014. [22] H. Alvarez, I. Aguinaga, and D. Borro, ‘‘Providing guidance for mainte-
nance operations using automatic markerless augmented reality system,’’
in Proc. 10th IEEE Int. Symp. Mixed Augmented Reality, Oct. 2011,
pp. 181–190. DEDY ARIANSYAH DEDY ARIANSYAH worked previously as a
Research Fellow at Cranfield University, under
the EPSRC-funded project DigiTOP, where his
research focused on how immersive technologies
like augmented reality can improve industrial pro-
ductivity and digital twin development to sup-
port decision-making. He is currently involved
in the AI Research Team, where he focuses on
the embedded AI in immersive technologies and
knowledge capture to improve task productivity,
increase the efficiency of knowledge transfer, and data-driven process opti-
mization. His Ph.D. degree was focused on the virtual reality driving
simulator for driver behaviour analysis, where he developed and integrated
physiological data and driving performance to study different human cogni-
tive operations when interacting with driver assistance systems. [23] T. V. Nguyen, B. Mirza, D. Tan, and J. Sepulveda, ‘‘ASMIM: Aug-
mented reality authoring system for mobile interactive manuals,’’ in
Proc. 12th Int. Conf. Ubiquitous Inf. Manag. Commun., Jan. 2018,
pp. 1–6. [24] T. V. Nguyen, D. Tan, B. Mirza, and J. Sepulveda, ‘‘MARIM: Mobile
augmented reality for interactive manuals,’’ in Proc. 24th ACM Int. Conf. Multimedia, Oct. 2016, pp. 689–690. [25] M. E. C. Santos, A. Chen, T. Taketomi, G. Yamamoto, J. Miyazaki,
and H. Kato, ‘‘Augmented reality learning experiences: Survey of proto-
type design and evaluation,’’ IEEE Trans. Learn. Technol., vol. 7, no. 1,
pp. 38–56, Jan. 2014. [26] J. Wang, Y. Feng, C. Zeng, and S. Li, ‘‘An augmented reality based
system for remote collaborative maintenance instruction of complex prod-
ucts,’’ in Proc. IEEE Int. Conf. Autom. Sci. Eng. (CASE), Aug. 2014,
pp. 309–314. 8420 VOLUME 11, 2023 R. Palmarini et al.: FARA: Fast Creation of AR Step-by-Step Procedures for Maintenance Operations RAJKUMAR
ROY
received the B.Eng. and
M.Eng. degrees in production engineering from
Jadavpur University, India, and the Ph.D. degree
in computing from the University of Plymouth,
U.K. He started his career as an Engineer at Tata
Motors; pioneered research in Through-life Engi-
neering Services (TES) with Rolls-Royce, BAE
Systems, Bombardier Transportation, the Min-
istry of Defence, and Babcock International; and
established an internationally known TES Centre. He joined the City, University of London from Cranfield University, where
he was the Director of Manufacturing. His cost engineering and obsolescence
research has transformed contemporary understanding of the engineering
effort required to design, make and support high-value products, resulting in
tools used by BAE Systems, Airbus, the Ministry of Defence, Rolls-Royce,
and Ford Motor Company. DEDY ARIANSYAH He is the Founding Editor-in-Chief of the Applied
Soft Computing journal (Elsevier); and a fellow of CIRP (International
Academy for Production Engineers), the Institute of Engineering Designers
(IED), and the Higher Education Academy (HEA). SAMIR KHAN (Member, IEEE) received the
B.Eng. and Ph.D. degrees from Loughborough
University. He is currently a Senior Lecturer
often collaborating with Rolls-Royce, Jaguar Land
Rover, BAE Systems, and MoD. His research
interests include intelligent monitoring, AI-based
control, perception, and planning in the context
of autonomous systems and service robots, with a
focus on the embedded hardware implementation
of machine learning techniques for anomaly detec-
tion and prognostics and system health management, development of the IoT
technologies, digital twins for maintenance decision making, and intelligent
monitoring of intermittent failures and false alarms in electronic systems. He is a Chartered Engineer and a member of IET. JOHN AHMET ERKOYUNCU received the Ph.D. degree in uncertainty modeling for maintenance
from Cranfield University, in 2011. He is currently
the Head of the Digital Engineering and Manu-
facturing Centre. He is active with Innovate U.K. and EPSRC-funded projects around research top-
ics: digital twins, augmented reality, digitization
(degradation assessment), and simulation of com-
plex manufacturing and maintenance procedures. He is a Chartered Engineer, an Associate Member
of CIRP, and the Co-Chair of the Through-life Engineering Services Council. 8421 VOLUME 11, 2023
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English
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The artists’ materials of P. S. Krøyer: An analytical study of the artist’s paintings and tube colours by Raman, SEM–EDS and HPLC
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Heritage science
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Abstract An analytical study of two paintings by P. S. Krøyer and the tube colours found in his painting cabinet has been carried
out in order to investigate the artists’ materials used by Krøyer in the latter part of his oeuvre. The study has given a
valuable knowledge about Krøyer’s choice of materials and their composition that may be helpful in e.g. preservation
issues. The pigments and dyes were analysed by micro-Raman spectroscopy (MRS), scanning electron microscopy-
energy dispersive X-ray spectroscopy (SEM–EDS), and high-performance liquid chromatography with diode-array
detection (HPLC–DAD). The technical analyses were supplemented with archival studies of Danish nineteenth century
paint shops. The results show that Krøyer used commercially available artists’ materials typical for that period in time. Being a wealthy artist, he seems to have favoured expensive pigments such as cobalt blue, viridian and cadmium yel-
low. Based on the analyses, the main development in Krøyer’s palette is the substitution of lead white with zinc white
that appears to have taken place after 1886. The results also show that the contents of the tube colours in some cases
differ from what was expected based on the trade names. This is particularly observed for the tube colours named
“Oxide rouge” of which only one contains the expected hematite pigment, while all the remaining contain cadmium
sulfide and natural organic red lakes. A rare tube colour named “Laque verte clair” was characterised to be mixture
of Prussian blue and synthetic Indian yellow. The study therefore also illustrates that it was not always obvious to the
artists what they bought from their colour merchant. It hence relates to existing international studies of nineteen
century artists’ materials, which highlight the complexity of tube colours from this particular period in time. Keywords: P. S. Krøyer, Paintings, Tube colours, Trade names, Pigments, Dyes with the tobacco manufacturer and art collector Heinrich
Hirschsprung (1836–1908), secured Krøyer a consider-
able income and made him a prosperous painter [1, 2]. With Hirschsprung’s support Krøyer was able to make
numerous study trips around Europe, which came to be
of great influence on his career. In particular the grand
tour of 4 years from 1877 to 1881 marked a turning point
in his artistic style and working methods. During this
tour Krøyer stayed for longer periods in Paris, where he
attended classes at the atelier of the French artist Léon
Bonnat (1833–1922) [3, 4]. © 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. Christiansen et al. Herit Sci (2017) 5:39
DOI 10.1186/s40494-017-0153-2 Christiansen et al. Herit Sci (2017) 5:39
DOI 10.1186/s40494-017-0153-2 Open Access Abstract After his return to Denmark
Krøyer began to spend the summer months working in
Skagen, a small fishing village in the northernmost part of
Denmark that became a meeting point for Nordic artists
during the 1880s [3]. The Skagen painters, as they came *Correspondence: kps@kadk.dk
1 School of Conservation, Esplanaden 34, 1263 Copenhagen K, Denmark
Full list of author information is available at the end of the article © 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. Background Peder Severin Krøyer (1851–1909), known as P. S. Krøyer,
is one of the most famous Danish painters, and a central
figure in the Nordic artists’ colony established in Ska-
gen at the end of the nineteenth century. Krøyer gradu-
ated in 1870 from the The Royal Danish Academy of Fine
Arts, and early in his career he proved to have a unique
talent as a portrait painter. His oeuvre counts numerous
portrayals of scenes from local life and social gatherings,
as well as commissioned portraits of prominent figures
in the industry and the cultural life [1–3]. The commis-
sioned works, along with his patronage and friendship *Correspondence: kps@kadk.dk
1 School of Conservation, Esplanaden 34, 1263 Copenhagen K, Denmark
Full list of author information is available at the end of the article Christiansen et al. Herit Sci (2017) 5:39 Page 2 of 11 Fig. 1 Top P. S. Krøyer. By the fireside. Portrait of Holger Drachmann
with a red fez, 1903–1907. Oil on canvas, 106 × 146 cm. Art Museums
of Skagen. Photo: Art Conservation Center. Bottom Michael Ancher. Krøyer painting Drachmann in the latters atelier, 1907. Oil on canvas,
140 × 100 cm. Art Museums of Skagen. Photo: Art Museums of
Skagen to be known, found inspiration in the surrounding nature
and local population in Skagen, and benefitted from the
social and professional community among the artists. Their motives were most often painted “en plein air” with
a clear influence from Naturalism and Realism. Today
extensive collections of Krøyer’s paintings are exhibited
at the Art Museums of Skagen, and at The Hirschsprung
Collection in Copenhagen [1, 3]. The latter is also exhib-
iting Krøyer’s painting cabinet. g
y
g
Despite Krøyer’s recognition as an artist, very little
is known about his painting technique and the artists’
materials he used. Gaining such knowledge is essential
in understanding how the works of art were made and
how to preserve them in the best possible way. The
current study is based on a technical investigation of
P. S. Krøyer’s painting technique and artists’ materi-
als, which included an examination of two of Krøyer’s
paintings and the contents of the artist’s painting cabi-
net. No comprehensive study on this subject has hith-
erto been carried out, and this is the first paper giving
an insight into the tube colours used by Krøyer [4]. The analyses were carried out on samples taken from
the two paintings as illustrated in Figs. 1 and 2, and on
samples taken from the tube colours. Analyses of cross
sections of the paint samples were performed by SEM–
EDS and Raman spectroscopy in order to obtain infor-
mation about the pigments used. Samples from the
tube colours were analysed by SEM–EDS and Raman
for identification of the pigments, and by HPLC for
identification of the dyes. Fig. 1 Top P. S. Krøyer. By the fireside. Portrait of Holger Drachmann
with a red fez, 1903–1907. Oil on canvas, 106 × 146 cm. Art Museums
of Skagen. Photo: Art Conservation Center. Bottom Michael Ancher. Krøyer painting Drachmann in the latters atelier, 1907. Oil on canvas,
140 × 100 cm. Art Museums of Skagen. Photo: Art Museums of
Skagen The two paintings, By the fireside. Portrait of Holger
Drachmann with a red fez (hereinafter, Drachmann) and
Christoffer mending nets in his living room (hereinafter,
Christoffer), were painted by Krøyer in Skagen in 1903–
1907 and 1886 respectively, and hence belong to the latter
part of his oeuvre. Drachmann (Fig. 1, top) is one of many
portrayals Krøyer did of his friend, the Danish writer and
painter Holger Drachmann (1848–1908) [1]. Krøyer’s
friend and fellow Skagen-painter Michael Ancher (1849–
1927) depicted Krøyer while working on the final stages
of the painting (Fig. 1, bottom). It took Krøyer 4 years to
complete the large portrait, which was unusual given the
fact that Krøyer was known for his ability to work rapidly. At the time being though, he was struggling with mental
illness, and was hospitalised several times [1]. In contrast,
the earlier portrait Christoffer from 1886 (Fig. 2, left) is an
example of a rapidly executed painting, depicting a local
fisherman working in a dark interior. The portrait clearly
shows the influence of the realistic model painting and
the concept “Valeur”, to which Krøyer was introduced
during his stay at Léon Bonnat’s atelier. Krøyer gave the
portrait as a gift to his friend, the French painter Ernest Baillet (1853–1902) as evidenced by the inscription in
the upper left corner (Fig. 2, right). Both of the paintings
are today a part of the collection at The Art Museums of
Skagen.hi The painting cabinet (Fig. 3) has been a fixture in
Krøyer’s ateliers in Copenhagen and Skagen, and
it contains, among other things, 32 well preserved
tube colours presumably dating from the latter part
of the nineteenth century. Today it belongs to the
Hirschsprung Collection who received it in 1911 as a
gift from Krøyer’s estate. Experimental Description of the paintings and tube coloursh p
p
g
The paintings investigated are depicted in Figs. 1 and 2
and are: By the fireside. Portrait of Holger Drachmann
with a red fez (1903–1907), and Christoffer mending nets
in his living room (1886). The locations where the samples
were taken are shown in Figs. 1 and 2. For ethical reasons Christiansen et al. Herit Sci (2017) 5:39 Page 3 of 11 Fig. 2 Left P. S. Krøyer. Christoffer mending nets in his living room, 1886. Oil on canvas, 47 × 39 cm. Art Museums of Skagen. Right detail of signature
and dedication in the upper left corner Fig. 2 Left P. S. Krøyer. Christoffer mending nets in his living room, 1886. Oil on canvas, 47 × 39 cm. Art Museums of Skagen. Right detail of signature
and dedication in the upper left corner Fig. 2 Left P. S. Krøyer. Christoffer mending nets in his living room, 1886. Oil on canvas, 47 × 39 cm. Art Museums of Skagen. Right detail of signature
and dedication in the upper left corner samples were only obtained from the tacking edges and
from areas with paint losses, hence not all colours could
be investigated. The results of the paintings analysed are
given in Table 1. Table 2 lists the trade names and brands of the 32
tube colours found in Krøyer’s painting cabinet (Fig. 3). A detailed description of the tube colours is given in
“Results and discussion” (tube colours). Fig. 3 P. S. Krøyer’s painting cabinet. The Hirschsprung collection Fig. 3 P. S. Krøyer’s painting cabinet. The Hirschsprung collection Christiansen et al. Herit Sci (2017) 5:39 Page 4 of 11 Table 2 List of trade names, durability, brands of the tube
colours, and number of tubes
The trade names listed are the English translations of the Danish names. The
original French or Italian names on the labels are noted in brackets. * Designates
tubes described as “slowly drying” on the label. The tube colours from Vilhelm
Pacht are listed with the designation HH (completely durable), H (durable) or
without a letter (uncertain) based on the information found in the supplier’s
price lists
Trade name
Brand
Nos. Cadmium yellow dark (Jaune de cadmium
foncé) HH
Vilhelm Pacht
2
Cadmium yellow light (Jaune de cadmium
clair) HH
Vilhelm Pacht
2
Brilliant yellow (Jaune brillant jaunâtre) HH
Vilhelm Pacht
2
Cadmium lemon yellow (J. Experimental de cadmium
citron) H
Vilhelm Pacht
1
Chromium yellow light (Jaune de chrome
clair)
Vilhelm Pacht
1
Viridian (Vert émeraude) HH
Vilhelm Pacht
2
Viridian (Vert émeraude)* HH
Vilhelm Pacht
2
Chrome green light (Vert de chrome clair)
HH
Vilhelm Pacht
1
Chrome green medium (Vert de chrome
moyen) HH
Vilhelm Pacht
1
Cobalt blue (B. de cobalt vivace surfin) HH
Vilhelm Pacht
2
Ivory black (Noir d’ivoire) HH
Vilhelm Pacht
3
Snow white (Blanc de neige)* HH
Vilhelm Pacht
1
Green lake light (Laque verte clair)
Hermann Averhoff
1
Madder lake golden rose (Laque garance
rose doré)* H
Vilhelm Pacht
1
Madder lake deep rose (Laque g. rose
intense) HH
Vilhelm Pacht
2
Madder lake rose (Laque de garance rose)
Felice Alman (Torino)
1
Carmine lake ordinary (Laque carminée
ordinaire)
Lefranc et Cie (Paris)
1
Red oxide (oxide rouge) H/HH
Vilhelm Pacht
1
Red oxide (oxide rouge)
Hermann Averhoff
5 Table 1 Pigments and fillers identified by SEM–EDS
and Raman in the ground layer and paint layers of the two
paintings; a: Drachmann, and b: Christoffer
() Designates that the pigment was identified in a very limited extent, and ? that the result is uncertain. The sample numbers refer to the locations shown in
Figs. 1 and 2
Pigment/filler
a
b
Sample number
Ground layer
Lead white
+
+
All samples
Calcite
+
+
All samples
Aluminium silicate
+
+
S1–S5, S8–S13
Paint layers
Lead white
(+)
+
(S2–S4), S5, S9–S13
Zinc white
+
S1–S8
Ochre
+
S9?, S12, S13
Cadmium yellow
+
+
S1–S5, S8, S9
Chromium orange
+
S2, S3, S5
Vermilion
+
+
S1–S6, S8–S11, S13
Minium
+
S2, S3, S5
Alizarin
+
S2, S3, S5
Cobalt blue
+
+
(S2), S3–S7, S10, S11, S13
Ultramarine
+
S10
Viridian
+
+
S5, S6, S8, S9
Carbon black
+
+
S3, S10, S13 Table 1 Pigments and fillers identified by SEM–EDS
and Raman in the ground layer and paint layers of the two
paintings; a: Drachmann, and b: Christoffer Table 2 List of trade names, durability, brands of the tube
colours, and number of tubes () Designates that the pigment was identified in a very limited extent, and ? that the result is uncertain. The sample numbers refer to the locations shown in
Figs. 1 and 2 Cross sections Paint samples taken from the two paintings during con-
servation were made into cross sections by embedding
in Technovit 2000 LC light curing resin placed in Easy-
Sections. The resin was cured for 10 min with blue light
under a flow of nitrogen gas in a light-curing oven (Egger
EL 1 plus N2). Finally the cross sections were polished
wet with silicon carbide and then dry with Micro-Mesh
(granularity 2400–12,000). The trade names listed are the English translations of the Danish names. The
original French or Italian names on the labels are noted in brackets. * Designates
tubes described as “slowly drying” on the label. The tube colours from Vilhelm
Pacht are listed with the designation HH (completely durable), H (durable) or
without a letter (uncertain) based on the information found in the supplier’s
price lists The trade names listed are the English translations of the Danish names. The
original French or Italian names on the labels are noted in brackets. * Designates
tubes described as “slowly drying” on the label. The tube colours from Vilhelm
Pacht are listed with the designation HH (completely durable), H (durable) or
without a letter (uncertain) based on the information found in the supplier’s
price lists Optical microscopyh adhesive carbon tabs using a Hitachi S-3400 N scanning
electron microscope with an energy dispersive X-ray
spectrometer (SEM–EDS) operating at an acceleration
voltage of 20 kV and low vacuum mode at 25 Pa. The EDS
system, QUANTAX 200, was equipped with two 30 mm2
SDD X-ray detectors (XFlash® 6|30 Silicon Drift Detec-
tor) from Bruker instruments. The samples and cross sections were studied with an
optical microscope Axio Imager M1 (Carl Zeiss) under
polarised white light and ultraviolet radiation with mag-
nifications between 100 and 500× and the use of Del-
taPix Insight. Raman MRS analyses were carried out with a Renishaw inVia
dispersive micro-Raman spectrometer equipped with a
785 nm laser. The laser power was kept between 0.1 and
1 mW by neutral density filters to avoid thermal degrada-
tion. A detailed description can be found in ref [5]. ATR‑FTIR Attenuated total reflectance-Fourier transform infrared
spectroscopy (ATR-FTIR) was used as supporting tech-
nique. ATR-FTIR spectra were recorded on a Perkin
Elmer Spectrum One FTIR-spectrometer, fitted with a
Universal ATR sampling accessory having a one-bounce
composite zinc selenide and diamond crystal. Spectra of
the samples were recorded over the range 4000–650 cm−1
with a resolution of 4 cm−1 and 4 accumulations. Results and discussion
Paintingsh The results from the pigment analysis carried out on sam-
ples taken from the two paintings are listed in Table 1. In
the table a distinction is made between pigments found
in the ground and paint layers, and the two paintings are
noted with the letter a or b, designating the portrait of
Drachmann and the portrait of Christoffer, respectively. Dark blue grains of cobalt blue were likewise consist-
ent in the samples taken from the two paintings. In one
of the samples from of the portrait of Christoffer, cobalt
blue was found in a mixture with minor amounts of syn-
thetic ultramarine, the latter identified by Raman (Fig. 5e)
[11]. The sample was taken from the dark paint layers in
the background (S10), and the ultramarine pigment was
possibly applied as a substitute for black similar to the
practice known from impressionist painters [11]. Krøyer
did not, however, avoid using regular black pigments, and
carbon black was identified in both paintings by its char-
acteristic broad Raman-bands (Fig. 5f) [8]. f
Both paintings were painted on a commercially pre-
pared canvas with a pale white ground containing a
mixture of lead white, calcite and silicate. Krøyer gener-
ally bought his canvases ready-primed [4]. In the case of
Christoffer, a canvas stamp from the paint retailer Kul-
torvets Farvehandel was found on the back of the can-
vas (Fig. 4, left). The company was established in 1879
at Kultorvet in central Copenhagen, and was run by the
founder F. E. Kühle until 1890 when it was taken over by
H. Averhoff [7]. A later company label from Averhoff’s
time was seen on the stretcher in the portrait of Drach-
mann (Fig. 4, right), indicating that Krøyer could have
bought his canvases and stretchers from this particular
supplier over a longer period of time. The most notable difference in the palette used for the
two paintings was seen in the white pigments. While
Krøyer used lead white in the portrait of Christoffer, he
mainly used zinc white in the later portrait of Drach-
mann. Zinc oxide was identified in all paint layers in
the Drachmann portrait by SEM–EDS, and by optical
microscopy due to the pigment’s fluorescence under UV
(Fig. 7) [12]. HPLC–DAD pigments were identified by SEM–EDS. Regarding the
green and blue pigments, Krøyer seems to have favoured
the expensive pigments viridian and cobalt blue, since
these were widely represented in the analysed sam-
ples from both paintings. An example of Krøyer’s use
of viridian is given in Fig. 6, where a sample taken from
the Drachmann portrait is depicted. In polarised light
viridian was seen as a deep dark green pigment in the
upper paint layer, and in SEM the characteristic irregu-
lar rounded shape of the pigment grains was clearly vis-
ible [10]. SEM–EDS analysis furthermore showed how
the pigment was mixed with barium sulfate, probably in
order to make it easier to grind and disperse [10]. HPLC analyses were carried out after mild HF extrac-
tion (2 M aqueous HF/dimethylformamide/acetoni-
trile = 2/1/1). The HPLC–DAD system used for the dye
analyses was a Spetra-SYSTEM from ThermoScientific,
controlled by ChromQuest 4software. A detailed descrip-
tion can be found in ref [6]. HPLC analyses were carried out after mild HF extrac-
tion (2 M aqueous HF/dimethylformamide/acetoni-
trile = 2/1/1). The HPLC–DAD system used for the dye
analyses was a Spetra-SYSTEM from ThermoScientific,
controlled by ChromQuest 4software. A detailed descrip-
tion can be found in ref [6]. SEM–EDS SEM–EDS analyses were carried out on cross sections
and paint samples placed on double sided conductive Page 5 of 11 Christiansen et al. Herit Sci (2017) 5:39 Results and discussion
Paintingsh Analyses carried out on two of Krøyer’s early
oil sketches from 1875 and 1876 (not shown) showed that
lead white was used in the paint layers in both paintings,
while zinc white was not. This seems to suggest that zinc
white was introduced on Krøyer’s palette somewhere
between 1886 where he finished the portrait of Christof-
fer, and 1903 where he begun painting the Drachmann
portrait. The palette used for the two paintings consisted of a
mixture of traditional pigments and modern pigments
developed in the eighteenth and nineteenth century. An example was seen in the use of red, orange and yel-
low pigments in the portrait of Drachmann, where tra-
ditional pigments and dyes like vermilion, minium and
alizarin were used in combination with modern pigments
like chromium orange and cadmium yellow. Vermilion,
minium, alizarin, and chromium orange were identified
by Raman (Fig. 5a–d) [8, 9], while the cadmium sulfide Fig. 4 Left canvas stamp from Kultorvets Farvehandel (F. E. Kühle) on the back of the portrait of Christoffer. Right label from Kultorvets Farvehandel
(H. Averhoff) on the stretcher used for the portrait of Drachmann Christiansen et al. Herit Sci (2017) 5:39 Page 6 of 11 Fig. 5 Raman spectra of pigments identified in the two paintings Drachmann (a–d), and Christoffer (e–f). The spectra depicted are a vermilion, b
minium, c alizarin, d chromium orange, e ultramarine, and f carbon black. The spectra were assigned by comparison of the spectra to those in the
literature [8, 9] Fig. 5 Raman spectra of pigments identified in the two paintings Drachmann (a–d), and Christoffer (e–f). The spectra depicted are a vermilion, b
minium, c alizarin, d chromium orange, e ultramarine, and f carbon black. The spectra were assigned by comparison of the spectra to those in the
literature [8, 9] Fig. 5 Raman spectra of pigments identified in the two paintings Drachmann (a–d), and Christoffer (e–f). The spectra depicted are a vermilion, b
minium, c alizarin, d chromium orange, e ultramarine, and f carbon black. The spectra were assigned by comparison of the spectra to those in the
literature [8, 9] Fig. 5 Raman spectra of pigments identified in the two paintings Drachmann (a–d), and Christoffer (e–f). The spectra depicted are a vermilion, b
minium, c alizarin, d chromium orange, e ultramarine, and f carbon black. Results and discussion
Paintingsh The light blue paint layer (2) above the iso-
lation layer (1) and ground layer (0) contains mainly zinc white and cobalt blue. Due to the presence of zinc white, the paint layer fluoresces when
exposed to UV light Fig. 7 Detail of cross section S7 from Drachmann depicted in polarised light (left) and in UV light (right). The light blue paint layer (2) above the iso-
lation layer (1) and ground layer (0) contains mainly zinc white and cobalt blue. Due to the presence of zinc white, the paint layer fluoresces when
exposed to UV light Fig. 8 FTIR spectrum of a paint sample from the Drachmann portrait
identifying zinc carboxylates in the paint layers colour merchant Hermann Averhoff at the shop Kultor-
vets Farvehandel, where he also bought canvases (Fig. 4). Only two of the tube colours found in Krøyer’s paint-
ing cabinet were from foreign companies, these being
respectively the French Lefranc et Cie and the Italian
Felice Alman. An investigation of price lists from Danish nineteenth
century paint shops was carried out in the archives of the
Royal Library of Denmark. Based on the archival infor-
mation found on trade names and brands, it seems likely
that the majority of the tube colours in Krøyer’s paint-
ing cabinet were bought in the last decade of the nine-
teenth century or later. The trade names on the tube
colours were in most cases written on the label in both
Danish and French. While some were rather simple and
well-known names like “Cadmium yellow”, “Chromium
yellow” and “Cobalt blue”, others like “Jaune Brilliant”,
“Ivory black” and “Snow white” were more fanciful, but
still common, examples of trade names used for artists’
colours in the nineteenth century [17–19]. Finally there
were examples of trade names like “Green lake light”,
“Red oxide”, and “Cadmium lemon yellow”, where the
results showed no clear correlation between trade name
and contents. Fig. 8 FTIR spectrum of a paint sample from the Drachmann portrait
identifying zinc carboxylates in the paint layers vibrations. Compared to reference spectra for artifi-
cially produced zinc carboxylates i.e. zinc stearate, zinc
palmitate, and zinc oleate the carboxylate stretchings
are shifted towards higher wavenumbers. However, this
is often the case for zinc carboxylates formed in actual
paint layers where mixtures of both saturated and unsat-
urated zinc soaps are formed [15]. Results and discussion
Paintingsh The spectra were assigned by comparison of the spectra to those in the
li
[8 9] Fig. 6 Detail of cross section S8 from Drachmann depicted in polarised light (left) and in SEM (right). A green paint layer containing viridian (3) is
seen above a white paint layer (2), an isolation layer (1) and a white ground layer (0). In the SEM-image a section of the green paint layer is seen with
arrows pointing to the rounded shape of the viridian pigment grains Fig. 6 Detail of cross section S8 from Drachmann depicted in polarised light (left) and in SEM (right). A green paint layer containing viridian (3) is
seen above a white paint layer (2), an isolation layer (1) and a white ground layer (0). In the SEM-image a section of the green paint layer is seen with
arrows pointing to the rounded shape of the viridian pigment grains Fig. 6 Detail of cross section S8 from Drachmann depicted in polarised light (left) and in SEM (right). A green paint layer containing viridian (3) is
seen above a white paint layer (2), an isolation layer (1) and a white ground layer (0). In the SEM-image a section of the green paint layer is seen with
arrows pointing to the rounded shape of the viridian pigment grains Zinc white has previously been linked to a number of
conservation issues due to the pigment’s strong tendency
to form zinc carboxylates [13–15]. We therefore inves-
tigated the formation of zinc soap. By FTIR analysis of
samples taken from the Drachmann portrait zinc soap
formation as a result from reaction between the oil binder
and the zinc oxide pigment was clearly seen (Fig. 8). The stretching vibrations at 2919 and 2850 cm−1, and the
bending vibration at 1458 cm−1 are assigned to the long
chained CH2 groups, and the vibration at 1739 cm−1 to
the C=O stretching from the oil binder. The strong band
at 1548 cm−1 and the shoulder at 1593 cm−1 are assigned
to asymmetric COO− stretching vibrations, and the split
band around 1412 cm−1 to symmetric COO− stretching Christiansen et al. Herit Sci (2017) 5:39 Page 7 of 11 Fig. 7 Detail of cross section S7 from Drachmann depicted in polarised light (left) and in UV light (right). Results and discussion
Paintingsh An overview of the results from the analysis carried
out on the contents of the tube colours is presented
in Table 3. In many cases the trade name on the label
proved to correspond to the pigment(s) found in the tube
colour. An example was the analysis of the tube colour
named “Chromium yellow light”, where Raman identi-
fied chrome yellow as the primary pigment (Fig. 9a) [8,
9]. The small band seen at 1054 cm−1 in the Raman spec-
trum can be assigned to the presence of lead carbonate or
basic lead carbonate [9], which suggests that lead white
was added to the colour in order to obtain a lighter shade
of yellow. This, however, appears to be unusual since lead Tube colours
Th The tube colours from
Vilhelm Pacht were ranked in the price lists (Table 2)
as being either completely durable (HH), durable (H)
or uncertain (without a letter). sulfate has traditionally been the most common addition
to colours based on chrome yellow [20]. The tube colour
containing chrome yellow and the tube colour labelled
“Cadmium lemon yellow” which contained the extremely
rare cadmium chromate pigment [21], were the only two
tube colours where chromate pigments were found. All
the remaining yellow tube colours were cadmium sulfide
based colours. To what extent Krøyer has used chromate
pigments is unclear based on the two paintings analysed. Chromium orange, PbCrO4·PbO, was found in three
samples from the Drachmann portrait (S2, S3 and S5),
but a tube colour containing this pigment was not pre-
sent in the painting cabinet. Thus, more paintings have to
be investigated in order to find out, which role chromate
pigments played in Krøyer’s palette. The instability of the
chromate pigments was already in the nineteenth century
well known among artists [22–24], which makes it a pos-
sibility that Krøyer deliberately avoided using them to a
greater extent in his paintings. sulfate has traditionally been the most common addition
to colours based on chrome yellow [20]. The tube colour
containing chrome yellow and the tube colour labelled
“Cadmium lemon yellow” which contained the extremely
rare cadmium chromate pigment [21], were the only two
tube colours where chromate pigments were found. All
the remaining yellow tube colours were cadmium sulfide
based colours. To what extent Krøyer has used chromate
pigments is unclear based on the two paintings analysed. Chromium orange, PbCrO4·PbO, was found in three
samples from the Drachmann portrait (S2, S3 and S5),
but a tube colour containing this pigment was not pre-
sent in the painting cabinet. Thus, more paintings have to
be investigated in order to find out, which role chromate
pigments played in Krøyer’s palette. The instability of the
chromate pigments was already in the nineteenth century
well known among artists [22–24], which makes it a pos-
sibility that Krøyer deliberately avoided using them to a
greater extent in his paintings. Among the 32 tube colours in Krøyer’s painting cabi-
net, 6 tubes contained lake pigments indicated by their
trade names. Tube colours
Th The vast majority of the tube colours were produced by
the Danish colour merchant Vilhelm Pacht, whose col-
ours, among others, were also used by Edvard Munch
[16]. In the late nineteenth and early twentieth century
the tube colours from Vilhelm Pacht were mainly retailed
by the large paint shop A. Stelling, established in cen-
tral Copenhagen in 1860 [7]. Besides from Pacht, Krøyer
also bought tube colours from the competing Danish Christiansen et al. Herit Sci (2017) 5:39 Page 8 of 11 Table 3 Pigments, fillers and dyes identified in the tube colours by Raman, SEM–EDS, and HPLC
* Designates tubes described as “slowly drying” on the label
Trade name (Brand)
Nos. Raman/SEM–EDS
HPLC
Cadmium yellow dark (Pacht)
2
CdS orange
Cadmium yellow light (Pacht)
2
CdS yellow
Brilliant yellow (Pacht)
2
CdS yellow, ZnO
Cadmium lemon yellow (Pacht)
1
KCd2(CrO4)2(H3O2)
Chromium yellow light (Pacht)
1
PbCrO4, Pb3(CO3)(OH)2
Viridian (Pacht)
2
Cr2O3·2H2O, Al2O3
Viridian* (Pacht)
2
Cr2O3·2H2O, BaSO4, Si, carbon black
Chrome green light (Pacht)
1
Cr2O3·2H2O, CdS, ZnO, BaSO4
Chrome green medium (Pacht)
1
Cr2O3·2H2O, CdS, KZn2(CrO4)2(H3O2), ZnO, BaSO4
Cobalt blue (Pacht)
2
CoAl2O4·Al2O3
Ivory black (Pacht)
3
C,Ca5(PO4)3(OH)
Snow white* (Pacht)
1
ZnO, carbon black
Green lake light (Averhoff)
1
Fe4[Fe(CN)6]3, BaSO4, Al
Naphthol yellow S, auramin O, martius yellow
Madder lake golden rose (Pacht)
1
CaCO3, Mg3Si4O10(OH)2, Al, P
Purpurin, pseudopurpurin
Madder lake deep rose (Pacht)
2
CaCO3, BaSO4, Al, P
Purpurin, alizarin
Madder lake rose (Alman)
1
Al, P, S, Sn
Purpurin
Carmine lake ordinary (Lefranc)
1
CaCO3, Al, Sn
Carminic acid
Red oxide (Pacht)
1
CdS orange, Al
Purpurin, alizarin
Red oxide (Averhoff)
1
Fe2O3, BaSO4, Al, P
Not analysed
Red oxide (Averhoff)
4
CdS orange, Al, P
Not analysed Table 3 Pigments, fillers and dyes identified in the tube colours by Raman, SEM–EDS, and HPLC Naphthol yellow S, auramin O, martius yellow
Purpurin, pseudopurpurin
Purpurin, alizarin
Purpurin
Carminic acid
Purpurin, alizarin
Not analysed
Not analysed * Designates tubes described as “slowly drying” on the label the manufacturer changed the composition of the col-
our in order to create an alternative to the traditional
“Chrome green” based on more durable pigments. Similar examples have been reported by Townsend
et al. [26]. In general durability seems to have been an
important matter to Danish colour merchants in the
end of the nineteenth century. Tube colours
Th The peaks in b arising from the organic dyes has not
been assigned Fig. 9 Raman spectra of pigments identified in the tube colours. The
spectra depicted are a chrome yellow in “Chromium yellow light”, b
Prussian blue and organic dyes in “Green lake light”, c hematite in “Red
oxide” from Averhoff, and d alizarin in “Red oxide” from Pacht. The
spectra were assigned by comparison of the spectra to those in the
literature [8, 9]. The peaks in b arising from the organic dyes has not
been assigned Fig. 10 HPLC chromatograms of the tube colours a: “Green lake light”
(350 nm), b: “Madder lake deep rose” (495 nm), and c: “Carmine lake
ordinary” (495 nm). Peak identification is inserted Newton (reference sample archived at Laboratories of
IRPA/KIK since the 1950s), hence the “Green lake light”
could be described as a mixture of Prussian blue and syn-
thetic Indian yellow. indicates the extraction method of cochineal, but its
significance is unclear.i i
Six tube colours, one from Vilhelm Pacht and five
from Hermann Averhoff, were all labelled “Red oxide”. Despite having identical trade names and almost iden-
tical colours, the analysis showed a considerably varia-
tion in the contents of the tubes. Only one of the five
tube colours from Averhoff was found to contain hema-
tite as the main pigment as expected by the trade name
(Fig. 9c). The remaining four all contained cadmium
sulfide, and hence showed a discrepancy between trade
name and contents. In addition all five tube colours
contained red lakes based on the identification of typi-
cal mordants Al, P, and Sn [28, 30, 31]. The “Red oxide”
from Pacht likewise contained a mixture of cadmium
sulfide and a red lake identified by Raman as alizarin
(Fig. 9d) and by HPLC as purpurin and alizarin (not
shown). Four tube colours had trade names for variant shades
of red madder i.e. “Madder lake golden rose”, “Mad-
der lake deep rose”, and “Madder lake rose”. In the first
mentioned HPLC-analysis identified the organic col-
ourants to be purpurin and pseudopurpurin, which
could be interpreted as Kopp’s purpurin [27] precipi-
tated onto a substrate containing calcite and talc. Pur-
purin and alizarin were identified in the latter two,
confirming a madder-based product best described as
garancine [27, 28]. In the tube labelled “Carmine lake
ordinary”, carminic acid was identified (Fig. Tube colours
Th One tube labelled “Green lake light” showed
a surprising result, whereas the remaining five tubes with
trade names for various shades of red madder and car-
mine contained the expected dyes based on their names
and the artists’ materials available in this period. The single tube colour labelled “Green lake light”
contained a mixture of yellow dyes precipitated on alu-
minium and barium sulfate containing substrate, and
a blue pigment identified as Prussian blue (Fig. 9b) by
Raman analysis (2155, 2092, 538, and 278 cm−1) [8]. By
HPLC the yellow dyes were identified as Naphthol yel-
low S together with a smaller amount of Auramin O and
Martius yellow (Fig. 10a). Naphthol yellow S precipitated
on aluminium and barium sulfate was found to be simi-
lar to a synthetic Indian yellow prepared by Winsor and In relation to the stability of chromate pigments
the analysis of the two tube colours labelled “Chrome
green light” and “Chrome green medium” should
be noticed. The name “Chrome green” has typi-
cally been used for the mixture of chrome yellow and
Prussian blue [20, 25], but in this case the tubes con-
tained a mixture of viridian, zinc yellow, cadmium
sulfide and barium sulfate. It seems very likely, that Christiansen et al. Herit Sci (2017) 5:39 Page 9 of 11 Fig. 9 Raman spectra of pigments identified in the tube colours. The
spectra depicted are a chrome yellow in “Chromium yellow light”, b
Prussian blue and organic dyes in “Green lake light”, c hematite in “Red
oxide” from Averhoff, and d alizarin in “Red oxide” from Pacht. The
spectra were assigned by comparison of the spectra to those in the
literature [8, 9]. The peaks in b arising from the organic dyes has not
been assigned Fig. 10 HPLC chromatograms of the tube colours a: “Green lake light”
(350 nm), b: “Madder lake deep rose” (495 nm), and c: “Carmine lake
ordinary” (495 nm). Peak identification is inserted Fig. 9 Raman spectra of pigments identified in the tube colours. The
spectra depicted are a chrome yellow in “Chromium yellow light”, b
Prussian blue and organic dyes in “Green lake light”, c hematite in “Red
oxide” from Averhoff, and d alizarin in “Red oxide” from Pacht. The
spectra were assigned by comparison of the spectra to those in the
literature [8, 9]. Relation between the analysed paintings and tube colours Relation between the analysed paintings and tube colours
Given the fact, that most of the tube colours in Krøyer’s
painting cabinet were probably bought in the last dec-
ades of Krøyer’s life, it seemed reasonable to draw a line
between the tube colours and the paintings from the lat-
ter part of Krøyer’s career. Especially the large portrait of
Drachmann, finished only 2 years before Krøyer’s death
in 1909, seemed to be closely linked to the tube colours
found in the artist’s painting cabinet. Cadmium sulfide
pigments, viridian, cobalt blue and zinc white were all
examples of pigments found in both Krøyer’s portrait of
Drachmann and in a number of his tube colours. The tube colours found in Krøyer’s painting cabinet were
primarily from Danish colour merchants, with the majority
from the colour manufacturer Vilhelm Pacht. Krøyer’s tube
colours had trade names typical for artists’ paints used dur-
ing that period of time. In most cases the pigment(s) found
in the tubes matched what would be expected based on the
trade name. However, notable exceptions were found, such
as the rare cadmium chromate pigment present in the tube
colour “Cadmium lemon yellow” [21], as well as the “Green
lake light” and “Red oxides” described above. This illus-
trates that it was not always obvious to artists what they
bought from their colour merchant. The fact that some
pigments were found in the paintings, but not in any of the
tube colours in the painting cabinet and vice versa, empha-
sises that conclusions about an artist’s painting materials
cannot be drawn based on access to the materials alone
without including analyses of the paintings themselves. Zinc white appears to have been an important part of
Krøyer’s palette in the latter part of his career [4, 32]. In
the analysed samples from the Drachmann portrait, zinc
white was present in all paint layers where it was applied
in mixtures with a wide variety of other pigments. More-
over the colour “Snow white” containing zinc white
pigment was the only white tube colour present in the
painting cabinet. A part from the pure white tube colour,
zinc white was only identified in one other tube colour,
this being “Jaune brilliant” where it was mixed with cad-
mium sulfide. Author details
1
h
l f 1 School of Conservation, Esplanaden 34, 1263 Copenhagen K, Denmark. 2 Art Conservation Center, ARoS Alle 2, 8000 Århus C, Denmark. 3 Laboratories
of IRPA/KIK, Parc du Cinquantenaire 1, 1000 Brussels, Belgium. Authors’ contributions
All
th
t ib t d t Authors’ contributions
All authors contributed to data interpretation and to finalizing the manuscript. All authors read and approved the final manuscript. Conclusionsh The analyses carried out on the samples from the two
paintings and the tube colours have given an insight in
the painting materials used by Krøyer in the latter part of
his career, e.g. the pigments applied on his palette, and his
favourite brand in tube colours and colour merchants. In Acknowledgements The authors would like to thank The Art Museums of Skagen and The
Hirschsprung Collection for kindly allowing investigation and sampling of
Krøyer’s paintings and tube colours respectively. Furthermore, we wish to
thank Cecile Glaude and Alexia Coudray from IRPA/KIK for assistance with the
instrumental analyses, and curator Mette Bøgh Jensen from The Art Museums
of Skagen for reviewing the art historical contents of the article. Finally, we
thank Foundation Idella, Marie Månsson’s Foundation, and the School of
Conservation Firenze grant, for making first authors research stay at IRPA/KIK in
Brussels, Belgium possible. Relation between the analysed paintings and tube colours It thus looks as if Krøyer himself intention-
ally blended zinc white into his colours on the palette in
order to lighten the variant shades, as zinc white was pre-
sent in all areas of the portrait of Drachmann. Some pigments like chromium yellow and cadmium
chromate [21] were only found in the tube colours. It is
uncertain to what extent Krøyer has actually used these
pigments in his paintings. Further analyses of other
paintings by the artist could possibly provide information
with regard to this question. In contrast the pigments
vermilion, minium and chromium orange were all iden-
tified in the Drachmann portrait, whereas they were not
present in any of the tube colours in the painting cabinet. This emphasises that conclusions about an artist’s paint-
ing materials cannot be drawn based on access to the
materials alone without including analyses of the paint-
ings themselves. Tube colours
Th 10c) show-
ing that this lake was actually prepared from cochi-
neal insect [29]. Together with carminic acid, another
unknown red dye compound was found, which perhaps Christiansen et al. Herit Sci (2017) 5:39 Page 10 of 11 general Krøyer used the commercially produced artists’
materials that became available from colourmen during
the nineteenth century. The canvases were bought ready
grounded, either on the stretcher or by the metre, and
the composition of the ground consisted of lead white,
calcite and silicate. Krøyer’s palette consisted of both
traditional pigments and modern ones developed in the
eighteenth and nineteenth century, among these, several
pigments containing chromium, cadmium and cobalt. In general the pigments found in Krøyer’s paintings and
tube colours were expensive reflecting that Krøyer was a
well-established and wealthy artist, who did not have to
choose the cheaper alternatives. A noticeable change in
Krøyer’s palette seems to have taken place somewhere in
between 1886 and 1903, where lead white was replaced
with zinc white.h The cases of the “Green lake light” and the “Red oxide”
are just a few examples of the complexity that relates to
artists’ colours from the 19th century [26]. A clear cor-
relation between trade name and contents is not always
seen, and consequently artists have often not had a sound
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Pilot study comparing simulation-based and didactic lecture-based critical care teaching for final-year medical students
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BMC anesthesiology
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© 2015 Solymos 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: Simulation-based medical education has rapidly evolved over the past two decades, despite this, there
are few published reports of its use in critical care teaching. We hypothesised that simulation-based teaching of a
critical care topic to final-year medical students is superior to lecture-based teaching. Methods: Thirty-nine final-year medical students were randomly assigned to either simulation-based or lecture-based
teaching in the chosen critical care topic. The study was conducted over a 6-week period. Efficacy of each teaching
method was compared through use of multiple choice questionnaires (MCQ) - baseline, post-teaching and 2 week
follow-up. Student satisfaction was evaluated by means of a questionnaire. Feasibility and resource requirements were
documented by teachers. Results: Eighteen students were randomised to simulation-based, and 21 to lecture-based teaching. There were no
differences in age and gender between groups (p > 0.05). Simulation proved more resource intensive requiring specialised equipment, two instructors, and increased duration of
teaching sessions (126.7 min (SD = 4.71) vs 68.3 min (SD = 2.36)). g
Students ranked simulation-based teaching higher with regard to enjoyment (p = 0.0044), interest (p = 0.0068), relevance
to taught subject (p = 0.0313), ease of understanding (p = 0.0476) and accessibility to posing questions (p = 0.001). Both groups demonstrated improvement in post-teaching MCQ from baseline (p = 0.0002), with greater improvement
seen among the simulation group (p = 0.0387), however, baseline scores were higher among the lecture group. The Both groups demonstrated improvement in post-teaching MCQ from baseline (p = 0.0002), with greater improvement
seen among the simulation group (p = 0.0387), however, baseline scores were higher among the lecture group. The
results of the 2-week follow-up MCQ and post-teaching MCQ were not statistically significant when each modality
were compared. Discussion: Simulation was perceived as more enjoyable by students. Although there was a greater improvement in
post-teaching MCQ among the simulator group, baseline scores were higher among lecture group which limits
interpretation of efficacy. Simulation is more resource intensive, as demonstrated by increased duration and personnel
required, and this may have affected our results. Conclusions: The current pilot may be of use in informing future studies in this area. care environment, the nature of which leads to limited op-
portunities for bedside teaching given dangers of exposing
this vulnerable patient subpopulation to large numbers of
students. Thus, critical care teaching frequently relies on
provision of tutorial and didactic lecture sessions. Pilot study comparing simulation-based
and didactic lecture-based critical care
teaching for final-year medical students Orsolya Solymos1, Patrick O’Kelly2 and Criona M. Walshe1* * Correspondence: crionawalshe@rcsi.ie
1Department of Anaesthesia and Critical Care Medicine, Royal College of
Surgeons in Ireland, Smurfit Building, Beaumont Hospital, Dublin, Ireland
Full list of author information is available at the end of the article Solymos et al. BMC Anesthesiology (2015) 15:153
DOI 10.1186/s12871-015-0109-6 Solymos et al. BMC Anesthesiology (2015) 15:153
DOI 10.1186/s12871-015-0109-6 Methods Ethics approval was obtained from the RCSI Research
Ethics Committee (REC). The study population comprised
final-year medical students rotating through a 1-week
Anaesthesia and Critical Care attachment as part of their
final-year medicine curriculum within the RCSI. Resource requirements
Th
i The resource requirements for each teaching method
were assessed by looking at the time required for didac-
tic lecture preparation and delivery versus preparation
and delivery of simulation scenarios, in addition to
personnel and equipment resources required for each
mode of teaching. The aim of this study is to examine whether simulation-
based teaching of a critical care topic to final-year medical
students is superior to lecture-based teaching. We utilised a high fidelity, event driven simulator. This
required two instructors, one to engage within the
scenario with the students, to protect against patient
demise in the event of poor student performance, and
a second to coordinate computer driven physiological
responses dependent on intervention implemented by
students within the scenario. Questionnaires and MCQ tests Pearson Chi-squared test was used to compare the
difference in gender composition between the two groups. We compared the efficacy of the two teaching methods
through the use of multiple choice questionnaire papers
(MCQ). Each MCQ comprised five questions with five
true or false stems, and results were marked out of a
maximum of 25. Prior to the teaching session each stu-
dent completed the baseline multiple choice questionnaire
(baseline MCQ). Students completed a second MCQ
immediately following the teaching session (post teaching
MCQ). Participants and setting Finally, qualitative assessment was analysed by means of
a questionnaire filled out by the students following each
teaching session. These were designed to assess clarity of
teaching, student enjoyment, accessibility of learner to
pose questions etc. to gain student perceptions of the
teaching style. Students are assigned to the Department of Anaesthesia in
groups of five to eight students per week. Following in-
formed consent, each group was randomised to one of two
teaching methods, simulation-based teaching or didactic
lecture. The chosen topic was recognition of the critically
ill patient at ward level with a focus on sepsis. Each method
of delivery of this teaching session was carefully prepared
to deliver the same key concepts to allow the student to
recognise the deteriorating patient at ward level, diagnose
and manage sepsis, as per the Surviving Sepsis Guidelines
[3]. All teaching was delivered by the same RCSI Clinical
Lecturer, and Consultant in Anaesthesia and Critical Care. Background Critical care medicine poses challenges for the education
of both medical students and junior doctors. There
exists an inherent difficulty in imparting knowledge on
complex topics that are difficult to grasp for medical
students who often have had minimal exposure to direct
patient care. This is particularly true within the critical Simulation involves the creation of an artificial depiction
of complex clinical situations which are infrequently
encountered. If the fidelity of the simulator is sufficient,
then learning is facilitated through immersion, reflection
and feedback but carries the benefit of protection of both
patient and learner from risks associated with these * Correspondence: crionawalshe@rcsi.ie
1Department of Anaesthesia and Critical Care Medicine, Royal College of
Surgeons in Ireland, Smurfit Building, Beaumont Hospital, Dublin, Ireland
Full list of author information is available at the end of the article Page 2 of 5 Solymos et al. BMC Anesthesiology (2015) 15:153 participation in this study, all students were offered the
opportunity to return to avail of the teaching delivered
by the teaching method to which they were not ran-
domised. This session was held 2 weeks following the
initial teaching session. Prior to this supplementary
teaching session they were asked to complete the
third and final MCQ (2-week follow up MCQ). The
results of all MCQ examinations were fed back to
each participating student upon completion of this
final teaching session and MCQ examination. clinical situations [1]. Simulation-based medical education
is gaining popularity with much discussion in the litera-
ture of its proposed merits [2]. Critical care teaching may
be particularly suited to simulation-based education as it
is often not feasible to demonstrate live critical care scenar-
ios to medical students in the hospital setting. A com-
pletely safe version mimicking such complex scenarios can
easily be acted out in the Simulation Laboratory. And yet
our literature research has revealed few studies of its use in
the education of medical students in critical care. The installation of two Laerdal SimMan 3G mannequins
by the Royal College of Surgeons in Ireland (RCSI) for the
training purposes of medical students has facilitated simu-
lation sessions to be delivered in the medical discipline,
the surgical discipline and in basic and advanced cardiac
life support. Statistical analysis Two-sample Wilcoxon rank-sum (Mann–Whitney) test
was used to evaluate the results of the student satisfaction
questionnaires and to compare the difference in results of
the pre- and post-teaching multiple choice questionnaires
between the two groups. Wilcoxon signed-rank test was used to compare the
results of the multiple choice questionnaires within the
two groups. Results Forty-one final year medical students comprised the
study population. Two students were excluded from the
study due to non completion of the pre- or the post-
teaching MCQ. Median ages (interquartile range IQR) were 24 (24–25)
in the simulation group and 24 (23–27) in lecture-based
teaching group (p = 0.7). Sex distribution in the study To ensure that the participants were not at a disadvan-
tage or that they did not perceive a disadvantage by Solymos et al. BMC Anesthesiology (2015) 15:153 Page 3 of 5 Page 3 of 5 Table 2 Duration of preparation and delivery of each mode of
teaching
Method of
teaching
Mean duration of
teaching session
Duration of preparation
Simulation
126.7 min
Training of teachers on simulator: 240 min
(SD = 4.71)
Scenario Preparation: 180 min
Simulator Start-up: 10 min
Lecture
68.3 min
Lecture Preparation: 180 min
(SD = 2.36)
Laptop/projector Start-up: 3 min
SD standard deviation Table 2 Duration of preparation and delivery of each mode of
teaching population was not statistically significant, 43.6 % female
(17/39), 56.4 % male (22/39), (p = 0.1). All students included in study population completed
baseline and post teaching MCQs. Sixteen students
returned to avail of the learning method to which they
were not randomised and completed the 2-week follow
up MCQ; 5 of the simulation-based teaching group
(28 % (5/18)) and 11 of the lecture-based teaching group
(52 % (11/21)). Results of all MCQ papers are depicted
in Table 1. There was a significant difference in the
improvement from baseline and post teaching MCQ in
the simulation group compared to lecture 6.8 (21.1 - 14.3)
vs. 4.5 (21.5 - 17), p = 0.0387. The results of 2-week follow
up MCQ were lower in both groups than post teaching
results. Although this margin was smaller in the simula-
tion group 1.3 (19.8-21.1) vs. 3.6 (17.9 - 21.5), this was not
statistically significant (p = 0.167). SD standard deviation use of the simulator to teach medical emergencies, with
varied results. Ruesseler et al. studied the use of simula-
tion training in medical emergencies in a group of final
year medical students and found performance in OSCE
stations superior among simulation students, compared
to controls [9]. In a study comparing didactic lecture to
use of simulation to teach perioperative ultrasound
higher
test
scores
were
demonstrated
among
the
simulation group [10]. Daniels et al. Results demonstrated
significantly
higher
performance
in
the
simulation
group among residents and nurses being taught ob-
stetrical emergencies compared to didactic teaching
and Hallikainen et al. demonstrated an improved task
performance among students taught anaesthesia in-
duction among the simulator group, as compared to
those learning by means of observed practice [11]. In
a study by Morgan et al., simulation was compared to
video based learning to teach a number of medical
emergencies, however, the investigators determined no
differences in quantitative assessments between each
group, although students found the simulator sessions
more enjoyable and valuable [12]. Tan et al. demonstrated
equal efficacy between screen based simulation and con-
ventional lecture in a group of students taught medical
emergency management [13]. However, we could not
identify studies examining the use of the simulator for the
purposes of critical care education. Duration of preparation and delivery of each teaching
session are outlined in Table 2. In terms of financial outlay,
the SimMan simulator cost Euro 60,000. There was no
financial implication for the provision of the didactic
lecture as all necessary equipment was made freely avail-
able by RCSI and Beaumont Hospital. Tables 3 and 4 depict student perceptions of the teaching
experience. Table 1 Results of MCQ at baseline, post teaching and at 2 week follow up. Scores are marked out of a total of 25, results are
depicted as mean +/−standard deviation for each group
Method
Baseline MCQ
Mean(+/−SD)
Post-teaching MCQ
Mean(+/−SD)
Two-week follow up MCQ
Mean(+/−SD)
Simulation
14.3(2.2)
21.1(1.8)
19.8(3)
Lecture
17(3)
21.5(3.1)
17.9(2.5) Discussion Students were asked to rank the teaching
experience from 1 – 5 (one strongly disagree; five strongly agree) for each of the following in written test scores among groups of radiology trainees
receiving didactic lecture versus simulation based training
in management of contrast reaction, although perform-
ance among simulation based training was superior when
tested in a contrast reaction simulation scenario. In the
current study, sudents who received simulation-based
education demonstrated significantly improved perform-
ance in post teaching MCQ from baseline, compared to
those who received didactic lecture (Table 1). However,
baseline MCQ scores were lower among the simulator
group. These results may support the evidence outlined
above, that simulation may be more efficacious, although
randomisation failure cannot be excluded and further
study is warranted. traditional medical education methods including the
provision of a safe environment for both teacher and
student during training in risky procedures, unlimited
exposure to rare but complicated and important clin-
ical events, the ability to plan and shape training op-
portunities rather than waiting for a suitable situation
to arise clinically, the ability to provide immediate
feedback, the opportunity to repeat performance as
well as the opportunity for team training. We could
find no study in the literature exploring the use of
simulation education on this topic, sepsis, to either
medical students, or junior doctors. Simulator based teaching proved to be more resource
intensive. In addition to the cost of purchasing the
Simulator are the as yet unknown ongoing maintenance
costs beyond the 5-year warranty period. In terms of
preparation, the simulator required longer time for
teachers to be trained in its operation and use, an
addition to scenario preparation 420 min vs. 180 min for
didactic lecture preparation (Table 2). Similarly, delivery
of simulation based teaching proved more resource inten-
sive taking on average twice as long compared to didactic
lecture delivery, 126.7 vs. 68.3 min, and necessitating
presence of two teachers. Although our results demon-
strate a trend toward improved efficacy as demonstrated
by greater increase in MCQ scores from baseline to post
teaching, there remains the possibility that some of this
was due to the greater time spent teaching the topic
among the simulation group, for which it is difficult to
control, and this may have influenced our results. We compared the sustainability of the acquired know-
ledge through the completion of an MCQ 2 weeks follow-
ing the teaching session. Discussion The use of simulators in medical education has vastly
increased in recent years [4], and there are now available
a wide variety of commercially available products. In this
pilot study, we utilised a high fidelity simulator to
engage the students in a teaching session which focused
on recognising a patient at ward level who is critically ill,
with sepsis as the cause of clinical deterioration, and
explore the merits and resources required to run
simulation-based teaching of a critical care topic to medical
students. Critical care medicine lends itself particularly well to
simulation training [5], given the inherent difficulties of
teaching large numbers of students on rare and serious
clinical events in a vulnerable patient population [6, 7]. Despite this, there are limited studies examining the use
of the simulator in critical care. Schroedl et al. examined
simulation based education as a means to teach topics
pertinent to medical ICU and demonstrated higher
scores in skills assessment among simulation training as
compared to controls [8]. Others have investigated the In terms of the critical care topic utilised in this study,
we chose the deteriorating patient at ward level, because
this is a particularly pertinent clinical scenario fre-
quently encountered by doctors in their first year of
clinical practice. The focus was sepsis because of the
time sensitive nature of interventions that have been
shown improve patient outcomes [14]. The use of
simulation training has a number of advantages over Table 1 Results of MCQ at baseline, post teaching and at 2 week follow up. Scores are marked out of a total of 25, results are
depicted as mean +/−standard deviation for each group
Method
Baseline MCQ
Mean(+/−SD)
Post-teaching MCQ
Mean(+/−SD)
Two-week follow up MCQ
Mean(+/−SD)
Simulation
14.3(2.2)
21.1(1.8)
19.8(3)
Lecture
17(3)
21.5(3.1)
17.9(2.5) Page 4 of 5 Page 4 of 5 Solymos et al. BMC Anesthesiology (2015) 15:153 Table 3 Summary of student perceptions of the experience of each mode of teaching. Students were asked to rank the teaching
experience from 1 – 5 (one strongly disagree; five strongly agree) for each of the following
Method of teaching
Organisation
Amenable to questions
Pace
Duration
Addressed subject matter
Simulation
4.9
4.9
4.5
2.3
4.8
Lecture
4.6
4.6
4.2
2.5
4.3
p value
0.037
0.011
0.703
0.077
0.019 Table 3 Summary of student perceptions of the experience of each mode of teaching. Discussion This was offered as an entirely
optional session, and unfortunately only a small number
of students obliged (28 % of simulator group returned for
follow up compared to 52 % of the lecture group). We
found that both simulation and lecture based groups had
lower scores at 2-week follow up compared to post teach-
ing, and although the margin of this deterioration was
smaller in the simulation group (1.3 vs. 3.6), this did not
achieve significance, (p = 0.1670). The non significance of
this result may be due, at least in part, to the small num-
bers of students involved, and there is the possibility of
bias given the discrepancy between return rates for each
group, and high attrition rate given the small numbers in
each
group. Other
investigators
have
demonstrated
improved knowledge retention among simulation based
education compared to didactic lecture, with follow at
2–3 weeks [10]. This needs to be further studied. In this study we utilised MCQ as a means of assessing
the efficacy of education sessions provided, but further
study is warranted to explore whether the assessment of
skills acquired in carrying out practical tasks such as
resuscitation of critically ill patients with sepsis would be
more accurately achieved through use of the simulator
as a means of assessment. In a study among Radiology
trainees [15], Wang et al. demonstrated no improvement Students were asked to rate the teaching experience and
there was a statistically significant difference in favour of
the simulation-based teaching with regard to organisation,
and adequate address of chosen subject matter and how
amenable the session proved to students in terms of Table 4 Summary of student perceptions of quality of each mode of teaching. Students were asked to rank their thoughts on the
quality of the teaching out from 1–10 (one being the worst ten the best) strong for each of the following
Method of teaching
Enjoyment
Interest
Relevance
Ease of understanding
Simulation
8.7
9.2
9.7
9.6
Lecture
7.2
8
9
9.2
p value
0.0044
0.0068
0.0313
0.0476 Page 5 of 5 Page 5 of 5 Solymos et al. BMC Anesthesiology (2015) 15:153 posing questions (Table 3). Students also ranked enjoy-
ment, interest, relevance and ease of understanding of the
teacher higher among the simulation teaching (Table 4). That the students enjoyed simulation based education has
been commented upon by other investigators, such as the
study mentioned above by Morgan et al. Competing interests
f
d No funding was received for this study, and the authors would like to
declare no conflict of interest. 15. Wang CL, Schopp JG, Petscavage JM, Paladin AM, Richardson ML, Bush WH. Prospective randomized comparison of standard didactic lecture versus
high-fidelity simulation for radiology resident contrast reaction
management training. Am J Roentgenol. 2011;196(6):1288–95. Discussion [12], and a study
by Paskins et al. which evaluated students views on simu-
lation based teaching [16]. Received: 8 May 2015 Accepted: 17 September 2015 Received: 8 May 2015 Accepted: 17 September 2015 Received: 8 May 2015 Accepted: 17 September 2015 The purpose of this pilot was to establish feasibility of
the use of the simulator to deliver critical care education
to final year medical students, and to establish study
population required for further randomised control trial. We acknowledge the limitation of our use of an MCQ as
the assessment tool, where Simulation based assessment
sessions would likely have proved superior; however
resource implications obviated our ability to perform such
assessments. Further limitations of this small pilot study
include the differences in duration of each teaching ses-
sion, with the simulation session being almost twice as
long in duration and the lower student:teacher ratios
which may have influenced our results. Conclusions
Th
d f 7. Wong N. Medical education in critical care. J Crit Care. 2005;3(20):270–3. 8. Schroedl CJ, Corbridge TC, Cohen ER, Fakhran SS, Schimmel D, McGaghie WC,
et al. Use of simulation-based education to improve resident learning and
patient care in the medical intensive care unit: a randomized trial. J Crit Care. 2012;27(219):e7–13. The need for simulation-based medical education in
clinical practice, especially in critical care medicine, is
likely to continue or increase given the proposed merits
for both students and teachers. Given that simulation
based education is both time-consuming and resource in-
tensive, its long-term merits with regard to retaining
knowledge and translating into improved patient care
need to be further studied and confirmed. While acknow-
ledging the limitations of the current study as outlined
above, this study demonstrated increased student enjoy-
ment of simulator based teaching compared to didactic
lectures and may be used to inform further studies investi-
gating this important area. 9. Ruesseler M, Weinlich M, Müller MP, Byhahn C, Marzi I, Walcher F. Republished: Simulation training improves ability to manage medical
emergencies. Postgrad Med J. 2012;88(1040):312–6. 10. Ramsingh D, Alexander B, Le K, Williams W, Canales C, Cannesson M. Comparison of the didactic lecture with the simulation/model approach fo
the teaching of a novel perioperative ultrasound curriculum to
anesthesiology residents. J Clin Anesth. 2014;26(6):443–54. 11. Hallikainen J, Väisänen O, Randell T, Tarkkila P, Rosenberg PH, Niemi-Murola L. Teaching anaesthesia induction to medical students: comparison between full-
scale simulation and supervised teaching in the operating theatre. Eur J
Anaesthesiol. 2009;26(2):101–4. 12. Morgan PJ, Cleave-Hogg D, McIlroy J, Devitt JH. Simulation technology: a
comparison of experiential and visual learning for undergraduate medical
students. Anesthesiology. 2002;96(1):10–6. 13. Tan GM, Ti LK, Tan K, Lee T. A comparison of screen-based simulation and
conventional lectures for undergraduate teaching of crisis management. Anaesth Intensive Care. 2008;36(4):565–9. Acknowledgements
h
h
ld l k The authors would like to acknowledge the enthusiasm demonstrated by
the final year students who eagerly participated in this pilot study. References 1. Ogden PE, Cobbs LS, Howell MR, Sibbitt SJ, DiPette DJ. Clinical simulation:
importance to the internal medicine educational mission. Am J Med. 2007;120:820–4. 1. Ogden PE, Cobbs LS, Howell MR, Sibbitt SJ, DiPette DJ. Clinical simulation:
importance to the internal medicine educational mission. Am J Med. 2007;120:820–4. 2. Grenvik A, Schaefer JJ, DeVita MA, Rogers P. New aspects on critical care
medicine training. Curr Opin Crit Care. 2004;10(4):233–7. 2. Grenvik A, Schaefer JJ, DeVita MA, Rogers P. New aspects on critical care
medicine training. Curr Opin Crit Care. 2004;10(4):233–7. 3. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM. Surviving
Sepsis Campaign Guidelines Committee including the Pediatric Subgroup. Surviving sepsis campaign: international guidelines for management of
severe sepsis and septic shock. Crit Care Med. 2013;41(2):580–637. 4. Ker J, Bradley P. Simulation in medical education. Edinburgh: Association for
the Study of Medical Education (ASME). 2007 4. Ker J, Bradley P. Simulation in medical educati
the Study of Medical Education (ASME). 2007 y
5. Murphy JG, Torsher LC, Dunn WF. Simulation medicine in intensive care and
coronary care education. J Crit Care. 2007;22(1):51–5. 6. Bion J, Heffner J. Challenges in the care of the acutely ill. Lancet. 2004;941(3):970–7. 7. Wong N. Medical education in critical care. J Crit Care. 2005;3(20):270–3. 8
S h
dl CJ C b id
TC C h
ER F kh
SS S hi
l D M G
hi W Abbreviations MCQ: Multiple choice questionnaire; SD: Standard deviation; RCSI: Royal
College of Surgeons in Ireland. 14. Levy MM, Dellinger RP, Townsend SR, Linde-Zwirble WT, Marshall JC, Bion J,
et al. Surviving Sepsis Campaign. The Surviving Sepsis Campaign: results of
an international guideline-based performance improvement program
targeting severe sepsis. Crit Care Med. 2010;38(2):367–74. 14. Levy MM, Dellinger RP, Townsend SR, Linde-Zwirble WT, Marshall JC, Bion J,
et al. Surviving Sepsis Campaign. The Surviving Sepsis Campaign: results of
an international guideline-based performance improvement program
targeting severe sepsis. Crit Care Med. 2010;38(2):367–74. Authors’ information
Not applicable Not applicable Availability of data and materials
Not applicable Authors’ contributions OS submitted Ethics application, participated in provision of teaching
sessions, collected data and prepared manuscript. POK carried out
compilation of data and performed statistical analysis. CMW designed study,
assisted with ethics application, participated in design and delivery of
teaching sessions and supervised preparation of manuscript writing. All
authors read and approved the final manuscript. 16. Paskins Z, Peile E. Final year medical students’ views on simulation-based
teaching: a comparison with the Best Evidence Medical Education
Systematic Review. Med Teacher. 2010;32(7):569–77. Author details
1 1Department of Anaesthesia and Critical Care Medicine, Royal College of
Surgeons in Ireland, Smurfit Building, Beaumont Hospital, Dublin, Ireland. 1Department of Anaesthesia and Critical Care Medicine, Royal College of
Surgeons in Ireland, Smurfit Building, Beaumont Hospital, Dublin, Ireland. 2Department of Nephrology, Beaumont Hospital, Beaumont Road, Dublin 9,
Ireland. 2Department of Nephrology, Beaumont Hospital, Beaumont Road, Dublin 9,
Ireland. Received: 8 May 2015 Accepted: 17 September 2015 Availability of data and materials Not applicable
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A investigação da construção de modelos no estudo de um tópico de física utilizando um ambiente de modelagem computacional qualitativo
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A investiga¸c˜ao da constru¸c˜ao de modelos no estudo de um t´opico de
F´ısica utilizando um ambiente de modelagem computacional qualitativo
(Investigation of model building in the study of a physics issue using a qualitative computer model environment) A investiga¸c˜ao da constru¸c˜ao de modelos no estudo de um t´opico de
F´ısica utilizando um ambiente de modelagem computacional qualitativo
(Investigation of model building in the study of a physics issue using a qualitative computer model environment) Thi´eberson Gomes1 e La´ercio Ferracioli2 Laborat´orio de Tecnologias Interativas Aplicadas a Modelagem Cognitiva, Departamento de F´ısica,
Universidade Federal do Esp´ırito Santo,Vit´oria, ES, Brasil
Recebido em 16/11/2005; Aceito em 28/4/2006 Laborat´orio de Tecnologias Interativas Aplicadas a Modelagem Cognitiva, Departamento de F´ısica,
Universidade Federal do Esp´ırito Santo,Vit´oria, ES, Brasil
Recebido em 16/11/2005; Aceito em 28/4/2006 A integra¸c˜ao de tecnologias de informa¸c˜ao e comunica¸c˜ao no contexto educacional tem sido tema de diversos
congressos e simp´osios ao redor do mundo e no Brasil. Neste sentido, v´arios estudos tˆem sido realizados com o
objetivo de se obter metodologias que tornem efetivo o emprego das novas tecnologias no ensino. Este artigo
mostra um estudo que investigou a intera¸c˜ao entre estudantes universit´arios da ´area de ciˆencias exatas e um am-
biente de modelagem computacional qualitativo em atividades de modelagem expressiva. Os resultados obtidos
mostram que os estudantes foram capazes de criar e modificar o modelo do sistema proposto a partir de suas
pr´oprias concep¸c˜oes. Palavras-chave: ambientes de modelagem, modelos, modelos mentais, modelagem qualitativa, atividades ex-
pressivas, modelagem, cognitiva, autˆomatos celulares. The integration of information and communication technologies into the educational context has been the
subject of several symposiums and conferences around the world and also in Brazil. Many studies have been
carried out for developing methodologies that turn successful their application in the learning process. This
paper shows a study that investigate the interaction between graduate students from physics and engineering
areas and activities of a qualitative computer modelling environment expressive modelling activities. Results
show that the students were able to build up and modify the model from the system proposed, starting from
their own ideas. Keywords: modelling environments, models, mental models, qualitative modelling, expressive activities, cogni-
tive modelling, cellular automata. 1. Introdu¸c˜ao ambientes de modelagem computacional no contexto
da educa¸c˜ao em ciˆencias e tecnologia e, em particular,
apresenta resultados relativos ao estudo de um t´opico
de F´ısica. A investiga¸c˜ao sobre a integra¸c˜ao da tecnologia da in-
forma¸c˜ao e comunica¸c˜ao no contexto educacional tem
sido o foco de diversos estudos nos diferentes n´ıveis de
escolaridade e em diferentes partes do mundo. No Bra-
sil, os Parˆametros Curriculares Nacionais (PCN) res-
saltam a importˆancia deste fato apontando para a ade-
qua¸c˜ao dos curr´ıculos `a realidade contemporˆanea e aos
avan¸cos tecnol´ogicos. Os PCN indicam que a inte-
gra¸c˜ao do computador no contexto educacional pode
ser realizada de diferentes formas, tais como, plani-
lhas eletrˆonicas, editores de texto, jogos e softwares de
conte´udos espec´ıficos. Al´em destas formas, ´e ainda su-
gerida a utiliza¸c˜ao de modelos representativos dos mais
variados sistemas da natureza [1]. Neste sentido, o pre-
sente artigo descreve um estudo sobre a integra¸c˜ao de Copyright by the Sociedade Brasileira de F´ısica. Printed in Brazil. Revista Brasileira de Ensino de F´ısica, v. 28, n. 4, p. 453-461, (2006)
www.sbfisica.org.br Revista Brasileira de Ensino de F´ısica, v. 28, n. 4, p. 453-461, (2006)
www.sbfisica.org.br Revista Brasileira de Ensino de F´ısica, v. 28, n. 4, p. 453-461, (2006)
www.sbfisica.org.br 1E-mail: thieberson@gmail.com.
2E-mail: laercio@npd.ufes.br. g
2E-mail: laercio@npd.ufes.br. 1E-mail: thieberson@gmail.com. Revista Brasileira de Ensino de F´ısica, v. 28, n. 4, p. 453-461, (2006)
www.sbfisica.org.br 2.
Referencial te´orico Modelar significa representar um objeto, sistema ou
fenˆomeno, entre outras coisas, atrav´es de met´aforas e
analogias [2]. Os cientistas buscam explicar estruturas
que v˜ao desde escalas microsc´opicas, como o ´atomo, at´e
as escalas macrosc´opicas, como as ´orbitas dos planetas. Na maioria das vezes estas estruturas s˜ao inalcan¸c´aveis,
devido a limita¸c˜oes da tecnologia, incitando cientistas
a criarem modelos para estud´a-las [3]. Neste contexto,
as teorias podem ser entendidas como um conjunto de
abstra¸c˜oes que s˜ao mapeadas em um mundo imagin´ario 454 Gomes e Ferracioli Gomes e Ferracioli • Modelos mentais s˜ao inst´aveis: as pessoas esque-
cem os detalhes do sistema que est˜ao usando,
principalmente quando esses detalhes n˜ao foram
usados por um bom tempo; atrav´es da utiliza¸c˜ao dos modelos [4]. Um modelo pode
ser visto como um intermedi´ario entre as abstra¸c˜oes da
teoria e as a¸c˜oes concretas da experimenta¸c˜ao; e que
ajuda a fazer predi¸c˜oes, guiar a investiga¸c˜ao, resumir
dados, justificar resultados e facilitar a comunica¸c˜ao
[2]. Assim, devido `a sua versatilidade e tratamento, os
modelos s˜ao amplamente utilizados em todas as ´areas
de conhecimento. • Modelos mentais n˜ao tˆem uma fronteira definida:
dispositivos e opera¸c˜oes similares se confundem
umas com as outras; No contexto educacional, v´arios estudiosos, entre
eles Johnson-Laird [5], acreditam que as estruturas in-
ternas `a mente de um indiv´ıduo podem ser consideradas
como modelos, denominados modelos mentais, que s˜ao
criados a partir das intera¸c˜oes deste indiv´ıduo com o
mundo que o cerca. Por´em, para que se possa falar
sobre modelos mentais, Gilbert e Boulter [2] afirmam
que ´e preciso fazer distin¸c˜ao entre: Sistema-Alvo - sis-
tema real que se deseja modelar, ou seja, o objeto da
representa¸c˜ao; Modelo Consensual - modelo expresso
que foi submetido a testes por um grupo social, por
exemplo, pertencente `a comunidade cient´ıfica, e sobre
o qual se concorda que apresenta algum m´erito; Modelo
Pedag´ogico - modelo especialmente constru´ıdo e usado
para auxiliar na compreens˜ao de um modelo consen-
sual; Modelo Mental - representa¸c˜ao pessoal e ´ıntima
do sistema-alvo; e Modelo Expresso - vers˜ao do modelo
mental que ´e expressa atrav´es da a¸c˜ao, da fala ou da
escrita. 2.
Referencial te´orico • Modelos mentais s˜ao n˜ao-cient´ıficos: as pessoas
incluem, em seus modelos, comportamentos su-
persticiosos mesmo quando sabem que esses com-
portamentos n˜ao s˜ao necess´arios: custa pouco es-
for¸co f´ısico e reduz o esfor¸co mental; • Modelos Mentais s˜ao econˆomicos: freq¨uentemente
as pessoas fazem muito mais opera¸c˜oes mecˆanicas
do que planejamento mental que as permitiria eli-
minar estas a¸c˜oes. Pode-se perceber, atrav´es destes resultados, que
modelos mentais n˜ao precisam ser tecnicamente apu-
rados, mas devem ser primordialmente funcionais, caso
contr´ario ele s˜ao descartados pelo indiv´ıduo [4]. Assim, ao se construir um modelo das concep¸c˜oes
de um estudante ´e necess´ario cautela, uma vez que,
na verdade, est´a se construindo uma vis˜ao destas con-
cep¸c˜oes, a qual pode n˜ao estar de acordo com o ver-
dadeiro modelo mental do estudante [6]. Contudo, a
utiliza¸c˜ao da modelagem como forma de acesso a estes
modelos pode ser promissora: o aluno, quando solici-
tado a construir seu pr´oprio modelo sobre determinado
t´opico, externaliza seus modelos mentais e os trans-
forma em uma estrutura concreta que pode ser anali-
sada e testada. As atividades de modelagem podem ser
realizadas utilizando-se materiais que v˜ao desde papel
e l´apis at´e as tecnologias interativas, como o computa-
dor. Deste modo, o presente trabalho est´a centrado em
atividades de modelagem que utilizam o computador,
ou seja, na modelagem computacional. Desta forma, dentro de um conte´udo espec´ıfico um
professor determina um sistema-alvo a ensinar e, ap´os
a verifica¸c˜ao dos modelos consensuais relacionados a
ele, constr´oi um modelo pedag´ogico. Este modelo pe-
dag´ogico ser´a utilizado como base para levar o estu-
dante a construir um modelo mental - do sistema-alvo -
que seja o mais pr´oximo poss´ıvel do modelo consensual
do sistema-alvo. Esta abordagem pode ser representada
pela Fig. 1. Figura 1 - Esquema de rela¸c˜oes entre os Modelos em Educa¸c˜ao. 2.3.
Ambientes de modelagem computacional Os modelos matem´aticos s˜ao muito utilizados para ex-
plicar os fenˆomenos da natureza nas diversas ´areas de
conhecimento e amplamente utilizados no contexto edu-
cacional. Contudo, aprender o ferramental matem´atico
para entender estes fenˆomenos n˜ao ´e uma tarefa f´acil
para a maioria dos estudantes. Ogborn [9] afirma que a
utiliza¸c˜ao de ambientes de modelagem computacional
pode ser uma alternativa mais eficiente para introdu-
zir o estudante ao aprendizado de t´opicos de ciˆencias,
pois envolve a cria¸c˜ao de modelos sem que o ferramen-
tal matem´atico seja exigido. Muitos fenˆomenos da na-
tureza podem ser analisados, inicialmente, pelas suas
tendˆencias de varia¸c˜ao, j´a outros podem ser estudados
atrav´es da intera¸c˜ao entre os seus componentes b´asicos. Desta forma, pode-se distinguir entre trˆes tipos de am-
bientes de modelagem computacional [8]: Por´em, o Ambiente WorldMaker apresenta algumas
diferen¸cas em rela¸c˜ao aos autˆomatos celulares. A prin-
cipal delas ´e o fato das c´elulas n˜ao conterem valores,
mas objetos. Para que um modelo seja constru´ıdo no
WorldMaker ´e necess´ario inicialmente especificar quais
s˜ao os objetos relevantes para representar o sistema
em quest˜ao. Os objetos no WorldMaker podem ser
de dois tipos: Objetos e Objetos-cen´ario. Os objetos
representam todos os constituintes b´asicos do sistema
que podem se mover na rede, enquanto que os objetos-
cen´ario representam os locais por onde os objetos se
movem. Para exemplificar pode-se dizer que um obje-
to seria um coelho e um objeto-cen´ario seria a grama. Por defini¸c˜ao, uma c´elula s´o pode ocupar um objeto de
determinado tipo num mesmo tempo. Ap´os a especi-
fica¸c˜ao dos objetos ´e necess´ario especificar os eventos
que ocorrem no sistema e as regras que geram estes
eventos para, em seguida, proceder `a implementa¸c˜ao
do modelo no ambiente. • Ambientes de Modelagem Quantitativo Tamb´em denominados de ferramentas de modela-
gem matem´atica devido ao fato de demandarem a espe-
cifica¸c˜ao das vari´aveis relevantes de um sistema e suas
rela¸c˜oes alg´ebricas bem como seus poss´ıveis valores [10]; A interface do Ambiente WorldMaker ´e mostrada na
Fig. 2.1.
A modelagem computacional A modelagem computacional no contexto educacional
busca tanto representar modelos do mundo real quanto
acessar os modelos do estudante a partir de ferramen-
tas computacionais, aqui denominadas de Ambientes
de Modelagem Computacional. No in´ıcio, a utiliza¸c˜ao
de ferramentas computacionais demandava o conheci-
mento de alguma linguagem de programa¸c˜ao por parte
do estudante. Nos dias de hoje, com os avan¸cos da
computa¸c˜ao gr´afica, os ambientes de modelagem com-
putacional permitem que os modelos sejam criados de
forma icˆonica apenas com a utiliza¸c˜ao do mouse. Es-
ses avan¸cos permitiram o desenvolvimento dos mais va-
riados tipos de ambiente de modelagem computacional
e, conseq¨uentemente, diferentes tipos e abordagens de
modelagem. Figura 1 - Esquema de rela¸c˜oes entre os Modelos em Educa¸c˜ao. No entanto, modelos mentais podem ser considera-
dos como estruturas dinˆamicas que n˜ao cessam de se
desenvolver. Nesta perspectiva, Norman [6] descreve
alguns resultados de seus estudos sobre modelos men-
tais: • Modelos mentais s˜ao incompletos; • As habilidades das pessoas em “rodar” seus mo-
delos s˜ao limitadas; 455 nstru¸c˜ao de modelos no estudo de um t´opico de F´ısica utilizando um ambiente de modelagem computacional 455 A investiga¸c˜ao da constru¸c˜ao de modelos no estudo de um t´opico de F´ısica utilizando um ambiente de modelage 2.4.
O ambiente WorldMaker A id´eia para o desenvolvimento do Ambiente de Mo-
delagem WorldMaker surgiu a partir da afirma¸c˜ao de
Ogborn [9] de que as pessoas, inicialmente, criam mo-
delos do mundo em que vivem baseados nos objetos que
o comp˜oem e nos eventos que ocorrem com estes obje-
tos. Desta forma, o Ambiente WorldMaker permite que
determinados sistemas da natureza sejam representados
no computador atrav´es da especifica¸c˜ao dos objetos que
os constituem e das regras de intera¸c˜ao que regem o
comportamento destes objetos, gerando os eventos. • Atividades de Aprendizagem Explorat´oria O estudante ´e levado a observar o comportamento
de um modelo constru´ıdo por um especialista, n˜ao po-
dendo alterar a estrutura do modelo apresentado; • Atividades de Aprendizagem Expressiva O estudante ´e levado a criar modelos sobre o mundo
a partir de suas pr´oprias concep¸c˜oes. Podem, ainda, existir atividades de modelagem in-
termedi´arias onde o professor apresenta, inicialmente,
um modelo pronto e, ap´os o estudante ter interagido
exploratoriamente, permitir que ele modifique estrutu-
ralmente o modelo de acordo com seu entendimento,
caracterizando uma atividade semi–expressiva [8]. Este ambiente de modelagem foi constru´ıdo sobre a
id´eia dos Autˆomatos Celulares [12,13]. Um autˆomato
celular ´e uma ferramenta computacional utilizada para
modelar sistemas que podem ser representados como
unidades b´asicas que interagem entre si. Cada c´elula
de um autˆomato celular pode possuir um ´unico valor e
esse valor varia com o tempo de acordo com os valores
das c´elulas vizinhas. 2.2.
Tipos de atividades de modelagem com-
putacional em educa¸c˜ao utilizando-se o Ambiente de Modelagem Computacio-
nal Qualitativo WorldMaker. Baseando-se na intera¸c˜ao do aluno com o ambiente de
modelagem computacional, Mellar e Bliss [7] distin-
guem dois modos de atividades de modelagem compu-
tacional. S˜ao elas as: 2.3.
Ambientes de modelagem computacional 2 e ´e composta das seguintes estruturas: a Grade
dos Mundos, local onde os objetos s˜ao inseridos para in-
teragirem entre si e que permite a visualiza¸c˜ao do com-
portamento dos objetos do mundo e conseq¨uentemente
o comportamento do modelo como um todo; a Barra de
Ferramentas de Arquivo, onde se encontram os bot˜oes
de trabalho com os arquivos dos mundos; os Bot˜oes de
Execu¸c˜ao do Mundo: Voltar, Parar, Avan¸car, Passo-a-
passo, Avan¸co r´apido; os Pain´eis dos Objetos, onde os
objetos e objetos-cen´ario s˜ao definidos; Painel das Re-
gras, onde s˜ao listadas as regras de intera¸c˜ao associadas
a cada entidade do mundo. O ambiente WorldMaker
permite, ainda, que seja associada uma probabilidade
de execu¸c˜ao para cada regra. • Ambientes de Modelagem Semiquantitativo • Ambientes de Modelagem Semiquantitativo Ambiente que tem seu enfoque nas rela¸c˜oes entre
as vari´aveis e suas respectivas tendˆencias de varia¸c˜ao
e n˜ao na an´alise matem´atica ou quantifica¸c˜ao de
vari´aveis [11]; • Ambientes de Modelagem Qualitativa Uma terceira abordagem ´e o estudo de certos
fenˆomenos da natureza atrav´es da modelagem sem a
utiliza¸c˜ao da matem´atica e baseados na tomada de de-
cis˜ao ou na id´eia de que o mundo pode ser estudado
atrav´es da intera¸c˜ao entre os seus constituintes b´asi-
cos [8]. O presente trabalho relata um estudo realizado 456 Gomes e Ferracioli Gomes e Ferracioli Gomes e Ferracioli Figura 2 - Interface gr´afica do ambiente de modelagem computa-
cional qualitativo WorldMaker. veria representar o fenˆomeno da difus˜ao de um g´as em
um sistema fechado. O conjunto de procedimentos necess´arios para a
constru¸c˜ao de um modelo em um ambiente de mode-
lagem computacional ´e aqui denominado de Processo
Constru¸c˜ao do Modelo - PCM. O processo utilizado
neste estudo foi adaptado de Camiletti e Ferracioli [11],
que sugeriram uma seq¨uˆencia de sete passos para a
constru¸c˜ao do modelo em um ambiente de modelagem
computacional semiquantitativa. Dessa forma, o PCM
para esta investiga¸c˜ao passou a ser constitu´ıdo de oito
passos: Figura 2 - Interface gr´afica do ambiente de modelagem computa-
cional qualitativo WorldMaker. 1◦Passo: Defini¸c˜ao do sistema a ser estudado; 2◦Passo: Escolha do fenˆomeno de interesse; O Ambiente de Modelagem WorldMaker permite
que os modelos sejam criados de forma icˆonica com
o aux´ılio do mouse. Desta forma, ´e poss´ıvel utiliz´a-
lo para a realiza¸c˜ao de atividades de modelagem com
adolescentes e crian¸cas devido `a sua versatilidade na
constru¸c˜ao de modelos [9]. Os problemas a serem abor-
dados com esse ambiente de modelagem podem abran-
ger t´opicos de F´ısica, Biologia, Qu´ımica, Ciˆencias Am-
bientais, entre outros. 3◦Passo: Listagem dos elementos - objetos - rele-
vantes; 4◦Passo: Classifica¸c˜ao dos elementos listados em
Objetos-Cen´ario e Objetos; 5◦Passo: Constru¸c˜ao do modelo atrav´es de regras
de intera¸c˜ao entre os elementos; 6◦Passo:
Representa¸c˜ao das Intera¸c˜oes no Am-
biente WorldMaker; 7◦Passo: Simula¸c˜ao; Para que uma atividade de modelagem possa ser
realizada adequadamente ´e necess´ario que ela seja
estruturada considerando-se: as caracter´ısticas do am-
biente de modelagem computacional, o sistema a ser
estudado e o n´ıvel de escolaridade dos estudantes que a
executar˜ao. 8◦Passo: Valida¸c˜ao do modelo. • Ambientes de Modelagem Semiquantitativo Para o desenvolvimento da atividade de modelagem
expressiva no segundo m´odulo, foi disponibilizado um
material constitu´ıdo do texto base sobre o problema a
ser modelado e dos passos para constru¸c˜ao do modelo
que continham espa¸cos em branco para que os estu-
dantes pudessem escrever suas id´eias. Os dois m´odulos
do curso foram v´ıdeo filmados e, posteriormente os
di´alogos foram transcritos para serem analisados. As-
sim, a base de dados para an´alise consistiu das filma-
gens, dos di´alogos, do material escrito e dos modelos
constru´ıdos pelos estudantes. Atividades de Modelagem Expressiva utilizando o
Ambiente WorldMaker. Atividades de Modelagem Expressiva utilizando o
Ambiente WorldMaker. As atividades de modelagem expressiva do segundo
m´odulo abordaram dois t´opicos:
um relacionado `a
F´ısica e outro relacionado `a Biologia. O primeiro foi
o fenˆomeno da difus˜ao de um g´as e o segundo o sistema
predador-presa. A escolha destes t´opicos para a inves-
tiga¸c˜ao foi baseada na sua diferen¸ca de complexidade e
familiaridade dos estudantes com os mesmos. Este ar-
tigo abordar´a somente o estudo do fenˆomeno da difus˜ao
de um g´as, caracterizado por uma sala isolada contendo
um frasco de perfume fechado. Os estudantes foram so-
licitados a desenvolver um modelo que representasse o
que aconteceria ap´os o frasco de perfume ser aberto. Assim, o modelo a ser constru´ıdo pelos estudantes de- 3.
Concep¸c˜ao do estudo Este estudo foi desenvolvido com o objetivo de investi-
gar a intera¸c˜ao entre estudantes e o ambiente de mo-
delagem qualitativo WorldMaker em atividades de mo-
delagem expressiva. A coleta de dados foi feita atrav´es
da aplica¸c˜ao de um Curso de Extens˜ao com a dura¸c˜ao
de 3 horas, dividido em 2 m´odulos: Para a realiza¸c˜ao do estudo foram convidados de-
zesseis estudantes dos cursos de F´ısica e de Engenha-
ria El´etrica da Universidade Federal do Esp´ırito Santo. Esses estudantes foram distribu´ıdos em oito duplas e
o Curso de Extens˜ao foi realizado separadamente para
cada uma delas. • Primeiro M´odulo (1 hora) • Primeiro M´odulo (1 hora) Introdu¸c˜ao `a Met´afora de Objetos e Eventos e ao
Ambiente WorldMaker; Depois de terminada a coleta de dados, estes foram
analisados utilizando-se a t´ecnica das redes sistˆemicas
[14] apropriada para an´alise de dados de natureza pu-
ramente qualitativa. • Segundo M´odulo (2 horas) 4.2.
Aspectos da descri¸c˜ao do processo de mo-
delagem computacional comuns `as duplas A partir destes elementos foi constru´ıda a rede
sistˆemica que enfocou o Processo de Modelagem dos
estudantes atrav´es do Ambiente de Modelagem Com-
putacional Qualitativo WorldMaker. A Descri¸c˜ao do PCM foi dividida nos 5 aspectos re-
presentados na Fig. 4. Os aspectos apresentados s˜ao
In´ıcio da Atividade, que descreve a seq¨uˆencia inicial de
desenvolvimento da atividade de modelagem; Postura
de Trabalho, relacionado `a maneira com a qual os estu-
dantes trabalham a constru¸c˜ao dos modelos; Elementos
de Modelagem, que mostra como os estudantes inseri-
ram os elementos no modelo; Vers˜ao do Modelo, relacio-
nado ao processo de valida¸c˜ao do modelo pela dupla e
Modelo Final, que diz respeito `a vers˜ao final do modelo
constru´ıdo. 4.
An´alise dos dados As redes sistˆemicas [14] s˜ao utilizadas para avaliar da-
dos qualitativos atrav´es da categoriza¸c˜ao de seus prin-
cipais aspectos. Esta categoriza¸c˜ao ´e feita utilizando-se
os elementos b´asicos colchete e chave, onde • um colchete ´e usado para representar qualquer
conjunto de escolhas exclusivas; • uma chave ´e usada para representar um conjunto
de escolhas que ocorrem simultaneamente. u¸c˜ao de modelos no estudo de um t´opico de F´ısica utilizando um ambiente de modelagem computacional 457 A investiga¸c˜ao da constru¸c˜ao de modelos no estudo de um t´opico de F´ısica utilizando um ambiente de modelage 4.1.
A rede sistˆemica sobre o processo de mo-
delagem computacional A rede sistˆemica representa o comportamento dos estu-
dantes ao longo de todo o processo de constru¸c˜ao de
modelos no desenvolvimento da atividade de modela-
gem expressiva. Os aspectos inclu´ıdos na rede refletem
o conjunto de comportamentos observados em todas as
duplas. No entanto, isso n˜ao significa que necessaria-
mente todas as duplas apresentaram todos estes com-
portamentos. Para isso foi utilizado um recurso t´ecnico
das redes sistˆemicas denominado recurs˜ao e represen-
tado pela nota¸c˜ao. Esse recurso foi utilizado no aspecto
Regras, mostrado na Fig. 4, e significa que se deve
passar pelo mesmo colchete quantas vezes forem ne-
cess´arias at´e se obter a descri¸c˜ao desejada da situa¸c˜ao
antes de seguir adiante na rede [14]. O aspecto In´ıcio da Atividade, representado pela se-
gunda chave da Fig. 4, mostra que todos os estudantes
iniciaram a atividade pela leitura do texto e que quatro
duplas percorreram os passos de constru¸c˜ao do modelo
no papel, representado pelo primeiro colchete, expondo
suas id´eias sobre os elementos relacionados ao sistema. Os elementos listados por eles e apresentados no se-
gundo colchete foram Probabilidades: Dessa forma, a rede sistˆemica foi constru´ıda a par-
tir de dois aspectos mais gerais: a Descri¸c˜ao, onde s˜ao
abordados aspectos que descrevem o que foi realizado
pelos estudantes no processo de desenvolvimento da ati-
vidade de modelagem expressiva e a An´alise, onde s˜ao
abordados aspectos relativos a como os estudantes in-
teragiram com a atividade no processo de constru¸c˜ao de
modelos. Estes dois aspectos s˜ao retratados pela chave
representada na Fig. 3. • ...deixa do jeito que t´a que o equil´ıbrio a gente vˆe
pela probabilidade • ...deixa do jeito que t´a que o equil´ıbrio a gente vˆe
pela probabilidade Figura 3 - Rede sistˆemica para a an´alise dos dados. Figura 3 - Rede sistˆemica para a an´alise dos dados. O presente artigo se restringe aos resultados da in-
vestiga¸c˜ao da intera¸c˜ao entre estudantes e o ambiente
WorldMaker a partir dos aspectos da Descri¸c˜ao do Pro-
cesso de Constru¸c˜ao do Modelo. A Fig. 4 mostra Rede Sistˆemica da Descri¸c˜ao, e
observa-se que, partindo-se dos aspectos mais gerais `a
esquerda e seguindo para a direita, o n´ıvel de deta-
lhamento vai aumentando at´e atingir os termos mais
`a direita que representam informa¸c˜oes mais pr´oximas
dos dados brutos. A contagem dos s´ımbolos chave e
colchete ´e realizada da esquerda para a direita e de
cima para baixo. Assim, o aspecto Vers˜ao do Modelo
´e representado pela sexta chave e o aspecto Passos, re-
presentado pelo primeiro colchete da Fig. 4. O quadro-
resumo anexado `a rede mostra como foi o comporta-
mento das duplas durante o Processo de Constru¸c˜ao do
Modelo (PCM): a leitura por colunas esclarece qual foi
o comportamento de cada dupla e a leitura por linhas
proporciona a visualiza¸c˜ao de quais aspectos da rede
foram comuns entre as duplas. Figura 4 - Rede sistˆemica dos aspectos da descri¸c˜ao do processo
de modelagem computacional. Comportamento dos objetos: • ...l´a tem que ter v´arias part´ıculas, umas em cima
mais r´apidas e outras em baixo mais devagar Figura 4 - Rede sistˆemica dos aspectos da descri¸c˜ao do processo
de modelagem computacional. Eventos: Eventos: • as part´ıculas do g´as v˜ao estar colidindo e depois
de um tempo elas v˜ao tomar conta de toda sala. 458 Gomes e Ferracioli consensual do sistema associado a este fenˆomeno no
Ambiente WorldMaker ´e constitu´ıdo de: Observa-se que,
apesar de introduzido na rede o
aspecto Regras, nenhuma dupla abordou verbalmente
este aspecto no in´ıcio da atividade. • Dois objetos: parede e part´ıcula do g´as; O segundo aspecto da Descri¸c˜ao, Postura de Tra-
balho, representado pela terceira chave da Fig. 4, mos-
tra que todas as duplas optaram por trabalhar a lista-
gem das regras no papel. ´E tamb´em poss´ıvel observar
que, apesar da Dupla 01 ter expressado verbalmente
id´eias relacionadas a probabilidades no In´ıcio da Ativi-
dade, nenhuma dupla trabalhou com esse conceito nem
no papel nem no ambiente. • Trˆes eventos: part´ıcula se move aleatoriamente,
colis˜ao part´ıcula-part´ıcula e part´ıcula-parede. A Dupla 01 construiu um modelo de g´as como se fosse
uma nuvem que se espalha, associando a id´eia de um
efeito macrosc´opico - como uma nuvem. Os principais
argumentos utilizados por esta dupla para a constru¸c˜ao
do modelo foram: Em rela¸c˜ao aos Elementos de Modelagem - quarta
chave - observa-se que todas as duplas especificaram
corretamente os objetos, exceto pela Dupla 02 que espe-
cificou o objeto “parede” do recipiente como um objeto-
cen´ario. Contudo, os estudantes desta dupla, durante a
simula¸c˜ao observaram que algo estava errado com o mo-
delo e perceberam que deveriam modificar a “parede”
para objeto. Ocorre que o modelo do sistema abordado
n˜ao necessita da especifica¸c˜ao de objetos-cen´ario. Na
rede sistˆemica foi introduzida a possibilidade de apare-
cimento de dois tipos de regras: como evento e como
geradora de evento. O primeiro tipo ocorreria quando
a dupla, ao listar a regra, escrevesse o evento resultante
da regra, tais como, a part´ıcula se move e a part´ıcula
colide na parede. J´a o segundo tipo ocorreria quando a
dupla listasse a regra na estrutura se ... ent˜ao, ambos
representados pelo oitavo colchete. Observa-se atrav´es
da rede que todas as duplas especificaram as regras
como geradoras de evento. • O g´as se multiplica e toma conta de todo o espa¸co; • Se uma part´ıcula encontra espa¸co vazio ela se
multiplica; • Se part´ıcula encontra uma parede ela para. Na constru¸c˜ao do modelo no ambiente, os estudan-
tes criaram uma regra de multiplica¸c˜ao das part´ıculas
do g´as com o objetivo de simular seu espalhamento. Eventos: Ap´os a simula¸c˜ao, quando perguntados se o sistema
apresentava o comportamento de acordo com o que eles
esperavam, afirmaram que, em parte, sim. Os estudan-
tes defenderam dizendo que o g´as est´a se comportan-
do como uma nuvem e que fica um efeito mais ma-
crosc´opico. Por´em, ap´os observarem a simula¸c˜ao da
primeira vers˜ao do modelo e refletirem sobre o seu com-
portamento, assumiram que o modelo n˜ao estava com-
pleto e modificaram a regra de movimento da part´ıcula. Ap´os nova simula¸c˜ao perceberam que o comportamento
n˜ao havia sido alterado e complementaram que seria
necess´ario inserir alguma regra que fizesse o g´as se tor-
nar rarefeito quando afirmaram para gerar a rarefa¸c˜ao
´e necess´ario criar regras para destruir mol´eculas. No
entanto, essa altera¸c˜ao n˜ao foi implementada e termi-
naram a atividade. A sexta chave representa o aspecto Vers˜ao do Mo-
delo e mostra os procedimentos de Simula¸c˜ao, An´alise e
a verifica¸c˜ao da Evolu¸c˜ao do modelo constru´ıdo. Pode-
se observar atrav´es deste aspecto que todas as duplas
realizaram a simula¸c˜ao do modelo. O quadro resumo
mostra, ainda, que, a partir das simula¸c˜oes, quatro du-
plas realizaram modifica¸c˜oes ao modelo. O ´ultimo aspecto da descri¸c˜ao, denominado Modelo
Final, mostra como ficou a ´ultima vers˜ao do modelo
de cada dupla em rela¸c˜ao ao n´umero de elementos de
modelagem - objetos e regras -, especificando quais du-
plas conseguiram construir um modelo que apresentasse
o comportamento esperado. Observa-se, atrav´es deste
aspecto, que para o modelo constru´ıdo apresentasse tal
comportamento era necess´ario que tanto o n´umero de
objetos inseridos quanto as regras implementadas esti-
vesse de acordo com o modelo consensual esperado. A Dupla 02 realizou uma ´unica modifica¸c˜ao e uti-
lizou os seguintes argumentos durante a constru¸c˜ao da
primeira vers˜ao do modelo: • As part´ıculas do g´as se movem em linha reta caso
encontrem espa¸co vazio; • As part´ıculas do g´as colidem entre si e com as
paredes do recipiente; • As part´ıculas, ao colidirem, retornam na dire¸c˜ao
contr´aria. 4.3.
O aspecto vers˜ao do modelo:
an´alise da
evolu¸c˜ao dos modelos Percebe-se, atrav´es destes excertos, que os estu-
dantes n˜ao possu´ıam clareza sobre o movimento das
part´ıculas de um g´as, pois indicaram que elas se mo-
vem em linha reta. A dupla apresentou uma concep¸c˜ao
sobre o fenˆomeno de colis˜ao. Inicialmente, a dupla con-
cebeu o objeto “parede” como objeto-cen´ario. Durante
a primeira simula¸c˜ao, os estudantes observaram que as
part´ıculas atravessavam a “parede” e perceberam esta Como mencionado anteriormente, este artigo aborda os
aspectos da Descri¸c˜ao do PCM no estudo do fenˆomeno
da difus˜ao de um g´as com o Ambiente WorldMaker. As-
sim, a partir do aspecto Vers˜ao do Modelo, da Fig. 4,
pode-se analisar a poss´ıvel evolu¸c˜ao dos modelos de
cada dupla em separado, ressaltando que um modelo 459 A investiga¸c˜ao da constru¸c˜ao de modelos no estudo de um t´opico de F´ısica utilizando um ambiente de modelage nstru¸c˜ao de modelos no estudo de um t´opico de F´ısica utilizando um ambiente de modelagem computacional 459 conceitua¸c˜ao n˜ao era adequada. A partir da´ı, realiza-
ram a modifica¸c˜ao da “parede” para objeto e na se-
gunda simula¸c˜ao observaram que aquela situa¸c˜ao havia
sido corrigida e fizeram o seguinte coment´ario: devido ao fato de terem sido concebidos com a con-
cep¸c˜ao apenas de que as part´ıculas se moviam em linha
reta. As demais duplas conseguiram construir modelos
que apresentaram o comportamento esperado. A Dupla 04 realizou apenas uma modifica¸c˜ao e uti-
lizou os seguintes argumentos: • Eu acho que realmente algumas part´ıculas batem
e ficam ali nas proximidades das paredes. N˜ao
ficam sempre se movimentando pra l´a e pra c´a. • As mol´eculas do g´as v˜ao colidir entre si no canto
do recipiente e ap´os um tempo v˜ao estar em todos
os lugares; Dessa forma, apesar do modelo ter sido constru´ıdo
a partir do pressuposto das part´ıculas se deslocarem
em linha reta, os estudantes afirmaram que o modelo
apresentava o comportamento esperado por eles e n˜ao
realizaram mais modifica¸c˜oes. • As mol´eculas ao andar em linha reta e ap´os a
colis˜ao v˜ao andar aleatoriamente; Esta dupla construiu uma primeira vers˜ao do mo-
delo estabelecendo um movimento retil´ıneo para as
mol´eculas do g´as e esperando que a partir das co-
lis˜oes entre as mol´eculas todo o espa¸co seria ocupado. • ... elas est˜ao distribu´ıdas homogeneamente; Observa-se que os estudantes associaram concep¸c˜oes
sobre colis˜oes no primeiro e terceiro argumentos e, no
segundo, eles admitem que as part´ıculas possuem movi-
mento pr´oprio bastando haver um local vazio para que
elas se movimentem. Finalizada a primeira vers˜ao do
modelo, os estudantes realizaram a simula¸c˜ao e, na vi-
sualiza¸c˜ao do seu comportamento, verificaram que as
part´ıculas n˜ao se moviam como esperavam. Isso foi ex-
presso pelo excerto o movimento retil´ıneo das part´ıculas
est´a incorreto. Ent˜ao, modificaram a regra de movi-
mento da part´ıcula e o modelo passou a apresentar o
comportamento esperado. Ao final os estudantes co-
mentaram o movimento aleat´orio das part´ıculas torna
o modelo mais parecido com a realidade, o que pode ser
interpretado como o estabelecimento da rela¸c˜ao entre o
que ele estava visualizando no ambiente de modelagem
e sua concep¸c˜ao do sistema real. 4.3.
O aspecto vers˜ao do modelo:
an´alise da
evolu¸c˜ao dos modelos Ap´os a simula¸c˜ao eles observaram que o movimento das
mol´eculas n˜ao permitia que elas se espalhassem homo-
geneamente e argumentaram que elas n˜ao devem andar
em linha reta e que o comportamento das mol´eculas
deve ser aleat´orio. Assim, realizaram a modifica¸c˜ao no
movimento da part´ıcula, tornando-o aleat´orio. Ap´os
a visualiza¸c˜ao do comportamento do g´as a dupla ale-
gou que as mol´eculas est˜ao mais espalhadas. O modelo
desta dupla apresentou o comportamento esperado e a
dupla terminou a atividade. A Dupla 03 n˜ao realizou nenhuma modifica¸c˜ao no
modelo mesmo n˜ao tendo chegado ao modelo consen-
sual esperado. Os principais argumentos desta dupla
durante a constru¸c˜ao do modelo foram: Ap´os a simula¸c˜ao eles observaram que o movimento das
mol´eculas n˜ao permitia que elas se espalhassem homo-
geneamente e argumentaram que elas n˜ao devem andar
em linha reta e que o comportamento das mol´eculas
deve ser aleat´orio. Assim, realizaram a modifica¸c˜ao no
movimento da part´ıcula, tornando-o aleat´orio. Ap´os
a visualiza¸c˜ao do comportamento do g´as a dupla ale-
gou que as mol´eculas est˜ao mais espalhadas. O modelo
desta dupla apresentou o comportamento esperado e a
dupla terminou a atividade. • Part´ıculas diferentes com velocidades diferentes,
umas mais r´apidas outras menos r´apidas; • Part´ıculas se movendo e colidindo entre si; • Cria¸c˜ao de part´ıcula a partir da colis˜ao entre
part´ıculas; Nestes excertos ´e poss´ıvel perceber que o estudante
faz referˆencia `as diferentes velocidades das part´ıculas
de um mesmo g´as ao inv´es de referenciar a abordagem
sistˆemica quando o enfoque ´e na velocidade m´edia das
part´ıculas. A id´eia do estudante de implementar uma
regra que crie uma part´ıcula a partir da colis˜ao entre
outras duas part´ıculas pode estar relacionada `a id´eia
de quebra de mol´eculas atrav´es da colis˜ao. Os prin-
cipais argumentos ap´os a simula¸c˜ao e visualiza¸c˜ao do
comportamento do modelo foram: A Dupla 05 tamb´em realizou apenas uma modi-
fica¸c˜ao e os argumentos expostos foram: • As part´ıculas podem colidir de v´arias formas
(ˆangulos) diferentes; • O movimento ´e intr´ınseco `as part´ıculas, e se a
part´ıcula n˜ao encontra nada ela se move; • Se a part´ıcula encontra qualquer objeto ela muda
de sentido. • ... elas est˜ao distribu´ıdas homogeneamente; • As part´ıculas saem do recipiente e, ap´os algum
tempo, retornam novamente ao recipiente. No primeiro argumento, mesmo observando que as
part´ıculas estavam se movendo em linha reta, a dupla
alegou que elas ocupavam todo o espa¸co. J´a o segundo
argumento parece estar relacionado `a id´eia de equil´ıbrio
dinˆamico que inclui a possibilidade das part´ıculas po-
derem atingir qualquer configura¸c˜ao, inclusive a con-
figura¸c˜ao inicial de retorno ao recipiente. Assumindo
que o modelo apresentava o comportamento esperado
por eles, os estudantes finalizaram a atividade. Como se pode observar, essas trˆes primeiras duplas
analisadas n˜ao conseguiram construir um modelo que
apresentasse o comportamento do modelo consensual
esperado. A Dupla 01 concebeu regras que implemen-
tavam a cria¸c˜ao e/ou destrui¸c˜ao de mat´eria, sendo que
as Duplas 02 e 03 constru´ıram modelos semelhantes que
n˜ao estavam adequados ao modelo consensual esperado O desenvolvimento da Dupla 06 foi diferente das de-
mais, uma vez que eles conseguiram chegar ao modelo 460 Gomes e Ferracioli Gomes e Ferracioli esperado logo na em sua primeira vers˜ao. Os principais
argumentos utilizados por esta dupla na constru¸c˜ao do
modelo foram: Por outro lado, Wilenski [15], utilizando um mo-
delo de g´as constru´ıdo no ambiente de modelagem com-
putacional StarLogo, observou que estudantes levados
a interagir com o modelo, foram capazes de levan-
tar hip´oteses e fazer inferˆencias sobre o seu compor-
tamento, al´em de estabelecer rela¸c˜oes com o sistema
real. O autor reporta, ainda, que o desafio maior desta
linha de pesquisa ´e desenvolver ambientes de modela-
gem e pedagogias que promovam a modelagem de forma
efetiva no contexto educacional. • As part´ıculas andam aleatoriamente; • Elas colidem umas com as outras e com as pare-
des; • As mol´eculas ir˜ao se espalhar por todo o recipi-
ente; Neste contexto, os PCN indicam que a introdu¸c˜ao
de modelos no ensino pode promover a constru¸c˜ao de
abstra¸c˜oes indispens´aveis ao conhecimento cient´ıfico,
al´em de abordar a importˆancia da introdu¸c˜ao de novas
tecnologias no contexto do ensino visando a integra¸c˜ao
das mais variadas formas de ensinar [1]. Entretanto,
a introdu¸c˜ao do conceito de modelos e a inser¸c˜ao de
novas tecnologias levantam quest˜oes relacionadas tanto
`a forma¸c˜ao de recursos humanos quanto ao desenvolvi-
mento de atividades curriculares adequadas a essa nova
realidade [16]. Assim, a investiga¸c˜ao da integra¸c˜ao de
ambientes de modelagem computacional no contexto
educacional tem por objetivo a busca de alternativas
que levem em considera¸c˜ao os dois aspectos acima ci-
tados. 5.
Considera¸c˜oes finais Os resultados deste estudo mostram que os estudan-
tes foram capazes de construir modelos no ambiente
de modelagem computacional qualitativo a partir de
suas pr´oprias concep¸c˜oes e tamb´em de modific´a-los. As
modifica¸c˜oes eram realizadas se, durante a visualiza¸c˜ao
do comportamento da vers˜ao do modelo constru´ıdo,
os estudantes observassem que este n˜ao apresentava o
comportamento esperado, de acordo com suas pr´oprias
concep¸c˜oes. Para quatro duplas, esta reflex˜ao levou os
estudantes a realizarem uma evolu¸c˜ao no modelo cons-
tru´ıdo, sendo que para uma dupla n˜ao houve evolu¸c˜ao
pelo fato do modelo esperado ter sido alcan¸cado em sua
primeira vers˜ao. Em rela¸c˜ao ao desenvolvimento de atividades cur-
riculares alternativas para a integra¸c˜ao do conceito de
modelagem no ensino, foi desenvolvida uma seq¨uˆencia
de passos, descrita no item 3, para a constru¸c˜ao de mo-
delos utilizando o ambiente de modelagem WorldMa-
ker. A seq¨uˆencia sugerida permitiu que os estudantes
dividissem o PCM em trˆes etapas: primeira etapa, a
constru¸c˜ao do modelo no papel; segunda etapa, a im-
plementa¸c˜ao do modelo no ambiente e terceira etapa,
a an´alise e modifica¸c˜ao do modelo. Na primeira os
estudantes puderam refletir sobre o sistema, represen-
tando no papel tudo o que conseguiam expressar sobre
ele. Na segunda os estudantes puderam implementar,
no ambiente de modelagem computacional, as id´eias
que haviam expressado no papel. J´a na terceira etapa,
eles puderam simular e analisar o modelo, conflitando o
comportamento apresentado pelo mesmo no ambiente
com o esperado por eles. Os resultados obtidos reve-
lam que esta metodologia foi adequada a esse tipo de
atividade, sendo sugerido a repeti¸c˜ao dos passos 3, 4 e
5 ap´os o t´ermino da atividade como forma de verificar
uma poss´ıvel evolu¸c˜ao das concep¸c˜oes dos estudantes. A evolu¸c˜ao do modelo dos estudantes atrav´es da
visualiza¸c˜ao de seu comportamento dinˆamico no am-
biente de modelagem computacional qualitativo cor-
robora os resultados relatados por Camiletti e Ferra-
cioli [10] que, na utiliza¸c˜ao de um ambiente de mo-
delagem computacional qualitativo, observaram que a
visualiza¸c˜ao da simula¸c˜ao dinˆamica permitiu aos estu-
dantes a reflex˜ao sobre aspectos e conceitos que n˜ao
haviam considerado anteriormente `a atividade. A si-
mula¸c˜ao dinˆamica pode representar uma alternativa
para a limitada capacidade das pessoas ‘rodarem’ seus
pr´oprios modelos apontados por Norman [6]. Foi
tamb´em poss´ıvel observar, em vers˜oes intermedi´arias
dos modelos considerados finais pelos estudantes, as
caracter´ısticas de incompletude, instabilidade e n˜ao-
cientificidade apontadas por Norman [6]. 5.
Considera¸c˜oes finais Dessa forma, estes resultados apontam para uma
promissora utiliza¸c˜ao do Ambiente de Modelagem Com-
putacional Qualitativo WorldMaker no levantamento
de concep¸c˜oes de estudantes sobre determinado t´opico
bem na an´alise da evolu¸c˜ao de modelos ao longo do
processo de sua constru¸c˜ao. • As part´ıculas saem do recipiente e, ap´os algum
tempo, retornam novamente ao recipiente. Desta forma, este artigo relata um estudo con-
textualizado na investiga¸c˜ao da constru¸c˜ao de modelos
sobre um t´opico de conte´udo espec´ıfico atrav´es da uti-
liza¸c˜ao de um ambiente de modelagem computacional
qualitativo. Observa-se que estes trˆes argumentos refletem uma
concep¸c˜ao na dire¸c˜ao da conceitua¸c˜ao cient´ıfica do
mesmo. Tendo constru´ıdo o modelo, a dupla realizou
a simula¸c˜ao e observou que este apresentava o compor-
tamento esperado e terminaram a atividade. Este mo-
delo, al´em de se comportar como o esperado, possu´ıa
uma estrutura semelhante ao modelo consensual espe-
rado. Munic´ıpio de Vit´oria, ES. Munic´ıpio de Vit´oria, ES. de Mestrado, Universidade Federal do Esp´ırito Santo,
Vit´oria, 2003. Agradecimento Trabalho parcialmente financiado pelo CNPq, CAPES
e pelo FACITEC – Fundo de Apoio `a Ciˆencia e Tecno-
logia do Conselho Municipal de Ciˆencia e Tecnologia do u¸c˜ao de modelos no estudo de um t´opico de F´ısica utilizando um ambiente de modelagem computacional 461 Munic´ıpio de Vit´oria, ES. Referˆencias [9] J. Ogborn, in Modeling and Simulation in Science and
Mathematics Education, edited by W. Feurzeig and N. Roberts (Springer-Verlag, New York, 1999). [1] Brasil, Minist´erio da Educa¸c˜ao. Secretaria de Educa¸c˜ao
M´edia e Tecnol´ogica. Ciˆencias da Natureza,
Ma-
tem´atica e suas Tecnologias. Bras´ılia (1999). Dispon´ıvel
em www.mec.gov.br. Acesso em 30 maio 2004. [10] G. Camiletti e L. Ferracioli, in Anais do IV Semin´ario
sobre Representa¸c˜oes e Modelagem no Processo de
Ensino-Aprendizagem,
Vit´oria,
2003,
p. 253. Dis-
pon´ıvel em www.modelab.ufes.br/ivseminario. Acesso
em 30 maio 2004. [2] J. Gilbert e C. Boulter, in, Modelos e Educa¸c˜ao em
Ciˆencias, editado por D. Colinvaux (Ravil, Rio de Ja-
neiro, 1998). Artigo traduzido do original que integra
a coletˆanea The Handbook of Science Education, orga-
nizada por B. Frazer e K. Tobin. [11] G. Camiletti e L. Ferracioli, Caderno Catarinense de
Ensino de F´ısica 18, 2 (2001). [3] R. Harr´e, Physics Education 13, 275 (1978). [12] T. Gomes e L. Ferracioli, Fractais e Autˆomatos Ce-
lulares – Uma Vis˜ao Geral (2001). Dispon´ıvel em
http://www.modelab.ufes.br/automato. Acesso em 30
maio 2004. [4] J. Gilbert, Models in Science and Science Education,
Exploring Models and Modelling in Science and Tech-
nology Education – Contribution From the MISTRE
Group (The College of Estate Management, Reading,
1997). [13] T. Gomes and L. Ferracioli, in Proceedings of X Inter-
national Organization for Science and Technology Edu-
cation Symposium, Foz do Igua¸cu, 2002. Dispon´ıvel em
www.modelab.ufes.br/xioste. Acesso em 30 maio 2004. [5] P.N. Johnson-Laird, Mental Models (Harvard Press,
Cambridge, 1983). [6] D.A. Norman, in Mental Models, edited by D. Gent-
ner and A.L. Stevens (Lawrence Erlbaum Associates,
Hillsdale, 1983). [14] J. Bliss, M. Monk e J. Ogborn, Qualitative Data Ana-
lysis for Educational Research: A Guide of Systemic
Networks. (Croom Helm, London, 1983), 1st ed. [7] H. Mellar and J. Bliss, in Learning with Artificial
Worlds: Computer Based Modeling in the Curriculum
edited by H. Mellar, J. Bliss, R. Boohan, J. Ogborn
and C. Tompsett (The Falmer Press, London, 1994). [15] U. Wilenski, in Modeling and Simulation in Science and
Mathematics Education, edited by W. Feurzeig and N. Roberts (Springer-Verlag, New York, 1999). [8] T. Gomes, A Modelagem Computacional Qualitativa
no Estudo de T´opicos de Ciˆencias: Um Estudo Ex-
plorat´orio com Estudantes Universit´arios. Disserta¸c˜ao [16] L. Ferracioli e F.F. Sampaio, Revista Brasileira de In-
form´atica na Educa¸c˜ao 8, 83 (2001).
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Pedagogika, roč. 72, č. 1, 2022, s. 47–76
http://pages.pedf.cuni.cz/pedagogika/ Pedagogika, roč. 72, č. 1, 2022, s. 47–76
http://pages.pedf.cuni.cz/pedagogika/ Výzkumná studie 1 Tento příspěvek vznikl za podpory interního grantu UJEP-SGS-2020-43-007-3. Nástroj zjišťující epistemická přesvědčení
učitelů v kontextu výuky historie1 Jaroslav Říčan, Ondřej Pešout, Vilém Zábranský Abstrakt: Předkládaná výzkumná studie je zaměřena na měření epistemických přesvědčení
praktikujících učitelů a studentů učitelství v kontextu výuky historie. Cílem šetření bylo přezkoumat faktorovou strukturu přeloženého nástroje Beliefs about His-
tory Questionnaire (BHQ) adaptovaného na české prostředí. Nástroj vychází z rámců historické-
ho myšlení podpořených kognitivními výzkumy zasazenými do sociálně konstruktivistického pří-
stupu a výzkumů explicitně usouvztažňujících problematiku epistemických přesvědčení (vývojové
modely) ve vyučování dějepisu, historie a vlastivědy. Z výsledků vyplývá, že teoreticky navržené dimenze dotazníku BHQ tvoří psychometricky
samostatné faktory. Nástroj dobře rozlišuje dimenze subjektivistického a kriteriálního přístupu
k povaze historických znalostí. Naopak dimenze napodobujícího přístupu nedisponuje přesvědči-
vými psychometrickými vlastnostmi. Zjištěné závěry je třeba interpretovat vzhledem k vybrané populaci. Výsledky jsou diskutová-
ny ve vztahu se zahraničními výzkumy jak v doménově obecném kontextu výzkumů epistemic-
kých přesvědčení, tak i v doménově specifi ckém. Na základě uvedených výsledků jsou navrženy
potenciální možnosti optimalizace nástroje, především položková úprava napodobujícího pří-
stupu, a potenciální možnosti dalších výzkumných směřování. Modifi kovaný nástroj disponuje
potenciálem k uplatnění jak v pregraduální přípravě studentů učitelství historie (včetně děje-
pisu a vlastivědy), tak i učitelů z praxe. Na základě předložených výsledků empirického šetření
a kontextu výzkumů zahraniční provenience autoři této studie diskutují význam epistemických
přesvědčení napříč vzdělávacími stupni. Klíčová slova: vyučování dějepisu, vzdělávání učitelů, vzdělávání studentů učitelství, epis-
temická přesvědčení, faktorová analýza 1. ZDŮVODNĚNÍ svou spolehlivostí. Feucht (2017, s. 8)
výstižně uvádí, že „nehledě na to, jaký
typ informací a za jakým účelem lidé
[tyto informace] absorbují, měli by veri-
fi kovat jejich důvěryhodnost a relevanci
před tím, než učiní rozhodnutí nebo než VÝZNAMNOSTI TÉMATU Současná doba je charakterizována
prakticky neomezeným přístupem k in-
formačním zdrojům, které se však liší DOI: 10.14712/23362189.2021.1960 Říčan, J., Pešout, O., Zábranský, V. je uloží do dlouhodobé paměti“. Bråten
a kolektiv (2011) zdůrazňují, že při kon-
struování znalostí z množství rozmani-
tých zdrojů je výchozím bodem právě
aplikace epistemických strategií (hod-
nocení důvěryhodnosti zdroje, validity
argumentů). Tyto strategie představují
esenciální bod pro budoucí chování je-
dince, jelikož následné jednání (např. příprava na test, zastávaná pozice při
diskusi) bude podmíněno tím, „jak“ je-
dinec příslušné informace vnímá, uklá-
dá do paměti a následně používá. Jed-
ná se o rozhodnutí, ve kterých jedinec
„něčemu“ přiřkne atribut „znalost“, při-
čemž v daném procesu zvažuje přijatel-
nou míru nejistoty nebo hodnotí míru
důvěryhodnosti zdroje (Trevors et al.,
2017). Epistemická přesvědčení2 v pe-
dagogicko-psychologickém
výzkumu
referují o přesvědčení jedinců o tom,
jak znalost a proces poznávání ovlivňuje
a je ovlivňován učebním procesem (Ho-
fer, 2004). Tato přesvědčení defi nuje
Pajares (1992, s. 316) jako „individu-
ální posouzení pravdy nebo nepravdy
tvrzení; soud, který je možný odvodit
pouze prostřednictvím kolektivního
porozumění toho, co lidské bytosti
říkají, zamýšlejí a dělají“. Empirické studie dokládají vazbu mezi epistemic-
kými přesvědčeními žáků a klíčovými
komponenty učebního procesu. Podtr-
háváme výsledky korelační metaanaly-
tické studie Greena et al. (2018) analy-
zujících 132 neexperimenálních studií
(N = 55 418) prokazujících sice nízkou
(r = 0,16), avšak statisticky významnou
(p < 0,001) korelaci mezi epistemickými
přesvědčeními a rozmanitými učebními
aspekty, přičemž silnější korelace byla
nalezena v rámci doménové specifi -
city než v rámci obecně doménového
přístupu. Považujeme proto za stěžejní
zkoumat a rozvíjet epistemická přesvěd-
čení jedince v kontextu charakteristik
příslušné disciplíny, vyučovacího před-
mětu. je uloží do dlouhodobé paměti“. Bråten
a kolektiv (2011) zdůrazňují, že při kon-
struování znalostí z množství rozmani-
tých zdrojů je výchozím bodem právě
aplikace epistemických strategií (hod-
nocení důvěryhodnosti zdroje, validity
argumentů). Tyto strategie představují
esenciální bod pro budoucí chování je-
dince, jelikož následné jednání (např. příprava na test, zastávaná pozice při
diskusi) bude podmíněno tím, „jak“ je-
dinec příslušné informace vnímá, uklá-
dá do paměti a následně používá. Jed-
ná se o rozhodnutí, ve kterých jedinec
„něčemu“ přiřkne atribut „znalost“, při-
čemž v daném procesu zvažuje přijatel-
nou míru nejistoty nebo hodnotí míru
důvěryhodnosti zdroje (Trevors et al.,
2017). Epistemická přesvědčení2 v pe-
dagogicko-psychologickém
výzkumu
referují o přesvědčení jedinců o tom,
jak znalost a proces poznávání ovlivňuje
a je ovlivňován učebním procesem (Ho-
fer, 2004). 2 V souladu s Muisovou (2007) nebo Schommer-Aikinsovou (2004) používáme dále v textu termín epistemická
přesvědčení (epistemic beliefs), přestože refl ektujeme existenci dalších termínů (osobní epistemologie – personal
epistemology – Hofer & Pintrich, 1997; epistemická kognice – epistemic cognition – Greene, Azevedo & Torney-
-Purta, 2008; epistemické zdroje – epistemic resources – Hammer & Elby, 2003; epistemické myšlení – epistemic
thinking – Kuhn & Weinstock, 2002). 3 Na základě Parsonova konceptu čtyř základních funkcí (AGIL) fungování společenského systému můžeme
vznik historického vědomí vymezit na základě těchto složek: A – prožitá historická zkušenost, G – ideologický
výklad dějin, I – vědění utvářené historickou vědou a dějepisectvím a L – kulturněhistorická kolektivní paměť.
S ohledem na tento koncept pak můžeme zdůraznit, že výuka historie by měla primárně stavět na vědění utváře-
ném historickou vědou, avšak v rámci vzdělávacího procesu je nutné brát v úvahu i kolektivní paměť. 2 V souladu s Muisovou (2007) nebo Schommer-Aikinsovou (2004) používáme dále v textu termín epistemická
přesvědčení (epistemic beliefs), přestože refl ektujeme existenci dalších termínů (osobní epistemologie – personal
epistemology – Hofer & Pintrich, 1997; epistemická kognice – epistemic cognition – Greene, Azevedo & Torney-
-Purta, 2008; epistemické zdroje – epistemic resources – Hammer & Elby, 2003; epistemické myšlení – epistemic
thinking – Kuhn & Weinstock, 2002).
3 Na základě Parsonova konceptu čtyř základních funkcí (AGIL) fungování společenského systému můžeme
vznik historického vědomí vymezit na základě těchto složek: A – prožitá historická zkušenost, G – ideologický
výklad dějin, I – vědění utvářené historickou vědou a dějepisectvím a L – kulturněhistorická kolektivní paměť.
S ohledem na tento koncept pak můžeme zdůraznit, že výuka historie by měla primárně stavět na vědění utváře-
ném historickou vědou, avšak v rámci vzdělávacího procesu je nutné brát v úvahu i kolektivní paměť. VÝZNAMNOSTI TÉMATU Tato přesvědčení defi nuje
Pajares (1992, s. 316) jako „individu-
ální posouzení pravdy nebo nepravdy
tvrzení; soud, který je možný odvodit
pouze prostřednictvím kolektivního
porozumění toho, co lidské bytosti
říkají, zamýšlejí a dělají“. Empirické Ve shodě s Bartonem a Levstikovou
(2004) se domníváme, že výuka his-
torie by jedincům měla umožnit roz-
voj schopností, které jsou považovány
za důležité v demokratické a pluralitní
společnosti. Výzkumně bylo prokázá-
no, že vnímání (reprezentace) minulos-
ti může ovlivnit to, jak člověk vnímá
přítomnost (vazba s konceptem histo-
rického vědomí – Körber, 2011; po-
případě Šubrt & Vinopal, 20133) a jak
interpretuje konfl ikty (Shreiner, 2014). 48 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie Přístup jedinců se sofi stikovanějšími4
epistemickými přesvědčeními „kore-
sponduje s poznávajícím oceňujícím
více interpretací minulosti, ale zároveň
zohledňujícím oborová kritéria potřeb-
ná pro hodnocení historických zázna-
mů“ (tzv. kriteriální přístup – CrS; Sto-
el et al., 2017a, s. 120). Naopak méně
sofi stikovaná epistemická přesvědčení
referují k vnímání sebe sama buď jako
pasivního příjemce informací a vnímá-
ní minulosti jako fi xované (objektivní,
neměnné) záležitosti (tzv. napodobující
přístup – CS), nebo jako sice aktivní-
ho, ale nekritického jedince vnímajícího
historické interpretace jako pouhé ná-
zory (tzv. subjektivistický přístup – SS;
ibid.). Podobně jako Štech (2013) kritic-
ky refl ektuje nedostatek pedagogicko-
-psychologických poznatků pro akcen-
taci kompetencí v poslední kurikulární
reformě v České republice (RVP), tak
o nedostatečném množství výzkumů
psychologicko-pedagogického charak-
teru ve vztahu k oborové didaktice děje-
pisu se již v devadesátých letech vyjád-
řil Jílek (1996). Předkládaná empirická
studie se snaží tuto mezeru částečně
doplnit. Konstrukt epistemických pře-
svědčení, ke kterému v tomto příspěv-
ku v souvislosti s vyučováním historie
centrujeme pozornost, představuje po-
dle našeho názoru jedno z esenciál- ních témat efektivního vyučování pro
21. století. Zároveň reagujeme na apel
VanSledrighta a Maggionové (2016), že
výzkumy zjišťování epistemických pře-
svědčení v doméně historie jsou limito-
vány malým výzkumným vzorkem a jen
malé množství bylo realizováno mimo
severoamerický kontext. 4 V kontextu teoretického rozlišení mezi více a méně rozvinutými epistemickými přesvědčeními užíváme termi-
nologii více vs. méně sofi stikovaná epistemická přesvědčení, přestože opět můžeme nalézt odlišnou terminologii
(naïve vs. developed – Mierwald et al., 2016; naïve vs. nuanced – Stoel et al., 2017a; more vs. less appropriate –
Wiley et al., 2020). 5 Též jako second-order substantive knowledge (VanSledright & Limón, 2006), advanced conteptual, principled
knowledge beliefs (Greene & Yu, 2014) nebo second-order procedural understandings (Lee & Ashby, 2000). 2. EPISTEMICKÁ PŘESVĚDČENÍ
V KONTEXTU VÝUKY HISTORIE Napříč modely his-
torického myšlení je možné identifi kovat
dva sdílené koncepty, a to užívání zdrojů
a argumentace (Reisman et al., 2019). Oba tyto koncepty bezprostředně souvisí
s konstruktem epistemologie v pedago-
gicko-psychologickém výzkumu. 2. EPISTEMICKÁ PŘESVĚDČENÍ
V KONTEXTU VÝUKY HISTORIE Platforma výzkumů mezi doménou
historie a pedagogicko-psychologických
výzkumů epistemických přesvědčení úzce
souvisí s myslicími návyky, které ovlivňují
schopnost jedince pracovat s rezidui mi-
nulosti (VanSledright & Reddy, 2014). Počátky sledování vývojových trajektorií
změny v uvažování žáků a studentů je
možné vystopovat do posledního kvar-
tálu minulého století (Amherst History
Project – Brown, 1996). Ohniskem zájmu
výzkumníků se stala snaha přiblížit škol-
ní dějepis skutečné mateřské disciplíně
(Wineburg, 1991b) s akcentem na tzv. metakoncepty5 (Reisman, Brimsek &
Hollywood, 2019), kam je možné zařadit
např. čas, změnu, příčinu, empatii, důka-
zy, pohnutky (Lee, 2005), názor, vysvět-
lení (VanSledright & Maggioni, 2016),
průkaznost tvrzení (Lee & Shemilt,
2003), signifi kanci (Seixas, 1997) a expli- 49 Říčan, J., Pešout, O., Zábranský, V. (Yilmaz, 2008). Oproti vědám budo-
vaným na exaktním základu (chemie,
fyzika) není možné objekt historického
zájmu empiricky pozorovat, a proto his-
torici nestudují minulost, ale stopy minu-
losti skrze prameny, které vytvářejí limity
badatelského pole. Tento fakt nezbytně
vede k situaci, že historici, přestože sta-
ví na ověřených pracovních metodách
a vhodně položených badatelských otáz-
kách, subjektivně tvarují to, co interpre-
tují. Učitelé však tendují prezentovat his-
torii v lineární podobě (Bohan & Davis,
1998), orientovat výuku na fakta (Yeager
& Davis, 2005), vnímat znalosti jako
hlavní výstup vzdělávání (nezohlednění
procesu jejich získávání – Yilmaz, 2008). Interpretativní pojetí je někdy dokonce
vnímáno jako morálně nejednoznačné
(a tedy škodlivé). Učitelé věří v existenci
hlavního příběhu, což je podle nich jed-
nodušší a také méně riskantní („nemusíš
se obávat, že naštveš rodiče“ – James,
2008, s. 195). Výsledkem takto pojaté
výuky je pasivní role žáků orientujících se
k externím zdrojům znalosti (encyclope-
dia epistemology – VanSledright, 2002a)
a tato silná fi xace inhibuje žákovu
schopnost přiblížit se myšlení histori-
ka (Lee & Shemilt, 2003). citně i epistemologii (Wineburg, 2001). Primárně metakoncepty dávají tvar histo-
rii a vedou k jejímu pochopení (Seixas &
Morton, 2013), přestože koncepty první-
ho řádu (též substantive concepts – histo-
rický fakt, např. morové epidemie) jsou
samozřejmě nedílnou součástí výuky his-
torie. Nicméně vybavování si informací
z paměti není synonymem pro historic-
ké myšlení (Smith, 2017), které zahrnuje
komplexní kognitivní procesy užívané
historiky při uvažování o minulosti (Pu-
ustinen & Khawaja, 2020), přičemž tyto
procesy vycházejí z pojetí historického
myšlení, tj. „konstruování obhajitelných
interpretací událostí v minulosti“ (Freed-
man, 2015, s. 357). 2.2 Diference mezi začátečníky
a experty-historiky terpretativní vědy, ale při výuce prezen-
tuje historii jako jeden narativ. Centrální kutikulární otázkou pro
výuku historie zůstává vyvážení mezi
deklarativními a procedurálními zna-
lostmi (VanSledright, 1996). Wansink
a kolektiv (2017) uzavírají, že historická
znalost by neměla být prezentována jako
faktická nebo subjektivní záležitost, ale
jako otevřený narativ založený na prů-
kaznosti, který může být zpochybňován
a refl ektován. V souvislosti s výukou his-
torie se tedy otázka primárně týká toho,
jak se epistemická přesvědčení žáků
a studentů (i učitelů) mohou transfor-
movat směrem k epistemickým pře-
svědčením expertů-historiků (Lee &
Ashby, 2000; VanSledright, 2004). Be-
neš (2005, s. 21) uvádí, že „školní dějepis
nemá vychovávat odborníky; má pro-
střednictvím dostupných prostředků …
vychovávat ke stylu myšlení potřebnému
pro porozumění sociální skutečnosti, kte-
rá má historickou povahu, a tím přispí-
vat k tvorbě myšlenkové kultury jedince
i společnosti“. Historik je schopný komparovat, uvá-
dět do kontrastu a kombinovat informace
z rozličných zdrojů pro vytvoření kohe-
rentního mentálního modelu historické
události na základě hodnocení pohnutek
autora prostřednictvím čtení implicit-
ních informací v textu (VanSledright,
2002a). Na příkladu výzkumu dvou his-
toriků, z nichž jeden byl expert na udá-
losti občanské války v USA a druhý ni-
koli, došel Wineburg (1998) k závěru,
že i když historik nebyl expert na danou
oblast, byl schopný formulovat argumen-
ty z předložených pramenů, jelikož pou-
žíval metodologické přístupy historické
vědy. VanSledright (2010) doplňuje, že
začátečníci přistupují k historii dekontex-
tualizovaně a autor historického sdělení
podle nich podává neutrální informace. Na základě výzkumu mezi středoškolský-
mi studenty a experty dospěl Wineburg
(1991a) k závěru, že experti se zaměřovali
na hledání implicitních informací v tex-
tu (text jako komunikace mezi autorem
a jeho čtenáři). Dokonce i zdatní studenti
se snažili o vytvoření přímočarého smy-
slu z textu a opomíjeli implicitní prvky
v textu. S textem manipulovali jako s ne-
utrálním a objektivním zdrojem o minu-
losti a opomíjeli kontext. Tento aspekt
podtrhává VanSledright (2004, s. 344):
„… možná nejpozoruhodnější je, že jejich
[začátečníků] epistemologie textu je čas-
to diametrálně odlišná od epistemologie
odborníků. Jinými slovy se domnívají,
že význam je v textu nezprostředkovaný Několik posledních dekád výzkum-
níci pro oblast matematiky, přírodních
věd a historie popisují kognitivní aspekty
v kontextu dané disciplíny tím, že stu-
dují uvažování expertů (Shreiner, 2014). Nechceme po žácích, aby začali uvažo-
vat jako historici, ale můžeme jim toto
myšlení přiblížit a vést jejich uvažování
podobným směrem. 2.1 Povaha historické znalosti
a její odraz v přístupu učitelů Historie je idiosynkratická, měkká
a multiparadigmatická věda (Muis, Ben-
dixen & Haerle, 2006; ill-structured do-
main – Goldman et al., 2016), která je
vystavěna na interpretativní povaze disci-
plíny v sociálním kontextu (Beneš, 2011). Interpretativní charakteristika historické
znalosti je důsledkem její proměnlivosti
(nové důkazy či metodologie), subjekti-
vity (zkušenost), jazykové/literární slož-
ky (jazykové kategorie), sociokulturního
zázemí, ale i empirické opodstatněnosti Jestliže učitelé nevnímají historické
znalosti jako interpretativní záležitost,
nemohou přijmout, že znalost je konstru-
ována, což zjevně ovlivňuje způsob výuky
(Wansink et al., 2017). Hartzler-Mille-
rová (2001) poukazuje na jistý paradox
dvojitého epistemického standardu (dou-
ble epistemic standard), kdy učitel může
zastávat pozici vnímání historie jako in- 50 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie 2.2 Diference mezi začátečníky
a experty-historiky Následující pasáže
věnujeme relevantním diferencím mezi
expertem a začátečníkem, přičemž ex-
pertní validizace nástroje BHQ je jednou
ze součástí empirické části této studie. 51 Říčan, J., Pešout, O., Zábranský, V. autorem a jejich úkolem je ho [význam]
správně extrahovat“. Pedagogicko-psychologický výzkum v ob-
lasti epistemických přesvědčení v kontex-
tu doménově specifi ckého přístupu k vý-
uce historie vzhledem k užitému nástroji
v empirické části této studie rozebíráme
v intencích vývojového modelu epistemic-
kých přesvědčení. Wineburg (2001) dodává, že přestože
někteří zdatní studenti disponují soubo-
rem dostatečných kognitivních a meta-
kognitivních strategií pro budování po-
rozumění (jednoho) textu, tak v případě,
kdy jsou tito studenti vystaveni většímu
množství historických zdrojů, selhávají
při budování porozumění. Rouetová a její
kolegové (1998) zase upozornili na rozdíl-
ná kritéria při hodnocení zdrojů. V jejich
výzkumu američtí a francouzští vyso-
koškolští studenti hodnotili dokumenty
ve smyslu jejich užitečnosti a důvěryhod-
nosti, svá hodnocení měli odůvodnit a na-
psat esej, do jaké míry je možné obhájit
americkou intervenci v panamské revolu-
ci. V momentě, kdy se v textu objevily ne-
srovnalosti, tak se u studentů snížila důvě-
ra v daný text. Naopak historici daný text
ocenili a použili více kritérií při jeho hod-
nocení (hledisko obsahu, kdo text napsal). Konfl iktní perspektivy mají být uchovány
spíše jako klíč pro to, jak má být historic-
ká událost vysvětlena, než jako signál, že
jeden zdroj je (automaticky) méně důvěry-
hodný (Wiley et al., 2020). 3. VÝVOJOVÝ MODEL Počátky zájmu o epistemická pře-
svědčení jsou spjaté s výzkumem Perryho
(1970). Také jeho pokračovatelé se převáž-
ně zaměřovali na longitudinální výzkum
fenomenologického charakteru (Juklová,
2020) s tím, že se začala ozývat kritika
mířící na linearitu rozvoje epistemické-
ho přesvědčení a na jednodimenzionální
pojetí pokračovatelů rozvíjejících Perryho
model (např. tříúrovňový model refl ek-
tivního uvažování – Kitchener & King,
1981; model argumentativního uvažová-
ní – Kuhn, Cheney & Weinstock, 2000). Vývojové modely v různých terminologic-
kých nuancích referují ke třem základním
epistemickým úrovním (Barzilai & Ka’a-
dan, 2017), které již uvádíme v doménově
specifi ckém vztahu výuky historie: Tuto subkapitolu uzavíráme zjištěním
Wineburga (1991a, 1991b), který na zákla-
dě výzkumu začátečníků a expertů identi-
fi koval tři heuristiky typické pro experty:
1. zdrojování (vazba na autora, datum,
adresáty a účel psaní), 2. kontextualizace
(umístění dokumentu do času a místa, tj. braní v potaz okolnosti obklopující udá-
lost, a jak souvislosti mohly ovlivnit obsah
dokumentu) a 3. potvrzování (porovnává-
ní událostí a důkazů napříč více zdroji). i
1. Objektivismus (realism; dualism;
absolutism; CS). Znalost je vnímána jako
objektivní a z faktů složený konstrukt,
který může být excerpován přímo ze zku-
šeností a vnější pozorovatelné reality. Tato
úroveň se obvykle přimyká k situacím,
ve kterých žák získává první zkušenos-
ti s výukou historie. Minulost a historie
jsou ztotožňovány. Artefakty minulosti
„říkají“, jak se „to“ skutečně stalo (existu- 52 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie je jeden správný příběh). Externí autority
(historik, učitel, učebnice) jsou vnímány
jako jediné kompetentní zdroje schopné
oddělit správné informace od těch chyb-
ných (Lee & Ashby, 2000; under-sociali-
zed – národní narativ, memorování faktů,
perspektiva vítězů – Young & Muller,
2010). Tento přístup umožňuje žákům
vnést určitou dávku jistoty do nejisté-
ho světa. V intencích historické vědy se
jedná o naivní myšlenku vědecké objek-
tivity (Kloppenberg, 1989). Problém na-
stává v situacích, kdy jsou žáci vystaveni
kontradiktorním výkladům (protichůd-
ná svědectví). Poznávající se kognitivně
zarazí, zachytí do mentální slepé uličky
a přestává rozumět. Školní výuka historie
tuto epistemickou úroveň vítá, jelikož lépe
koresponduje s technokratickými postu-
py. Ve velmi sporných situacích akceptuje
i radikální subjektivismus (viz níže), aby
se vyhnula konfrontaci (VanSledright &
Maggioni, 2016). 2011; přijetí závěru na základě většinového
hlasování – VanSledright, 2002b). V této
rovině se jedná o naivní subjektivismus
(kompletní relativismus – Kloppenberg,
1989) referující o historii jako fundamen-
tálně subjektivní záležitosti. 3. VÝVOJOVÝ MODEL Obě tyto
úrovně (1 a 2) reprezentují miskoncepční
přístup v kontextu historie jako vědecké
disciplíny i školního předmětu (Puustinen
& Khawaja, 2020). 2011; přijetí závěru na základě většinového
hlasování – VanSledright, 2002b). V této
rovině se jedná o naivní subjektivismus
(kompletní relativismus – Kloppenberg,
1989) referující o historii jako fundamen-
tálně subjektivní záležitosti. Obě tyto
úrovně (1 a 2) reprezentují miskoncepční
přístup v kontextu historie jako vědecké
disciplíny i školního předmětu (Puustinen
& Khawaja, 2020). 2011; přijetí závěru na základě většinového
hlasování – VanSledright, 2002b). V této
rovině se jedná o naivní subjektivismus
(kompletní relativismus – Kloppenberg,
1989) referující o historii jako fundamen-
tálně subjektivní záležitosti. Obě tyto
úrovně (1 a 2) reprezentují miskoncepční
přístup v kontextu historie jako vědecké
disciplíny i školního předmětu (Puustinen
& Khawaja, 2020). 3. Kriterialismus (contextualism; eva-
luativism; objectivism-subjectivism; CrS). Znalost je vnímána jako individuální
a společenský konstrukt, který však může
být objektivizován prostřednictvím dů-
kazů (VanSledright & Maggioni, 2016). Tento přístup představuje nejvyšší úro-
veň sofi stikovanosti. Multiperspektivita
je nadále oceňována, ale pohledy jsou
posuzovány na základě stanovených kri-
térií (VanSledright, 2002b). Přes přítom-
nost protichůdných důkazů tak mohou
být konstruovány i odůvodněné závěry. V intencích historické vědy představuje
tato pozice pragmatický hermeneutický
přístup, který podle Kloppenberga (1989)
nahrazuje vznešenou představu vědecké
objektivity a noční můru naprostého re-
lativismu. Koordinace objektivních infor-
mací a subjektivního přístupu stojí na me-
todologických základech historické
vědy určovaných zvolenými kritérii
(Seixas & Morton, 2013; VanSledright,
2010). 2. Subjektivismus (relativism; multi-
plism; SS). Zpočátku je znalost vnímána
jako relativní konstrukt (všechny pohledy
mají stejnou váhu, neexistuje absolutní
pravda), následně jedinec tenduje pod-
porovat jednu stranu. Interpretace jsou
vnímány jako záležitosti názoru, přece-
ňuje se role subjektu (svědek historické
události jako nejspolehlivější zdroj). Po-
znávající nenastavuje kritéria posuzování
(selektivní výběr informací; over-socialized
– znalost záleží na poznávajícím – Young
& Muller, 2010). Při kritice brání výběr
na základě tvrzení, že v demokratických
společnostech má každý právo na svůj
názor (VanSledright, Maggioni & Reddy, Vývojové trajektorie konceptuální
změny ve vnímání historie 7–14letých
žáků mapovali Lee a Ashby (2000)
nebo Lee a Shemilt (2003) v projek-
tu „CHATA“ (Concepts of History and 53 Říčan, J., Pešout, O., Zábranský, V. 4. OPERACIONALIZACE
VÝVOJOVÉHO MODELU
EPISTEMICKÝCH PŘESVĚDČENÍ
V DOMÉNĚ HISTORIE ZA ÚČELEM
MĚŘENÍ 54 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie Tab. 1 Přehled nálezů použitých podob nástroje BHQ v předchozích studiích
Studie
Užitý nástroj
Počet (N), profi l
respondentů, země
realizace výzkumu
Základní popis, faktorové složení a reliabilita
Maggioni et al.,
2004
BLTHQ
72, učitelé
1. stupně, USA
21 položek (z analýzy vyřazena 1 položka u obou faktorů)
Faktor 1 (vysoké skóre = CS vs. nízké skóre = CrS);
0,72 pro pre-test i post-test
Faktor 2 (SS); 0,63 pre-test, 0,70 post-test
Maggioni et al.,
2009
BLTHQ
58, učitelé
1. stupně, USA
Analýza 12 položek ze studie Maggioni et al., 2004
Doplněno o analýzu retenčního post-testu stejného
výzkumu (zahrnuty jen ty položky, které jednoznačně sytily
pouze jeden ze dvou faktorů, a zároveň ty, jejichž sycení
v předchozích měřeních nabývala vysoké úrovně)
Faktor 1 (CrS); 0,61, 0,76 a 0,71 (7 položek)
Faktor 2 (SS); 0,51, 0,60 a 0,62 (5 položek)
Maggioni, 2010
BHQ
66, učitelé
1. a 2. stupně a SŠ,
USA
Faktor 1 (7 položek SS + 4 položky CS); 0,78
Faktor 2 (8 položek CrS + 3 SS, ale s negativním vztahem);
0,72
Stoel et al., 2015
BHQ
74, 11. ročníky
(průměrný věk –
16,6 let), Nizozemí
Faktor 1 (9 položek CS a SS); 0,81/0,81 (pre-test/post-test)
Faktor 2 (6 položek CrS); 0,57/0,40 (pre-test/post-test
pro N = 43) – faktor vyjmut z hlavní studie
Mierwald et al.,
2016
BHQ
Studie 1
124, 11. a 12. roč-
níky (medán
– 16,51 let),
Německo
Studie 2
224, studenti
učitelství historie,
Německo
Studie 1
Faktor 1 (SS + CS; 14 položek); 0,71
Faktor 2 (CrS; 8 položek); 0,70
Studie 2 (modifi kovaná verze BHQ; čtyřstupňová škála)
CS (6 položek; 0,69)
SS (5 položek; 0,70)
CrS (8 položek; 0,68)
Namamba & Rao,
2016
BHQ
30, učitelé historie,
Tanzanie
Přidání jedné položky do CS
CS (6 položek; 0,61)
SS (9 položek; 0,83)
CrS (8 položek; 0,62)
Stoel et al., 2017b
BHQ
2 pilotní výzkumy
140, 74; 11. roční-
ky, Nizozemí
Hlavní studie
95, 11. 4. OPERACIONALIZACE
VÝVOJOVÉHO MODELU
EPISTEMICKÝCH PŘESVĚDČENÍ
V DOMÉNĚ HISTORIE ZA ÚČELEM
MĚŘENÍ Teaching Approaches), přičemž defi -
novali šest vývojových úrovní, které
je možné kategorizovat do třech epis-
temických přístupů uvedených výše. Ve shodě s těmito autory zdůrazňuje-
me, že jednotlivé metakoncepty (včetně
epistemického přesvědčení) se nevy-
víjejí paralelně a v kontextu výuky se
spíše jedná o transformaci žákovských
miskonceptů spíše než o postupnou
akumulaci správných myšlenek. Lee
a Shemilt (2003) doplňují, že např. roz-
voj žákovských představ o spolehlivosti
zdrojových důkazů (posuzování na zá-
kladě zvolených kritérií) může sloužit
jako příklad důležité konceptuální změ-
ny v jejich uvažování. Podle Kuhno-
vé a kolektivu (2000) nejsou studenti
na konci sekundárního vzdělávání ještě
schopni „koordinovat objektivní a sub-
jektivní dimenze poznávání a znalosti“
(s. 310). Naopak VanSledright (2002b)
kontruje, že již žáci 5. tříd mohou být
učeni interpretativní povaze historie
a investigativním způsobům. Jak podtr-
hávají VanSledright a Reddyová (2014),
rozvoj jedince je zapotřebí vnímat v ne-
lineární podobě. Gottlieb a Wineburg
(2012) doplňují, že jedince není možné
„umístit“ do určité epistemické pozice. Ukazuje se, že normou je spíše episte-
mická inkonzistence (switching – ibid.;
wobbling – VanSledright & Reddy,
2014), jelikož epistemické přístupy ne-
jsou izolované od kontextu, jedinec je
mění v závislosti na interagujících fak-
torech, což souvisí s jeho adaptabilitou
(Maggioni, VanSledright & Alexander, Za významný bod v oblasti empiric-
kého uchopení epistemického přesvěd-
čení v oblasti historie považujeme studie
z první dekády nového milénia (Maggioni,
Alexander & VanSledright, 2004; Maggi-
oni et al., 2009), kdy Maggioniová a její
kolegové defi novali tříúrovňový model
rozvoje epistemického přesvědčení v ob-
lasti historie (Beliefs in Learning and Tea-
ching History Questionnaire – BLTHQ),
původně určeného pro studenty učitelství
1. stupně, přičemž stavěli na předchozích
modelech epistemické kognice (Kitche-
ner & King, 1981; Kuhn et al., 2000)
a historického myšlení (Lee & Shemilt,
2003; VanSledright, 2011; Wineburg,
2001). Na základě optimalizace nástroje
BLTHQ vznikl výzkumný nástroj zamě-
řený na diagnostiku jedincova přesvědče-
ní o historii (Beliefs about History Questi-
onnaire – BHQ; Maggioni, 2010). Kromě
třífaktorového řešení přinesla řada navazu-
jících výzkumů rozporuplné nálezy, které
souhrnně prezentujeme v tabulce 1. Velmi
často se při tom jednalo o spojení CS a SS
do jednoho faktoru a (b) CrS do druhého
faktoru. Teoretickým argumentem pro
spojování položek původně přináležejících
do CS nebo SS je východisko, že oba pří-
stupy postrádají „kritéria, jak se vypořá-
dat s interpretací protichůdných tvrzení“
(ibid., s. 121). Naopak ve studii Stoela et al. 4. OPERACIONALIZACE
VÝVOJOVÉHO MODELU
EPISTEMICKÝCH PŘESVĚDČENÍ
V DOMÉNĚ HISTORIE ZA ÚČELEM
MĚŘENÍ ročníky,
Nizozemí
CS – nedostatečná reliabilita – vyňato z hlavní studie;
podobně 2 položky z CrS (nízké sycení)
SS (9 položek; pre-test 0,77 pro N = 90; post-test
0,85 pro N = 90)
CrS (6 položek; pre-test 0,65 pro N = 88; post-test
0,65 pro N = 92)
Stoel et al., 2017a
BHQ (7 položek)
+ autory vytvořené
položky
922; 11. a 12. roč-
níky (556 – Ø 17
let; 366 – Ø 18 let)
Nizozemí
Naïve scale (15 položek)
Nuanced scale (11 položek)
Pouze 1 faktor splnil kritéria spolehlivosti (nature of
knowing-nuanced, 6 položek, z toho 2 položky nástroje
BHQ: 1, 7) Tab. 1 Přehled nálezů použitých podob nástroje BHQ v předchozích studiích 55 55 Říčan, J., Pešout, O., Zábranský, V. ZŠ/SŠ, v případě našeho východiska bylo
zohledněno zejména aktivní zapojení partici-
pantů do výuky dějepisu/historie/vlastivědy
(bez ohledu na jejich vystudovaný studijní
program), nebo jejich aktuální studium uči-
telského studijního programu či Studium
v oblasti pedagogických věd. Náš přístup
tak primárně klade důraz na epistemická
přesvědčení všech učitelů, kteří pracují nebo
potenciálně po dokončení studia budou pra-
covat ve školní praxi s historickou látkou, což
v souhrnu věrněji odráží pedagogické milieu
působící na žáky a studenty. (2017a) autoři nedoporučovali CS a SS
spojovat do jedné kategorie, jelikož explo-
ratorní faktorová analýza (EFA) na vzorku
922 studentů 10. a 11. ročníků odhalila
pět faktorů, přičemž pouze jeden faktor
jednoznačně naplnil standardy spolehli-
vosti měřicího nástroje. 5.1 Cíl šetření, výzkumná
otázka a hypotézy Cílem šetření bylo přezkoumat fak-
torovou strukturu přeloženého nástroje
BHQ (Maggioni, 2010) adaptovaného
na české prostředí. Předpokladem bylo,
že nástroj prokáže stabilní třífaktorovou
strukturu, ve které každá dimenze bude
představovat samostatný faktor, a prokáže
se přijatelným koefi cientem spolehlivosti. Skupině respondentů dominují ženy
oproti mužům, což souvisí s feminizací škol-
ství. Podobně převažují respondenti s dokon-
čeným magisterským vzděláním v některém
programu zaměřeném na učitelství vůči re-
spondentům s jiným typem vzdělání. Z hle-
diska zaměření studijního programu měli
absolventi programu učitelství 1. stupně ZŠ
a programu učitelství 2. stupně ZŠ zastou-
pení shodně okolo 15 %. Největší zastoupení
pak měli absolventi programu učitelství 2. stupně ZŠ a SŠ, a to 40 % z dotazovaného
vzorku, přičemž podobně jako absolventi
pouze učitelství 2. stupně ZŠ převládali re-
spondenti, u kterých byl jeden ze studova-
ných oborů dějepis nebo historie. Poměrně
velkou skupinu tvořili prozatím nekvalifi ko-
vaní dotazovaní. Při sledování celkové doby
praxe nebyl do statistiky zahrnut aktuálně
běžící školní rok 2020/2021, a proto počet
respondentů bez praxe přesáhl 10 %. Přesně
polovinu dotazovaných tvořili učitelé s pra-
xí do deseti let. S rostoucí délkou praxe pak
četnost ve zkoumaném vzorku klesala. V pří-
padě průměrného týdenního počtu hodin
výuky dějepisu jsme zaznamenali výrazný 5.2 Výzkumný vzorek Šetření bylo realizováno v lednu 2021
prostřednictvím online dotazníku v aplikaci
Survio na vzorku 216 učitelů, přičemž bylo
vyřazeno 11 odpovědí z důvodu nedokonče-
ní celého dotazníku. Učitelé byli kontaktová-
ni e-mailem či prostřednictvím vedení škol,
které v minulosti spolupracovalo s autory
článku (dostupný výběr). Dotazník byl dále
šířen prostřednictvím zájmových sociálních
sítí, které mohly oslovit cílové učitele. Ko-
horta respondentů je poměrně heterogenní. Oproti výzkumu L. Maggioni (2009) a A. Namamba společně s C. Rao (2016), kteří
se zaměřili na učitele 1. stupně ZŠ, respek-
tive na učitele dějepisu/historie pro 2. stupeň 56 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie Z hlediska prostorové distribuce působení
jednotlivých respondentů podle krajů ČR
bylo s ohledem na velikost jednotlivých
krajů a hlavně s ohledem na aktivní vazby
na naši domovskou instituci nejvíce dotazo-
vaných z Ústeckého kraje (30 %), dále pak
z kraje Středočeského (13 %) a vzdálenějšího
Jihomoravského (10 %). Ostatní kraje měly
zastoupení v řádu jednotek procent. podíl v intervalu do čtyř hodin týdně. Je to
dáno právě tím, že do zkoumaného vzorku
respondentů byli zahrnuti učitelé 1. stupně
ZŠ vyučující vlastivědu, která není tolik ho-
dinově dotována, a především tím, že žádný
z vyučujících nebyl specialista pouze na tento
předmět. Na druhou stranu učitelů, jejichž
úvazek z větší části naplňovala výuka děje-
pisu/historie, byla výrazná menšina – 10 %. Tab. 2 Charakteristika výzkumného vzorku v kontextu demografi ckých údajů
Proměnná
Odpověď
Relativní
četnost (%)
Gender
Ženy
73
Muži
26
Nechci odpovídat
1
Jiné
0
Dosažené vzdělání
Mgr. 74
Ing. 4
Bc. 11
Jiné
11
Studijní program v Mgr. stupni studia
Vystudoval/a jsem studijní program učitelství
68
Vystudoval/a jsem neučitelský studijní program, mám dostudo-
vané „učitelské minimum“ (= SPV, dříve DPS)
8
Nemám vystudovaný program učitelství/učitelské minimum
a ani aktuálně nestuduji
2
Aktuálně studuji program učitelství nebo „učitelské minimum“
220
Zaměření studijního programu (kvalifi kace)
Učitelství 1. stupně ZŠ
16
Učitelství 2. stupně ZŠ (z toho s oborem dějepis/historie)
18 (81)
Učitelství 2. stupně ZŠ a SŠ (z toho s oborem dějepis/historie)
42 (95)
Aktuálně nekvalifi kovaný/á (studuji nebo nemám vystudovaný
program učitelství ani „učitelské minimum“)
24 Tab. 2 Charakteristika výzkumného vzorku v kontextu demografi ckých údajů Říčan, J., Pešout, O., Zábranský, V. 5.3 Popis nástroje, překlad,
procedura a distribuce ní význam výzkumného nástroje. V prv-
ním kroku jsme využili dva akademiky
(ISCED 8) s jazykovou úrovní C2 (post-
graduální studium anglického jazyka). Zpětný překlad opět učinili dva akademici
(ISCED 7 a 8, oba s jazykovou úrovní C2). Ve druhém kroku jsme oslovili bilingvního
překladatele (ISCED 7), který zároveň učí
na SŠ, dále odborníka na oblast pedagogic-
ké psychologie6 a historika (oba ISCED 8). Odborníci na oblast pedagogické psycholo-
gie a historie byli přítomni i v závěrečném,
třetím kroku, při kterém byl komuniko-
ván konstrukt epistemického přesvědčení
s expertem (doc.) věnujícím se vzdělávání
cizinců, pluringvismu, didaktice českého
jazyka, onomastice a frazeologii. Respon-
denti se vyjadřovali na šestistupňové Liker-
tově škále (1 = silně nesouhlasím, 6 = silně
souhlasím). Po překladu a úpravě zahra-
ničního nástroje proběhla pilotáž (prosinec
2020) u šesti respondentů (dva učitelé 1. stupně ZŠ, dva učitelé dějepisu 2. stupně
ZŠ, jeden student učitelství pro 1. stupeň
ZŠ a jeden student učitelství historie pro
SŠ). Hlavní sběr dat proběhl v lednu 2021
online formou (Survio.cz). Maggionová (2010) defi novala v ná-
stroji BHQ (22 položek) tři přístupy
(stances) kopírující úrovně ve vývojových
epistemických modelech: • CS (položky 5, 9, 16, 19 a 20) • CS (položky 5, 9, 16, 19 a 20) • CS (položky 5, 9, 16, 19 a 20)
Př.: Fakta hovoří sama za sebe. (16)
• SS (položky 2, 4, 6, 8, 10, 12, 14, 17 a 22)
Př.: Dobří žáci vědí, že dějiny jsou
ve své podstatě věcí názoru. (6)
• CrS (položky 1, 3, 7, 11, 13, 15, 18 a 21)
Př.: Porovnávání pramenů a chápání
autorova pohledu na věc jsou nezbytnými
složkami učení se dějepisu. (13) • CS (položky 5, 9, 16, 19 a 20)
Př.: Fakta hovoří sama za sebe. (16)
• SS (položky 2, 4, 6, 8, 10, 12, 14, 17 a 22)
Př.: Dobří žáci vědí, že dějiny jsou
ve své podstatě věcí názoru. (6)
• CrS (položky 1, 3, 7, 11, 13, 15, 18 a 21) Př.: Fakta hovoří sama za sebe. (16) Př.: Fakta hovoří sama za sebe. (16)
• SS (položky 2, 4, 6, 8, 10, 12, 14, 17 a 22) (p
y
)
Př.: Dobří žáci vědí, že dějiny jsou
ve své podstatě věcí názoru. (6)
• CrS (položky 1 3 7 11 13 15 18 a 21) p
Př.: Porovnávání pramenů a chápání
autorova pohledu na věc jsou nezbytnými
složkami učení se dějepisu. 5.2 Výzkumný vzorek Celková doba praxe
Bez praxe
11
1–5 let
36
6–10 let
14
11–15 let
9
16–20 let
8
21–25 let
7
26–30 let
7
31 a více let
8
Průměrný počet hodin výuky dějepisu (vlastivědy, historie) týdně
0
16
1–2
36
3–4
12
6–8
14
9–10
9
11 a více
13
Kraj působnosti na škole
Žádný
6
Mimo ČR
0
Hlavní město Praha
7
Středočeský kraj
13
Jihočeský kraj
2
Plzeňský kraj
3
Karlovarský kraj
6
Ústecký kraj
30
Liberecký kraj
3
Královéhradecký kraj
7
Pardubický kraj
2
Kraj Vysočina
2
Jihomoravský kraj
10
Olomoucký kraj
2
Moravskoslezský kraj
6
Zlínský kraj
1 58 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie 6 Druhý autor tohoto příspěvku. Úlohou příslušného experta bylo spolupodílet se na refl exi zachování interpre-
tační roviny pro zjišťování pedagogicko-psychologického konstruktu epistemických přesvědčení (konstruktová
validita). 5.3 Popis nástroje, překlad,
procedura a distribuce (13) Při překladu nástroje jsme se drželi
protokolu doporučovaného v rámci kros-
-kulturních výzkumů (Klassen et al.,
2009). Kros-kulturní validizace zahrnuje
zjištění, zdali nástroj, který vznikl v ur-
čitém sociokulturním kontextu, je smys-
luplně aplikovatelný, a tedy ekvivalentní
pro užití v jiném sociokulturním rámci
(Huang & Wong, 2014). Klassen a ko-
lektiv (2009) uvádějí tři kroky: 1. překlad
a zpětný překlad do původního jazyka
(translation – back-translation process), 2. zapojení bi- nebo multi-lingvních jedinců,
které lze považovat za experty v příslušné
výzkumné doméně (aby byl překlad správ-
ný nejen jazykově, nýbrž i platný ve smyslu
své podstaty), 3. refl exe, zda změny v pře-
kladech (změna větné stavby a formulací)
odrážejí pouze zohlednění sociokulturních
a jazykových rozdílů a nezkreslují původ- 5.4 Analýza dat V první fázi bylo zjišťováno, zda se
u položek nachází dostatečná variabilita
dat. Z tabulky 2 lze vysledovat, že u všech
položek se objevila dostatečná míra varia-
bility. Ve zkoumaném vzorku byly zastou-
peny u každé položky všechny hodnoty Říčan, J., Pešout, O., Zábranský, V. Tab. 3 Variabilita dat v kontextu položkové analýzy nástroje BHGQ
Položky
Průměr (SD)
Směrodatná odchylka
Šikmost
BHQ.1
4,83
1,26
–1,01
BHQ.2
3,49
1,30
0,01
BHQ.3
4,31
1,09
–0,40
BHQ.4
2,93
1,28
0,47
BHQ.5
3,24
1,47
0,22
BHQ.6
3,38
1,27
0,05
BHQ.7
4,96
1,12
–1,29
BHQ.8
2,78
1,23
0,46
BHQ.9
2,77
1,24
0,45
BHQ.10
2,53
1,47
0,66
BHQ.11
4,54
1,33
–0,82
BHQ.12
2,74
1,28
0,31
BHQ.13
4,86
1,10
–1,04
BHQ.14
3,10
1,40
0,25
BHQ.15
4,01
1,27
–0,29
BHQ.16
3,74
1,28
–0,19
BHQ.17
3,64
1,18
–0,36
BHQ.18
4,24
1,00
–0,55
BHQ.19
3,34
1,43
0,22
BHQ.20
3,63
1,51
–0,23
BHQ.21
4,64
1,09
–0,92
BHQ.22
1,96
1,13
1,52
P
U š h p l ž k b l d
ž
t é
í h h d
t ( i
1
6) K ýp čt šik
ti b l p
žit p Tab. 3 Variabilita dat v kontextu položkové analýzy nástroje BHGQ Pozn.: U všech položek bylo dosaženo extrémních hodnot (min. = 1; max. = 6). K výpočtu šikmosti byl použit upra-
vený Fisherův–Pearsonův standardizovaný momentový koefi cient. Pozn.: U všech položek bylo dosaženo extrémních hodnot (min. = 1; max. = 6). K výpočtu šikmosti byl použit upra-
vený Fisherův–Pearsonův standardizovaný momentový koefi cient. Likertovy škály, průměr se pohyboval ko-
lem středových hodnot (M = 1,96–4,96)
i směrodatná odchylka naznačila dosta-
tečnou variabilitu odpovědí (SD ≥ 0,99). ověřuje latentní faktorovou strukturu empi-
ricky získaných dat s předem zvolenou teorií
či předpokladem, které jsou často vyjádřeny
strukturálním modelem (Brown, 2006). V tomto případě jsme testovali dimenze do-
tazníku BHQ a jejich faktorovou strukturu K ověření validity testu byla použita
konfi rmační faktorová analý za (CFA). CFA 7 Autoři Lei a Shiverdecker otestovali a porovnali několik odhadovacích algoritmů na ordinálních datech s chybějícími
údaji vytvořených simulací Monte Carlo. Autoři přehledně shrnují čtyři testované odhadovací algoritmy, konkréně
WLSMV (Weighted Least Square Mean and Variance adjusted), MLR (Robust Maximum Likelihood), MLR-CAT
(Robust Maximum Likelihood s převedením ordinálních dat na kategorické) a ML-CAT (Maximum Likelihood
s převedením ordinálních dat na kategorické). Ačkoli v mnoha případech se ukázalo jako optimální využít MLR-CAT,
obzvlášť v případech, kdy posuzovací škály nabízely méně možností (2–3), v některých situacích se ukázaly vhodnější
jiné odhadovací algoritmy. Konkrétně v případě, kdy se ukazuje nízká velikost vzorku (N < 200) a počet nabízených
možností se rovná pěti, autoři doporučují použít MLR. Velikost vzorku v překládané studii (N = 216) se blíží „kritic-
ké“ hranici (N < 200) podobně jako počet možností v kontextu Likertovy škály (6). 6. VÝSLEDKY Naopak položka BHQ.19 původně přiřa-
zená k dimenzi CS vykázala překvapivě
akceptovatelnou zátěž. Vzhledem k historii
prolínání faktorů v předchozích studiích
jsme tuto položku do dimenze také zařadili
(Maggioni, 2010). Po důsledné revizi polo-
žek, kde byly z modelu vynechány polož-
ky BHQ.2, BHQ.14 a BHQ.22 a naopak
přidána položka BHQ.19, byl nalezen ještě
vhodnější model než v původní verzi dotaz-
níku, který nabídl velmi dobré vlastnosti,
2/df (5) = 1,49; CFI = 0,98; TLI = 0,97;
RMSEA = 0,05; SRMR = 0,04. Vnitřní
konzistence byla velmi dobrá ( 0,81;
0,81) a z toho důvodu byl tento model
pro příslušnou dimenzi přijat. Nejdříve byly metodou CFA posuzo-
vány jednotlivé tři subškály BHQ dotaz-
níku samostatně, abychom ověřili jejich
jednodimenzionalitu. Na základě fakto-
rových zátěží položek u jednotlivých di-
menzí a přijatelnosti indexů shody byly
položky jednotlivých dimenzí označeny
a revidovány. Očekávaným předpokladem
bylo doložit originální model navzájem
korelovaných dimenzí (obr. 1). U dimenze CS byly zaznamenány vel-
mi nízké faktorové zátěže téměř u všech
testovaných položek (< 0,50) s výjimkou
položky BHQ.16 (0,54). Hodnoty posu-
zující vhodnost modelu pro faktorovou
strukturu CS projevily vlastnosti 2/df (5)
= 1,70; CFI = 0,94; TLI = 0,87; RMSEA
= 0,06; SRMR = 0,04, které nakonec ne-
splnily podmínky dobrého modelu a ani
redukcí položek se nepodařilo vlastnosti
modelu vylepšit. Index vnitřní konzisten-
ce položek nezaručil spolehlivost měření
pro žádný z testovaných modelů ( 0,51;
0,51). Konečně i model dimenze CrS pro-
jevil po revizi položek (vyjmuté BHQ.13
a BHQ.15), z důvodu nízkých faktorových
zátěží přijatelné hodnoty 2/df (9) = 2,20;
CFI = 0,95; TLI = 0,92; RMSEA = 0,08;
SRMR = 0,04 a prokázal se také ucházející
vnitřní konzistencí ( 0,74; 0,75). Jelikož žádný z původních výzkumů
nenaznačil existenci latentního faktoru,
vytvořili jsme model obou korelovaných
faktorů SS a CrS (obr. 2). Tento model,
který byl kombinací dvou zachovaných
faktorů (SS a CrS), poukázal na výsledné
indexy shody (tab. 4) pohybující se na hra-
nici přijatelnosti modelu. Přestože někte- Dimenze SS zprvu ukázala hodnoty
akceptovatelného modelu 2/df (27) = 2,18;
CFI = 0,93; TLI = 0,91; RMSEA = 0,08;
SRMR = 0,05, nicméně u některých polo-
žek se ukázaly velmi nízké faktorové zátěže. Tab. 60 60 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie Obr. 1. Původní třídimenzionální model navržený Maggioniovou (2010) s čísly položek vztahují-
cích se k jednotlivým dimenzím Obr. 1. Původní třídimenzionální model navržený Maggioniovou (2010) s čísly položek vztahují-
cích se k jednotlivým dimenzím s ohledem na data získaná od vybrané popu-
lace českých učitelů. Pro kalkulaci CFA byl
použit statistický program R 4.0.3, balíček
„lavaan“ (verze 0.6-7). Vzhledem k povaze
dat byla zvolena robustní verze odhado- vacího algoritmu ML (Robust Maximum
Likelihood – MLR), který prokázal vhodné
vlastnosti při výpočtech s ordinálními daty
použitými v dotazníku BHQ (Lei & Shiver-
decker, 2020).7 61 Říčan, J., Pešout, O., Zábranský, V. 6. VÝSLEDKY 4 Indexy shody (goodness-of-fi t indices) pro fi nální testované modely metodou CFA
Model
2
df
2/df
CFI
TLI
RMSEA
SRMR
CS
3,611
2
1,70
0,96
0,89
0,06
0,03
SS
20,792
14
1,49
0,98
0,97
0,05
0,04
CrS
19,810
9
2,20
0,95
0,92
0,08
0,04
2F Model SS+CrS
124,638
64
1,95
0,91
0,88
0,07
0,08 Tab. 4 Indexy shody (goodness-of-fi t indices) pro fi nální testované modely metodou CFA Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie dějiny“, „Historie/Obecné dějiny“), byla
provedena expertní validizace výsledných
dimenzí dotazníku BHQ (podobně Stoel
et al., 2017a). Vycházíme při tom z tzv. ex-
pertního modelu učení (Ericsson, 2009),
tj. z rozdílů v uvažování mezi experty-his-
toriky a začátečníky. Teoreticky je možné
předpokládat vyšší úroveň sofi stikova-
nosti v oblasti epistemických přesvědčení
expertů-historiků (VanSledright, 2004). Očekávali jsme, že experti budou mít ten-
denci k vyšším skórům v dimenzi CrS,
která indikuje sofi stikovanější přístup
k povaze historických znalostí. Vzorek se
skládal z 10 žen a 10 mužů ve středním
věku (M = 38,65, SD = 8,43). V tabulce
6 uvádíme základní údaje o vzdělání ex-
pertního souboru. ré indexy nebyly vyhovující (TLI < 0,90),
v modelu spolu dimenze SS a CrS signi-
fi kantně nekorelovaly (r = –0,09, p = 35),
společně však všechny položky dosáhly
přijatelné vnitřní konzistence ( 0,74;
0,65) za podmínky, že jedna z dimenzí
byla skórována negativně. Model s neko-
relovanými dimenzemi dosahuje téměř
identických hodnot indexů shody. V ta-
bulce 5 jsou prezentovány faktorové zátěže
položek. 7. EXPERTNÍ VALIDIZACE 6 Přehled dosaženého akademického titulu u expertního souboru Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie V tabulce 7 se nacházejí podrobně-
ji rozepsané získané hodnoty expertů
na jednotlivých položkách vybraných di-
menzí, které naznačují, že vyšších hodnot
bylo dosahováno u dimenze CrS, zatímco
položky dimenze SS ukazovaly nižší hod-
noty. přístup (CrS; znalost je objektivizovatel-
ná prostřednictvím zvolených kritérií). V této studii bylo prostřednictvím CFA
možné nalézt optimální faktorovou struk-
turu pouze u dimenzí SS a CrS. Expertní
validizace podpořila předpoklady studie,
tedy že experti skórovali v dimenzi CrS
výše než v dimenzi SS. 7. EXPERTNÍ VALIDIZACE Na vzorku 20 vybraných vysokoškol-
sky vzdělaných odborníků (dále „exper-
tů“) v oboru, jehož název obsahoval ter-
mín „dějiny“ či „historie“ (např. „České 63
Obr. 2. Autory zkoumaný výsledný dvoudimenzionální model Obr. 2. Autory zkoumaný výsledný dvoudimenzionální model 63 63 Říčan, J., Pešout, O., Zábranský, V. Tab. 5 Standardizované hodnoty faktorových zátěží jednotlivých položek na faktory u výsledných
testovaných modelů
Faktorové
subškály
CS
SS
CrS
Dvoufaktorový
model SS+CrS
Std. koef. Std. chyba
Std. koef. Std. chyba
Std. koef. Std. chyba
Std. koef. Std. chyba
BHQ.5
0,492
0,161
–
–
–
–
–
–
BHQ.9*
0,323
0,120
–
–
–
–
–
–
BHQ.16
0,538
0,141
–
–
–
–
–
–
BHQ.19**
0,349
0,154
0,552
0,096
–
–
0,553
0,096
BHQ.20
0,443
0,171
–
–
–
–
–
–
BHQ.2*
–
–
0,487
0,097
–
–
–
–
BHQ.4
–
–
0,701
0,078
–
–
0,701
0,078
BHQ.6
–
–
0,588
0,096
–
–
0,565
0,097
BHQ.8
–
–
0,626
0,092
–
–
0,628
0,092
BHQ.10
–
–
0,520
0,104
–
–
0,523
0,105
BHQ.12
–
–
0,751
0,083
–
–
0,754
0,083
BHQ.14*
–
–
0,530
0,098
–
–
–
–
BHQ.17
–
–
0,636
0,092
–
–
0,631
0,095
BHQ.22*
–
–
0,442
0,108
–
–
–
–
BHQ.1
–
–
–
–
0,692
0,104
0,680
0,102
BHQ.3
–
–
–
–
0,524
0,099
0,531
0,098
BHQ.7
–
–
–
–
0,663
0,116
0,664
0,113
BHQ.11
–
–
–
–
0,641
0,099
0,644
0,095
BHQ.13*
–
–
–
–
0,437
0,094
–
–
BHQ.15*
–
–
–
–
0,418
0,096
–
–
BHQ.18
–
–
–
–
0,437
0,094
0,425
0,097
BHQ.21
–
–
–
–
0,418
0,096
0,427
0,093i Tab. 5 Standardizované hodnoty faktorových zátěží jednotlivých položek na faktory u výsledných
testovaných modelů Pozn.: Všechny uvedené položky ukázaly hodnoty p < 0,05; * položky byly z fi nálního modelu vyjmuty
(hodnoty faktorových zátěží pocházejí z posledního modelu, ve kterém byly použity před vyřazením);
** položka byla mezi dimenzemi přesunuta. Tab. 6 Přehled dosaženého akademického titulu u expertního souboru
Nejvyšší získaný titul
N
Rok získání titulu (medián)
Mgr. 3
2016
Ph.D. 14
2014
Doc. 3
2017 Tab. 6 Přehled dosaženého akademického titulu u expertního souboru
Nejvyšší získaný titul
N
Rok získání titulu (medián)
Mgr. 3
2016
Ph.D. 14
2014
Doc. 3
2017 Tab. 8. INTERPRETACE DAT
A DISKUSE Doporučená verze nástroje, která pro-
kázala přijatelné hodnoty, je rozdělená
na dvě dimenze. První dimenze je tvořená
šesti položkami SS a jednou položkou CS. Seskupování položek SS a CS do jednoho
faktoru je ospravedlnitelné v intencích
společného znaku obou přístupů – jedi-
nec nenastavuje (metodologická) kritéria
při analýze historických zdrojů zejména
v kontextu vytváření koherentní mentál-
ní reprezentace při práci s protichůdný-
mi tvrzeními, což je jeden z typických Tato studie si kladla za cíl validizovat
nástroj BHQ určený pro měření epis-
temických přesvědčení k povaze histo-
rických znalostí. Původní verze nástroje
BHQ je rozdělena do tří základních di-
menzí: napodobující přístup (CS; znalost
je neměnná a excerpovatelná z vnější ob-
jektivní reality), subjektivistický přístup
(SS; znalost je subjektivní) a kriteriální Tab. 7 Dosažené hodnoty expertů
Položky
Průměr
Směrodatná odchylka
SS
BHQ.4
3,00
0,92
BHQ.6
3,20
1,24
BHQ.8
2,45
1,10
BHQ.10
2,10
1,02
BHQ.12
2,70
1,08
BHQ.17
4,10
1,02
BHQ.19
3,40
1,43
CrS
BHQ.1
5,10
1,02
BHQ.3
4,75
0,72
BHQ.7
5,45
0,51
BHQ.11
5,45
0,60
BHQ.18
4,75
0,72
BHQ.21
4,80
0,70 Tab. 7 Dosažené hodnoty expertů 65 Říčan, J., Pešout, O., Zábranský, V. škála u vzorku středoškolských studentů
nevykázala dostatečnou reliabilitu (nízké
hodnoty rovněž – Mierwald et al., 2016;
nízké hodnoty na vzorku učitelů – Na-
mamba & Rao, 2016). znaků při práci s historickými zdroji
(Maggioni, 2010). Toto seskupování je
patrné i v dalších studiích (Maggioni,
2010; Mierwald et al., 2016; Stoel et al.,
2015). Přijatelná spolehlivost škály SS
byla potvrzena v předcházejících studi-
ích (studenti učitelství – Mierwald et al.,
2016; učitelé – Namamba & Rao, 2016;
středoškolští studenti – Stoel, van Drie
& Van Boxtel, 2017b). Druhá dimenze
se skládá z šesti položek CrS. Ve shodě
s výsledky této studie a dalších výzku-
mů cílených na respondenty z řad uči-
telů (Maggioni et al., 2009; Maggioni,
2010; Namamba & Rao, 2016) nebo
studentů učitelství (Mierwald et al.,
2016) vykazují dimenze dobré – nebo
alespoň dostatečné – výsledné hodnoty
spolehlivosti. Nižší hodnoty spolehlivos-
ti měření se v případě aplikace škály CrS
obvykle nacházely na vzorku středoškol-
ských studentů (Stoel et al., 2015, 2017b;
kromě položky BHQ.1 a 7 – Stoel et al.,
2017a). U dimenze CS jsme našli mírně
nedostatečnou podporu pro její faktoro-
vou strukturu a především nepřijatelnou
vnitřní konzistenci měření. 9. ZÁVĚR hledání vazeb mezi zjištěnými výsledky
a dalšími studiemi vždy částečně pro-
blematické. Výzkumný vzorek se skládal
z relativně heterogenního souboru (roz-
manitý dosažený stupeň vzdělání, stu-
dijní program a jeho zaměření). Nabízí
se realizace výzkumu na homogennějším
souboru s vyšším počtem respondentů
nebo například srovnání skupin učitelů
s vystudovaným oborem učitelství děje-
pisu/historie (potenciál přínosu hodno-
cení impaktu vysokoškolského oborové-
ho prostředí na utváření epistemického
přesvědčení, což ale nebylo předmětem
této studie). Přes kritiku Wileyové a jejích kolegů
(2020), že BHQ zjišťuje spíše přesvědčení
o tom, jak je historie vyučována a učena,
než že by se jednalo o epistemickou po-
vahu historické znalosti, je daný instru-
ment užíván v původní i modifi kované
verzi (vazba mezi strategickými znalostmi
druhého řádu a kauzálním zdůvodňová-
ním a CrS – Stoel et al., 2017b; koherence
mezi epistemickými úrovněmi v kontex-
tu historie jako vědy a výuky historie –
VanSledright & Reddy, 2014). Podle Wi-
neburga (1991a) práce s textem ve škole,
v kontextu vývojového modelu epistemic-
kých přesvědčení, produkuje a posiluje
objektivistickou úroveň, jelikož jsou uči-
teli akcentovány faktografi cké informace
(Yeager & Davis, 2005) místo poselství
autora (Affl erbach & VanSledright, 2001). Studenti si příliš nedělají starosti s tím, že
je jakkoli problematické informace z textu
extrahovat (defi nitivní, objektivní a spo-
lehlivý doklad o skutečných událostech
– VanSledright, 1997). Pamětní učení
a orientace na znalosti posiluje vnímání
znalosti jako souboru izolovaných jedno-
tek (místo konceptuální struktury) a jako
defi nitivní záležitosti (znalost je jistá,
nepodléhá času, místu, aktérům, meto-
dologii). Absence centrování pozornos-
ti na proces získávání znalostí (Yilmaz,
2008) inhibuje žákovu schopnost přiblí-
žit se myšlení historika (Lee & Shemilt,
2003). Žáci i středoškolští a vysokoškol-
ští studenti učitelství historie (dějepisu,
vlastivědy) by měli být učeni posuzovat
spolehlivost
zdrojů
jejich
užitečnost V navazujícím šetření v českém pro-
středí je možné replikovat zahraniční
výzkumy v kontextu epistemických pře-
svědčení učitelů a jejich pojetí vyučování. McCrum (2013) ve svém výzkumu dospěl
k závěru, že modernistické přesvědčení
(modernist beliefs – minulost je pozna-
telná a reprezentovaná v textech histori-
ků) souvisí s transmisivně-instruktivním
způsobem vedení výuky (teacher-centered
approach), zatímco postmodernistické
přesvědčení (post-modernist beliefs – zna-
lost jako konstrukce historiků, která dává
smysl v kontextu prostřednictvím dis-
kurzu) s přístupem výuky orientované
na dítě (pupil-centered approach). 8. INTERPRETACE DAT
A DISKUSE Ačkoli stále
shledáváme tento faktor smysluplným
a věříme, že v měření epistemických
přístupů k historii má své místo, upo-
zorňujeme na velmi omezenou možnost
interpretovat výsledky z této škály. Je
možné, že po revizi položek a znovu-
testování této škály na novém souboru
ukáže škála přijatelné hodnoty měření. Zjištění, že dimenze CS nabyla hodnot
těsně pod hranicí akceptovatelnosti,
nejblíže koresponduje s výsledky studie
Stoela a kolektivu (2017b), ve které tato V další fázi ověřování možnosti vy-
užití předkládaného nástroje bylo zjiš-
ťováno, zda dvě výsledné dimenze sdí-
lí společný latentní faktor. Faktorová
struktura nabídla hodnoty těsně pod
hranicí přijatelnosti. Dimenze spolu ne-
korelují a jako nejvhodnější se tedy nabí-
zí využívat škály odděleně a neslučovat je
dohromady. y
Z výše uvedených výsledků navrhuje-
me přijmout dva faktory pro měření epis-
temických přesvědčení učitelů SS obo-
hacené o jednu položku CS (BHQ.19)
a CrS. Dimenze CS vykázala nízké
hodnoty spolehlivosti a pochybnou jed-
nodimenzionální faktorovou strukturu,
proto doporučujeme tuto dimenzi revi-
dovat: revidovat znění některých polo-
žek, přidat položky (viz přidání jedné
položky do dimenze ve studii Namam-
ba & Rao, 2016) a analyzovat původní
položky v nové validizační studii. Záro-
veň musíme zdůraznit, že v řadě studií
se explicitně uvádí slovo „optimalizace“
daného nástroje („změna znění polož-
ky“, „doplnění položky“, „odstranění
položky“, „čtyřstupňová Likertova šká-
la“ – Mierwald et al., 2016; „pětistup-
ňová škála“ – Namamba & Rao, 2016),
avšak bližší specifi kace změny zejména
ve vztahu k jednotlivým položkám ne-
byly většinou uváděny. Změny obvykle
vyplývaly z adaptace na příslušné socio-
kulturní prostředí a z toho důvodu bude 66 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie 9. ZÁVĚR Podob-
ně Namamba a Rao (2016) na základě
regresní analýzy prokázali, že úrovně CS
a SS predikují transmisivně-instruktivní
způsob vyučování, a naopak CrS predi-
kuje na dítě orientované učení. Budoucí
studie mohou využít tyto konstrukty
ke kriteriální validizaci nástroje měřícího
epistemická přesvědčení. 67 Říčan, J., Pešout, O., Zábranský, V. již u mladších jedinců (před vstupem
na SŠ), což první vývojové modely ne-
předpokládají (Kuhn et al., 2000), avšak
mnoho autorů dokazuje opak (Foster &
Yaeger, 1999; VanSledright, 2002a). Zá-
roveň je nutné věnovat pozornost epis-
temickým přesvědčením učitelů, jelikož
ovlivňují užití organizačních forem a vy-
učovacích metod (Wansink et al., 2017). V kontextu edukačního procesu zdůraz-
ňujeme, že užití strategií a epistemický
rozvoj úměrně nezrají (Alpasan, Yalvac
& Willson, 2017) a epistemická přesvěd-
čení je možné cíleně ovlivňovat (Wiley et
al., 2020). a reprezentativnost, učit se konstruovat
a dekonstruovat historické narativy, vní-
mat, že se nejedná o přímé kopie minu-
losti, ale interpretace, přičemž by se měli
učit hodnotit platnost těchto interpretací
na základě stanovených kritérií (Seixas
& Morton, 2013; VanSledright, 2011),
což v kontextu vývojových epistemic-
kých modelů odpovídá teoreticky nejso-
fi stikovanější úrovni. V intencích užíva-
né terminologie Maggionové (2010) se
jedná o tzv. kriteriální přístup. Považujeme za stěžejní do učebních
aktivit implementovat aktivity rozví-
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Stoel, G., Drie, v. J., & Boxtel, v. C. (2015). Teaching towards historical expertise: Developing
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suring epistemological beliefs in history education: An exploration of naïve and nuanced Stoel, G., Drie, v. J., & Boxtel, v. C. (2015). Teaching towards historical expertise: Developing
a pedagogy for fostering causal reasoning in history. Journal of Curriculum Studies, 47(1), 49–76. Stoel, G., Logtenberg, A., Wansink, B., Huijgen, T., Boxtel, v. C., & Drie, v. J. (2017a). Mea-
suring epistemological beliefs in history education: An exploration of naïve and nuanced
beliefs. International Journal of Educational Research, 83, 120–134. 71 Říčan, J., Pešout, O., Zábranský, V. Stoel, G. L., Van Drie, J. P., & van Boxtel, C. A. (2017b). The effects of explicit teaching of
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of secondary education. Journal of Social Studies Research, 41(1), 11–24. 72 Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie Wiley, J., Griffin, T. D., Steffens, B., & Anne Britt, M. (2020). Epistemic beliefs about the
value of integrating information across multiple documents in history. Learning and
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Univerzita J. E. Purkyně v Ústí nad Labem, Pedagogická fakulta, Katedra psychologie;
e-mail: ondrej.pesout@ujep.cz Mgr. Vilém Zábranský, Ph.D.
Univerzita J. E. Purkyně v Ústí nad Labem, Filozofi cká fakulta, Katedra historie;
e-mail: vilem.zabransky@ujep.cz 9. ZÁVĚR (1991a). On the reading of historical texts: Notes on the breach between
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University Press. Yeager, E. A., & Davis, O. L. (1995). Between campus and classroom: Secondary student-teachers’
thinking about historical texts. Journal of Research and Development in Education, 29(1), 1–8. Yilmaz, K. (2008). Social studies teachers’ conceptions of history: Calling on historiography. The Journal of Educational Research, 101(3), 158–176. Young, M., & Muller, J. (2010). Three educational scenarios for the future: Lessons from the
sociology of knowledge. European Journal of Education, 45(1), 11–27. Mgr. Vilém Zábranský, Ph.D. Univerzita J. E. Purkyně v Ústí nad Labem, Filozofi cká fakulta, Katedra historie;
e-mail: vilem.zabransky@ujep.cz 73 73 Říčan, J., Pešout, O., Zábranský, V. ŘÍČAN, J., PEŠOUT, O., ZÁBRANSKÝ, V. An Instrument Measuring
Epistemological Beliefs about History Instruction Th is paper describes the instrument the Beliefs about History Questionnaire (BHQ),
designed to assess the epistemological beliefs of pre-service and in-service history teachers. Th e
instrument that was developed was based on developmental models of epistemological beliefs
that are implemented in the domain of history. Th e conceptual background of the BHQ is based
on empirical evidence from scientifi c studies about historical thinking, the social constructivists’
perspectives on learning, and particular empirical studies linking epistemological beliefs and the
use of history instruction in classrooms. Th e current study conducted confi rmatory factor analysis on the BHQ as translated and
adapted to the Czech educational environment. Th e results suggest that the three conceptualized
dimensions of the BHQ present qualitatively distinct factors. Specifi cally, the instrument
distinguishes very well the conceptualized, subjectivist, and criterialist stances towards knowledge
about history. However, the instrument did not manifest methodologically robust properties for
the concept of the copier stance dimension. Th e fi ndings are interpreted within the population
that was sampled and other empirical fi ndings in both domain-general and domain-specifi c
educational contexts. Keywords: history instruction, pre-service teacher education, in-service teacher education,
epistemic beliefs, factor analysis 9. ZÁVĚR Th e results that are presented outline the further research avenues to validate
and implement the instrument that is introduced; more specifi cally, a revision of the copier stance
dimension is recommended. Th e modifi ed BHQ instrument can potentially be used to improve
both the pre-service and in-service training of history teachers. Th e alignment of the evidence that
is presented with the documented research from other countries provides a context for furthering
the discussion on positioning epistemic beliefs within the current Czech national curriculum. Keywords: history instruction, pre-service teacher education, in-service teacher education,
epistemic beliefs, factor analysis Nástroj zjišťující epistemická přesvědčení učitelů v kontextu výuky historie Příloha 1 Vážený pane učiteli, vážená paní učitelko! Rádi bychom Vás požádali o vyplnění dotazníku mapujícího Vaše postoje o povaze historic-
kých znalostí a dějepisného (vlastivědného) vyučování (vyučování historie). Cílem šetření je zjistit
praktické uplatnění převzatého nástroje Maggioniové (2010). Po úvodních 8 otázkách je uveden
samotný nástroj skládající se z 22 položek. Prosíme, ke každé z těchto položek vyjádřete míru
svého souhlasu na šestistupňové škále od 1 (= silně nesouhlasím) po 6 (= silně souhlasím). Vyplnění
dotazníku Vám zabere přibližně 14–17 minut. Jaroslav Říčan, Vilém Zábranský a Ondřej Pešout Epistemická přesvědčení o povaze historických znalostí a dějepisného vyučování
Silně
nesou-
hlasím
Nesou-
hlasím
Spíše
nesou-
hlasím
Spíše
souhla-
sím
Souhla-
sím
Silně
souhla-
sím
** 1. Žáky je nezbytné učit,
aby své uvažování podporovali
důkazy. 1
2
3
4
5
6
2. Dějiny jsou jednoduše věcí
interpretace. 1
2
3
4
5
6
** 3. Poznatky z dějin jsou
svědomitým výsledkem bada-
telské činnosti. 1
2
3
4
5
6
* 4. Žáci, kteří čtou mnoho děje-
pisných knih, se učí, že minulost
je to, co historik udělá, aby byla. 1
2
3
4
5
6
5. Názorový rozpor nad udá-
lostí, která se odehrála v mi-
nulosti, se vždy děje z důvodu
nedostatku důkazů. 1
2
3
4
5
6
* 6. Dobří žáci vědí, že dějiny
jsou ve své podstatě věc názoru. 1
2
3
4
5
6
** 7. Žáky je třeba učit praco-
vat s protichůdnými důkazy. 1
2
3
4
5
6
* 8. Historická tvrzení jsou
neobhajitelná, jelikož se jed-
noduše jedná o interpretace. 1
2
3
4
5
6
9 Obecné dovednosti dobře
číst a porozumět čtenému sta-
čí pro to se dobře učit dějepis. 1
2
3
4
5
6 75 Říčan, J., Pešout, O., Zábranský, V. * 10 Jelikož neexistuje
způsob, jak zjistit, co se v mi-
nulosti skutečně stalo, mohou
žáci věřit jakémukoli příběhu,
který si vyberou. 1
2
3
4
5
6
** 11. Historie je kritické
zkoumání minulosti. 1
2
3
4
5
6
* 12. Minulost je taková, ja-
kou ji historik udělá, aby byla. 1
2
3
4
5
6
13. Porovnávání pramenů
a chápání autorova pohledu
na věc jsou nezbytnými slož-
kami učení se dějepisu. 1
2
3
4
5
6
14. Nelze s určitostí vědět
něco o dávné minulosti, jeli-
kož nikdo z nás tam nebyl. Příloha 1 1
2
3
4
5
6
15 Znalost historických me-
tod zkoumání je základem jak
pro historiky, tak i žáky. 1
2
3
4
5
6
16 Fakta hovoří sama za sebe. 1
2
3
4
5
6
* 17. Žáci si potřebují uvědo-
mit, že dějiny jsou v podstatě
věcí interpretace. 1
2
3
4
5
6
** 18. Lze uvést rozumná
vysvětlení, i když se objevují
rozporuplné důkazy. 1
2
3
4
5
6
* 19. Ani svědci očitých událos-
tí se vždy neshodnou na tom,
co se stalo, je tedy nemožné
vědět, co se v minulosti stalo. 1
2
3
4
5
6
20. Učitelé by neměli zpo-
chybňovat názory žáků o udá-
lostech v minulosti, pouze
zjišťovat, zda vědí fakta. 1
2
3
4
5
6
** 21. Dějiny jsou zdůvod-
něnou rekonstrukcí minu-
losti postavenou na základě
dostupných důkazů. 1
2
3
4
5
6
22. V dějinách neexistují
důkazy. 1
2
3
4
5
6
Pozn.: * Položky patřící do 1. dimenze (objektivismus a subjektivismus) v navrhovaném modelu autorů. ** Položky
ří í d 2 di
(k i
i li
)
h
é
d l
ů ky patřící do 1. dimenze (objektivismus a subjektivismus) v navrhovaném modelu autorů. ** Položky
dimenze (kriterialismus) v navrhovaném modelu autorů. Pozn.: * Položky patřící do 1. dimenze (objektivismus a subjektivismus) v navrhovaném modelu autorů. ** Položky
patřící do 2. dimenze (kriterialismus) v navrhovaném modelu autorů. 76
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Supplementary Methods SM3 from A Stepwise Integrated Approach to Personalized Risk Predictions in Stage III Colorectal Cancer
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SUPPLEMENTARY METHODS SM2 SUPPLEMENTARY METHODS SM2 1. Discovery cohort The protein expression of Procaspase-9, XIAP, SMAC and Procaspase-3 was determined by Reverse
Phase Protein Arrays (RPPAs). Inputs for apoptosis modelling-based signatures The input concentrations for the APOPTO-CELL signatures are summarized in Supplementary
Methods 2 Table SM2.1. A detail description of the measurements and data processing for each of
the cohorts examined in this study is provided below. Additionally, Procaspase-3 expression was used
as input for the APOPTO-CELL-PC3 signature. Supplementary Methods 2 Table SM2.1. Measurement types for APOPTO-CELL inputs. Supplementary Methods 2 Table SM2.1. Measurement types for APOPTO-CELL inputs. Supplementary Methods 2 Table SM2.1. Measurement types for APOPTO-CELL inputs. 1.1 Protein extraction Protein lysates were prepared from FFPE samples as previously described (1). In brief, samples were
histopathologically confirmed to contain at least 70% tumor and 3–20 serial 20 μm sections of tumor
were then adhered to uncharged slides using nuclease-free water. One additional adjacent 5 μm
section was hematoxylin and eosin (H&E) stained and the tumor area was outlined by an expert
histopathologist. Slide overlays with the H&E stained section then allowed to isolate tumor-dense
target areas with sterile razor blades. Isolated tumor material was deparaffinized using xylene/ethanol
and incubated in protein extraction buffer (20 mM Tris buffer (pH 9) containing 2% SDS and protease
inhibitors) for 2 hours at 80°C. 1.2 Reverse phase protein arrays, immunostaining and data acquisition Antibody specificity was
tested in a panel of human cancer cell lines that were proficient in all (HeLa, HCT-116 cells) or
deficient in expression of the respective proteins of interest (SMAC- and XIAP-deficient HCT-116
cells, MCF-7 cells endogenously deficient in Procaspase-3, Jurkat cells deficient in Procaspase-9; red
solid lines). Data represent mean ± S.D. of three technical replicates assayed on the same RPPA array
as the patient samples of the discovery cohort. Supplementary Methods 2 Figure SM2.1. RPPA antibody validation. Antibody specific Supplementary Methods 2 Figure SM2.1. RPPA antibody validation. Antibody specificity was
tested in a panel of human cancer cell lines that were proficient in all (HeLa, HCT-116 cells) or
deficient in expression of the respective proteins of interest (SMAC- and XIAP-deficient HCT-116
cells, MCF-7 cells endogenously deficient in Procaspase-3, Jurkat cells deficient in Procaspase-9; red
solid lines). Data represent mean ± S.D. of three technical replicates assayed on the same RPPA array
as the patient samples of the discovery cohort. Supplementary Methods 2 Figure SM2.1. RPPA antibody validation. Antibody specificity was
tested in a panel of human cancer cell lines that were proficient in all (HeLa, HCT-116 cells) or
deficient in expression of the respective proteins of interest (SMAC- and XIAP-deficient HCT-116
cells, MCF-7 cells endogenously deficient in Procaspase-3, Jurkat cells deficient in Procaspase-9; red
solid lines). Data represent mean ± S.D. of three technical replicates assayed on the same RPPA array
as the patient samples of the discovery cohort. 1.2 Reverse phase protein arrays, immunostaining and data acquisition RPPAs were prepared and analyzed as described previously (2). In brief, protein lysates were
concentration-normalized to 1 μg/μl, as assessed by bicinchoninic acid assay (DC Protein Assay, Bio-
Rad, CA, USA). Three parts of protein lysates were mixed with 1 part of an SDS buffer (40%
Glycerol, 8% SDS, 0.25 M Tris-HCl, pH6.8 plus Bond-Breaker TCEP Solution (Pierce
Biotechnology, IL, USA) at 1/10 of the volume) and boiled. Lysates were then serially diluted four-
fold with SDS buffer (10% Glycerol, 2% SDS, 0.0625 M Tris-HCl, pH6.8 plus Bond-Breaker TCEP
Solution (Pierce Biotechnology, IL, USA) at 1/40 of the volume). A QArray 2 arrayer (Molecular
Device, UK) was used to create a 378 sample array on Oncyte Avid nitrocellulose-coated slides
(Grace Bio-Labs, OR, USA). The slides were stored together with desiccant (Drierite, OH, USA) at
−20°C prior to immunostaining. In controls for antibody specificity, cell line lysates were spotted in
6-fold serial dilutions. Lysates included extracts from the following cell lines: HeLa, MCF-7
(Procaspase-3 deficient), Jurkat (Procaspase-9 deficient), HCT-116, HCT-116 XIAP0/-, HCT-116
SMAC-/- (3,4). Immunostaining was performed on an automated slide stainer (Dako Link 48, Dako, CA, USA)
according to the manufacturer's instructions (CSA kit, Dako, CA, USA). Each slide was incubated
with a single primary antibody at room temperature for 30 min. Primary antibodies were purchased
from Cell Signalling for Caspase-3 (cat. no. 9662), Caspase-9 (cat. no. 9502), SMAC (cat. no. 2954),
XIAP (cat. no. 2042) and from Sigma-Aldrich for β-actin (cat. no. A5441). The secondary antibodies
used were goat anti-rabbit IgG (1:7500, Vector Laboratories, CA, USA) or rabbit anti-mouse IgG
(1:10, Dako, CA, USA). Dako secondary antibodies were used as a starting point for tyramide signal
amplification according to the manufacturer's instructions (Dako, CA, USA). TIFF images of scanned slides were analyzed using Microvigene software version 5.1 (VigeneTech
Inc., MA, USA) to obtain spot signal intensities. For quantification, the QRPPA module of
Microvigene, using a 4 parameter logistic-log model ("SuperCurve" algorithm), was used (5). Signal
intensities were normalized to β-actin to correct for differences in loading. The specificity of the
signals obtain for the different antibodies was validated in cell line extracts deficient in expression of
the target proteins (Suppl. Methods 2 Fig. SM2.1). Supplementary Methods 2 Figure SM2.1. RPPA antibody validation. Antibody specificity was Supplementary Methods 2 Figure SM2.1. RPPA antibody validation. 2. Expansion cohort Protein measurements were not available and thus gene expression was used as surrogate. Gene
expression was measured in FF samples by Affymetrix U133 Plus 2.0 chips (6) and resulting data
processed by the original authors (file “GSE39582_series_matrix.txt.gz”) were downloaded from the
GEO repository (http://www.ncbi.nlm.nih.gov/geo/). The list of probes mapping to each of the genes
of interest was obtained via the “GeneAnnot” tool (http://www.genecards.org). We mapped multiple
probes measurements onto a single gene by applying Principle Component Analysis (PCA) and by
retaining the first component. Next, protein concentrations were estimated from the first component
via the pipeline established in the discovery cohort. Again, since the reference molar distributions
were obtained from stage II/III patients (7), we used data for stage II/III participants to determine the
cumulative distribution functions. 3. Validation cohort For the validation cohort, the protein measurements (performed by RPPA) were available only for
SMAC and XIAP, but not for Procaspase-3 and Procaspase-9. Thus, input data for APOPTO-CELL
systems modeling consisted of level 3 normalized RPPA protein expression data for SMAC and XIAP
and RSEM normalized gene expression data for caspase-3 and caspase-9. Transcript abundance was
assessed by two different Illumina platforms: HiSeq and GA. For the patients that also met the
inclusion criteria, mRNA expression was measured with HiSeq and GA platforms in n=104 and n=39
samples, respectively. For 7 patients, both measurements were available. For these, both caspase-9
and -3 transcripts were strongly correlated (Pearson ⍴≥0.70) and quantitatively similar between the
datasets (Suppl. Methods Fig. SM2.2A-D). The two datasets were therefore merged without any
platform-related batch adjustment. Duplicate measurements from both platforms were averaged. Notable batch effects attributable to batch ID, tissue source site, shipment date or plate ID were not
detected in the RPPA or RNA datasets, therefore no batch effect correction was applied (Suppl. Methods 2 Fig. SM2.2E). The workflow described for the discovery cohort was used to obtain
estimates for protein concentrations in the validation cohort. Analogously to the expansion cohort,
only stage II/III COAD measurements were used to define the cumulative distribution functions. These functions were then used to obtain protein estimates for the entire stage I-IV COAD dataset
(Suppl. Methods 2 Fig. SM2.3). Supplementary Methods 2 Figure SM2.2 Analysis of the RPPA and RNASeqV2 data in the Supplementary Methods 2 Figure SM2.2 Analysis of the RPPA and RNASeqV2 data in the
validation cohort. Supplementary Methods 2 Figure SM2.2 Analysis of the RPPA and RNASeqV2 data in the
validation cohort. validation cohort. A-D. Comparison of the RNASeqV2 transcripts in n=7 patient samples measured by both HiSeq and
GA platforms. High correlation (Pearson and Spearman, A, C) and agreement (Bland-Altman plots,
B, D) between mRNA measurements was identified. E. Batch effects analysis. The presence of potential batch effects was assessed both in RPPA and
RNASeqV2 expression data with respect to Batch ID, Tissue Source Site, Shipment date and Plate ID. Batch effects were not detected. Supplementary Methods 2 Figure SM2.3 Estimation of the absolute concentrations of the
APOPTO-CELL proteins in tumor samples of the validation cohort. Supplementary Methods 2 Figure SM2.3 Estimation of the absolute concentrations of the
APOPTO-CELL proteins in tumor samples of the validation cohort. 3. Validation cohort APOPTO-CELL proteins in tumor samples of the validation cohort. Level 3 normalized expression (RPPA and RSEM for XIAP/SMAC and Procaspase-3/Procaspase-9,
respectively) were acquired (i) and the corresponding absolute concentrations (iii) were estimated via a conversion function (ii) determined with the same pipeline developed for the discovery cohort. Further details on these procedures are provided in the Materials and Methods section. a conversion function (ii) determined with the same pipeline developed for the discovery cohort. Further details on these procedures are provided in the Materials and Methods section. References 1. Guo H, Liu W, Ju Z, Tamboli P, Jonasch E, Mills GB, et al. An efficient procedure for protein
extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays. Proteome
Sci. Proteome Science; 2012;10:56. 2. Hennessy BT, Lu Y, Gonzalez-Angulo AM, Carey MS, Myhre S, Ju Z, et al. A Technical Assessment
of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-
microdissected Human Breast Cancers. Clin Proteomics. 2010;6:129–51. 3. Cummins JM, Kohli M, Rago C, Kinzler KW, Vogelstein B, Bunz F. X-linked inhibitor of apoptosis
protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL)-mediated apoptosis in human cancer cells. Cancer Res. 2004;64:3006–8. 4. Kohli M, Yu J, Seaman C, Bardelli A, Kinzler KW, Vogelstein B, et al. SMAC/Diablo-dependent
apoptosis induced by nonsteroidal antiinflammatory drugs (NSAIDs) in colon cancer cells. Proc Natl
Acad Sci U S A. 2004;101:16897–902. 5. Hu J, He X, Baggerly KA, Coombes KR, Hennessy BTJ, Mills GB. Non-parametric quantification of
protein lysate arrays. Bioinformatics. 2007;23:1986–94. 6. Marisa L, de Reyniès A, Duval A, Selves J, Gaub MP, Vescovo L, et al. Gene expression
classification of colon cancer into molecular subtypes: characterization, validation, and prognostic
value. PLoS Med. 2013;10:e1001453. 7. Hector S, Rehm M, Schmid J, Kehoe J, McCawley N, Dicker P, et al. Clinical application of a
systems model of apoptosis execution for the prediction of colorectal cancer therapy responses and
personalisation of therapy. Gut. 2012;61:725–33.
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English
| null |
Missense variants in <i>ANKRD11</i> cause KBG syndrome by impairment of stability or transcriptional activity of the encoded protein
|
medRxiv (Cold Spring Harbor Laboratory)
| 2,021
|
cc-by
| 9,310
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Elke de Boer and Charlotte W. Ockeloen contributed equally.
Maggie M.K. Wong and Tjitske Kleefstra contributed equally.
*Correspondence and requests for materials should be addressed to Charlotte W. Ockeloen, Department of Genetics, Radboudumc, P.O. Box 9101, 6500
HB, Nijmegen The Netherlands. E-mail address: Charlotte.Ockeloen@radboudumc.nl
A full list of authors and affiliations appears at the end of the paper.
doi: https://doi.org/10.1016/j.gim.2022.06.007
1098-3600/© 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/). www.journals.elsevier.com/genetics-in-medicine Genetics in Medicine (2022) 24, 2051–2064 Genetics in Medicine (2022) 24, 2051–2064 Genetics in Medicine (2022) 24, A B S T R A C T Article history:
Received 27 January 2022
Received in revised form
20 June 2022
Accepted 21 June 2022
Available online 14 July 2022
Keywords:
ANKRD11
Genotype–phenotype study
KBG syndrome
Missense variants
Neurodevelopmental disorders Purpose: Although haploinsufficiency of ANKRD11 is among the most common genetic causes
of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized clinical, molecular, and functional spectra of ANKRD11 missense variants. Methods: We collected clinical information of individuals with ANKRD11 missense variants and
evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants through in
silico analyses and cell-based experiments. Article history:
Received 27 January 2022
Received in revised form
20 June 2022
Accepted 21 June 2022
Available online 14 July 2022 Results: We identified 20 unique, mostly de novo, ANKRD11 missense variants in 29
individuals, presenting with syndromic neurodevelopmental disorders similar to KBG syndrome
caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants
significantly clustered in repression domain 2 at the ANKRD11 C-terminus. Of the 10 func-
tionally studied missense variants, 6 reduced ANKRD11 stability. One variant caused decreased
proteasome degradation and loss of ANKRD11 transcriptional activity. Keywords:
ANKRD11
Genotype–phenotype study
KBG syndrome
Missense variants
Neurodevelopmental disorders Conclusion: Our study indicates that pathogenic heterozygous ANKRD11 missense variants
cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and
reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with
haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but
also poses diagnostic challenges because the KBG-associated phenotype may be mild and
inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting
stringent variant classification and careful phenotyping. © 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical
Genetics and Genomics. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). © 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical
Genetics and Genomics. This is an open access article under the CC BY license
h
i
li
b considered a clinically recognizable syndrome with macro-
dontia as its most defining trait,6 there is considerable clinical
variability and none of the KBG features is pathognomonic. Hence, despite being described as a clinical entity since
1975,1 KBG syndrome was underdiagnosed before causative
ANKRD11 variants were discovered.7 The exact prevalence
of KBG syndrome is not established but it is thought to be a
relatively common cause of genetic NDDs, with the Introduction KBG syndrome (OMIM 148050) is an autosomal dominant
neurodevelopmental disorder (NDD) typically characterized
by mild intellectual disability (ID) or developmental delay,
macrodontia of upper central permanent incisors, mild skel-
etal anomalies, behavioral disturbances, and distinctive
craniofacial
features.1-6
Although
KBG
syndrome
is 2052 E. de Boer et al. KBG syndrome, including reduced body size and craniofacial
abnormalities such as shortened snouts with deformed nasal
bones, wider skulls, and failure of cranial sutures to close.20 In
addition, Yoda mice show behavioral abnormalities reflective
of cognitive dysfunction.15 On the cellular level, the hetero-
zygous Yoda variant causes similar cellular perturbations of
abnormal neuronal precursor proliferation and localization of
neurons as seen for Ankrd11 knockdown,15 suggesting a loss-
of-function mechanism. However, a dominant-negative
mechanism has also been hypothesized to contribute to the
Yoda mouse phenotype.13 Ankrd11 was shown to mislocalize
to the nucleolus, possibly resulting from diminished degra-
dation.13 Because the N-terminal ankyrin repeats are unaf-
fected by the variant, dimerization of wild-type and mutant
Ankrd11 was hypothesized to result in decreased degradation
of both proteins, potentially implicating such dominant-
negative mechanism.13 So far, a dominant-negative mecha-
nism has not been confirmed in additional studies. In general,
consequences of ANKRD11 missense variants on clinical
phenotypes and protein function are largely unknown. associated gene (ANKRD11) in the top 3 of mutated genes in
NDD cohorts accounting for 0.5% to 1% of diagnoses.8,9 KBG syndrome is caused by heterozygous protein trun-
cating variants (PTVs) in ANKRD11 (encoding ANKRD11)
or by 16q24.3 microdeletions encompassing (part of)
ANKRD11. Introduction PTVs and microdeletions explain all cases in 4
previously described KBG cohorts,2-5 whereas in the general
population, ANKRD11 shows strong constraint against loss-
of-function variation (probability of being loss-of-function
intolerant [pLI] = 1; observed/expected [o/e] = 0.05 [0.02-
0.11]; gnomAD v2.1.1).10 Therefore, haploinsufficiency of
ANKRD11 is commonly accepted as mechanism of pathoge-
nicity for KBG syndrome.11 This is supported by observations
of reduced amounts of ANKRD11 messenger RNA and pro-
tein when the gene contains a PTV,12 suggesting that variants
trigger the nonsense-mediated decay (NMD) pathway,12
although PTVs leading to (partial) escape from NMD have
also
been
described.7,13
Also
consistent
with
hap-
loinsufficiency is the finding that ANKRD11 mutated with
p.(Lys1347del) or p.(Leu2143Val) shows reduced transcrip-
tional activity on the p21 promotor in cell-based systems, that
can be rescued by wild type but not mutated ANKRD11.14 We characterized genotypes, phenotypes, and functional
consequences associated with ANKRD11 missense variants
by describing a cohort of 29 individuals. Most individuals
exhibit both characteristic facial appearance and other KBG-
associated features, fitting well within the clinical spectrum
described for KBG syndrome. We showed that missense
variants in ANKRD11 significantly cluster in the C-terminal
RD2, with an overrepresentation of mutated arginine resi-
dues. Missense
variants
result
in
a
loss
of
normal
ANKRD11 function, either caused by reduced protein sta-
bility with normal or increased proteasome degradation or
caused by a loss of transrepression capacity with decreased
proteasome degradation. Our findings are consistent with
ANKRD11 haploinsufficiency, mechanistically underlying
KBG syndrome caused by PTVs or 16q24.3 microdeletions. ANKRD11 is ubiquitously expressed and localizes mainly
to the nucleus in a homogenous pattern. ANKRD11 is a crucial
regulator of neuronal development7,15 that interacts with
coactivators and corepressors of transcription,16 showing (co)
regulatory effects on various sets of genes. These include genes
encoding signaling molecules, chromatin remodelers, and
transcriptional regulators,15 controlling histone acetylation and
gene expression during neural development. ANKRD11 con-
tains 3 transcriptional regulatory domains: 1 activation domain
and 2 repression domains (RDs). The RDs, located at the N-
terminus (RD1) and C-terminus of ANKRD11 (RD2), func-
tionally outweigh the activation domain, because full-length
ANKRD11 functions as a repressor of ligand-dependent
transcription.17 Interaction of ANKRD11 with other proteins
and homodimerization are mediated through ankyrin repeats,
located
at
the
N-terminus.13
The
C-terminal
part
of
ANKRD11, containing (predicted) destruction box motifs
(D-boxes), was suggested to be critical for its degradation.13 Missense permutation analysis (Invitrogen) was used for nuclear staining, before mounting
with VECTASHIELD Antifade Mounting Medium (Vec-
torlab). Fluorescence images were obtained using an
LSM880 AxioObserved confocal microscope (Zeiss). For
images of single nuclei, the Airyscan unit (Zeiss) was used
with a 4.0 zoom factor. Images were analyzed using the
ImageJ “Analyze particle” plugin. To test whether the observed number of variants affecting
arginine residues was significantly greater than expected by
chance, ANKRD11 (ENST00000301030.10/NM_013275.6)
was mutated in silico and output was annotated using Ensembl
Variant Effect Predictor v104.26 To generate an expected
missense distribution, sets of 17 missense variants in RD2 and
8 missense variants outside RD2 (based on 25 independently
observed missense variants, excluding 4 familial variants)
were randomly sampled 100,000 times using per-nucleotide
mutation rates as weights.27 Number of missense variants
affecting arginine residues inside and outside RD2 were
counted per iteration. P values were computed using a per-
mutation test by ranking the observed number of variants
affecting arginine residues within the set of 100,000 expected
values. P values < .05 were considered significant. Clinical and In Silico characterization (
)
gg
g
Whereas PTVs in ANKRD11 are a well-recognized cause
of KBG syndrome, the role of rare missense variants remains
ambiguous. Contrary to what is seen for PTVs, constraint
metrics based on the general population indicate that missense
variants tend to be well-tolerated (z-score –0.55; o/e = 1.04
[1-1.08]).10 There are numerous entries of ANKRD11
missense variants in ClinVar (access date August 20, 2021)
but only approximately 6% are classified as (likely) patho-
genic and almost half as variants of uncertain significance.18
In the literature, approximately 2.6% of (de novo) variants
in ANKRD11 are missense variants,19 listed in Supplemental
Table 1. Missense variants are reported with varying levels of
evidence on pathogenicity, and functional studies have only
been performed for p.(Leu2143Val), showing a loss-of-
function effect.14 The Yoda mutant mouse (C3H.Cg-Ankr-
d11Yod/H, p.[Glu2502Lys]) carries an Ankrd11 missense
variant and shows phenotypic overlap with core features of Identification and clinical characterization of individuals and in
silico analyses of (likely) pathogenic ANKRD11 missense
variants are described in the Supplementary Methods. Variants
were annotated in the context of genome build GRCh37/Hg19,
using transcript reference sequence NM_013275.6 and protein
reference
sequence
NP_037407.4. Human
Phenotype
Ontology (HPO)-based21 clustering was performed as previ-
ously described22 using clinical data of 29 individuals with
ANKRD11
missense
variants
and
35
individuals
with
ANKRD11PTVsormicrodeletions(SupplementalTable2A-C,
Supplemental JSON) after grouping HPO-data on the basis of
semantic similarity (Supplemental Table 3A).23,24 Spatial
clustering of independently observed missense variants (25/29)
was performed as previously described,25 excluding 4 familial
variants. The Supplementary Methods contain details of both
clustering analyses. P values < .05 were considered significant. E. de Boer et al. 2053 Luciferase reporter assays We used firefly luciferase reporters: pGL2-p21 promoter-
Luc and WWP-Luc carrying the promoter region of
CDKN1A/P21. Reporters were gifts from Martin Walsh
(Addgene plasmid #33021; http://n2t.net/addgene:33021;
RRID:Addgene_33021)28 and Bert Vogelstein (Addgene
plasmid 16451; http://n2t.net/addgene:16451; RRID:Addg-
ene_16451),29 respectively. HEK293/T17 cells were seeded
in clear-bottomed white 96-well plates (Greiner Bio-One). Cells were cotransfected with 320 ng of firefly luciferase
reporter construct, 6.5 ng of pGL4.74 Renilla luciferase
normalization control, and 1000 ng of an EGFP-ANKRD11
expression construct or empty EGFP expression vector. After 48 hours, firefly luciferase and Renilla luciferase ac-
tivities were measured using a Dual-Luciferase Reporter
Assay System (Promega) following the manufacturer’s in-
structions on an Infinite F Plex microplate reader (Tecan). Statistical analysis was performed using a 1-way analysis of
variance followed by Dunnett’s post hoc test. P values < .05
were considered significant. DNA constructs and site-directed mutagenesis Full-length wild-type ANKRD11 construct fused to a C-ter-
minal Myc-DDK tag under a human cytomegalovirus (CMV)
promoter (pCMV-Entry-ANKRD11) was purchased from
Origene (RC211717). To generate an N-terminal EGFP-tag,
sequence encoding EGFP was cut from a pEGFP-C2 vector
(Clontech) and subcloned into the pCMV-Entry-ANKRD11
plasmid
using
KpnI/NdeI
restriction
sites. Constructs
(pCMV-Entry-EGFP-ANKRD11)
carrying
(in
frame)
ANKRD11 missense variants were generated using a Quik-
Change Lightning Multi Site-Directed Mutagenesis Kit
(Agilent) following the manufacturer’s protocol. All con-
structs were verified using Sanger sequencing. Supplemental
Table 4 lists primer sequences. Fluorescence-based quantification of protein
stability and degradation HEK293/T17 cells were transfected in triplicate in clear-
bottomed
black
96-well
plates
with
EGFP-tagged
ANKRD11
variants. After
48
hours,
cycloheximide
(Sigma Aldrich) at 50 μg/mL or MG132 (R&D Systems) at
5 μg/mL was added. Cells were incubated at 37◦C with 5%
carbon dioxide in the Infinite M200PRO microplate reader
(Tecan), and fluorescence intensity of EGFP (excitation:
503 nm, emission: 540 nm) was measured over 24 hours at
3-hour intervals. Statistical analysis was performed using
2-way analysis of variance followed by Dunnett’s correc-
tion for multiple testing. P values < .05 were considered
significant. Cell culture and transfection HEK293T/17 cells (CRL-11268, ATCC) were cultured in
Dulbecco's Modified Eagle Medium (Gibco) supplemented
with 10% fetal bovine serum (Gibco) and 100 U/mL
penicillin-streptomycin (Thermo Fisher Scientific) at 37◦C
and 5% carbon dioxide. For immunofluorescence analysis,
cells were seeded onto coverslips coated with 100 μg/mL
poly-D-lysine (Merck, Millipore). Transfections were per-
formed using GeneJuice (Merck, Millipore) following the
manufacturer’s instructions or polyethyenimine in 3:1 ratio
with total mass of DNA transfected. Phenotypes associated with ANKRD11 missense
variants fit the KBG-associated clinical spectrum On the basis of 4 large published cohorts together describing
135 individuals with KBG syndrome caused by PTVs or
16q24.3 microdeletions affecting ANKRD11,2-5 we assessed
phenotypic fit of each of the individuals in the missense cohort
to the clinical spectrum associated with KBG syndrome. The
majority of individuals (23/29 [79.3%]) exhibited dys-
morphisms fitting the characteristic facial gestalt by which
KBG syndrome can be recognized (Figure 2A and B;
Supplemental Table 2A and B, Supplemental Table 5). Indeed, 2 individuals in the cohort (individual 6 and 14) were
diagnostically evaluated through targeted ANKRD11 Sanger
sequencing because of a high clinical suspicion. Of the 29
individuals, 22 (75.9%) met the diagnostic criteria described
for KBG syndrome4 (Supplemental Table 2A). After review
of the observed phenotypes by expert clinicians, it was
concluded that almost all individuals fit the KBG-associated
phenotypic spectrum. These included many individuals not
fully meeting the diagnostic criteria, either because some
features of KBG syndrome not captured in the diagnostic
criteria (eg, delayed bone age and congenital heart defects)
were seen or because the characteristic facial appearance of
KBG syndrome was observed. Only for individuals 3 and 29,
carrying p.(Leu509Pro) and p.(Leu2605Arg), phenotypic fit
to the clinical spectrum of KBG syndrome was considered
poor. ANKRD11 missense variants cause syndromic
neurodevelopmental phenotypes Most frequent phenotypic
features were facial dysmorphisms (28/29 [96.6%]; fitting
characteristic dysmorphisms of KBG, 23/29 [79.3%];
Figure 2A), behavioral disturbances (25/28 [89.3%]),
neurodevelopmental delay (26/28 [92.9%]; speech delay,
23/26 [88.5%]; motor delay, 20/27 [74.1%]), mild to
moderate ID (22/27 [81.5%]; borderline, 2/27 [7.4%];
mild, 15/27 [55.6%]; moderate, 4/27 [14.8%]; unknown
severity,
3/27
[11.1%]),
abnormal
dentition
(21/26,
[80.8%]; macrodontia of upper central incisors, 13/24
[54.2%];
other
dental
abnormalities,
14/21
[66.7%];
Figure
2B),
and
hand
abnormalities
(20/25
[80%];
Figure 2C). Of note, individual 19 also carried a likely
pathogenic variant in ARID2, implicated in Coffin-Siris
syndrome 6 (OMIM 617808), and in the family of 5 in-
dividuals an additional ANKRD11 variant of uncertain
significance, p.(Pro61Ser), was observed in cis with the
pathogenic p.(Arg2579His) variant. ANKRD11 missense variants cause syndromic
neurodevelopmental phenotypes HEK 293T/17 cells grown on poly-D-lysine-coated cover-
slips were transiently transfected with 500 ng of pCMV-
EGFP-ANKRD11 constructs 24 hours after seeding. Cells
were fixated 48 hours after transfection using 4% para-
formaldehyde solution (Electron Microscopy Supplies Ltd)
for 20 minutes at room temperature. Hoechst 33342 Through international collaborations30,31 we identified 29
individuals with rare missense variants in ANKRD11
(Figure 1; Supplemental Table 2A). The cohort consisted 2054 E. de Boer et al. variants affecting arginine residues in RD2 are likely to be
pathogenic. variants affecting arginine residues in RD2 are likely to be
pathogenic. of 24 unrelated individuals with ANKRD11 missense
variants—of which 20 variants occurred de novo—and 1
family with 5 affected individuals from 3 generations
(Supplemental Figure 1). For 5 individuals, inheritance
status of the variant could not be established. The cohort
comprises 18 males and 11 females, with an age range of 7
months to 73 years. We observed syndromic neuro-
developmental phenotypes, summarized in Table 1 with
detailed data compiled in Supplemental Table 2A and B
and Supplementary Results. Most frequent phenotypic
features were facial dysmorphisms (28/29 [96.6%]; fitting
characteristic dysmorphisms of KBG, 23/29 [79.3%];
Figure 2A), behavioral disturbances (25/28 [89.3%]),
neurodevelopmental delay (26/28 [92.9%]; speech delay,
23/26 [88.5%]; motor delay, 20/27 [74.1%]), mild to
moderate ID (22/27 [81.5%]; borderline, 2/27 [7.4%];
mild, 15/27 [55.6%]; moderate, 4/27 [14.8%]; unknown
severity,
3/27
[11.1%]),
abnormal
dentition
(21/26,
[80.8%]; macrodontia of upper central incisors, 13/24
[54.2%];
other
dental
abnormalities,
14/21
[66.7%];
Figure
2B),
and
hand
abnormalities
(20/25
[80%];
Figure 2C). Of note, individual 19 also carried a likely
pathogenic variant in ARID2, implicated in Coffin-Siris
syndrome 6 (OMIM 617808), and in the family of 5 in-
dividuals an additional ANKRD11 variant of uncertain
significance, p.(Pro61Ser), was observed in cis with the
pathogenic p.(Arg2579His) variant. of 24 unrelated individuals with ANKRD11 missense
variants—of which 20 variants occurred de novo—and 1
family with 5 affected individuals from 3 generations
(Supplemental Figure 1). For 5 individuals, inheritance
status of the variant could not be established. The cohort
comprises 18 males and 11 females, with an age range of 7
months to 73 years. We observed syndromic neuro-
developmental phenotypes, summarized in Table 1 with
detailed data compiled in Supplemental Table 2A and B
and Supplementary Results. ANKRD11 missense variants are predicted
deleterious and cluster at C-terminal RD2 entation of ANKRD11 (UniProt: Q6UB99, having 100% sequence identity with NP_037407.4) indicating the location
ed in this study. De novo variants are indicated by black disks, inherited variants by gray disks, variants with unknown inh
disks, and variants marked with # are included in functional assays. The ANKRD11 protein sequence (2663 amino acids) c
n-repeat domain (ANK; purple; amino acids 133-296), 4 PEST sequences (gray; amino acids 286-324, 1796-1806, 2146
297), 4 bipartite nuclear localization signals (NLS; black; amino acids 1184-1200, 1208-1236, 1358-1374, 1640-1656),
or domains (RD) (RD1; blue; amino acids 318-611 and RD2; green; amino acids 2369-2663), and an activator domain (A
acids 2076-2145). An overview with variant details per subject is provided in Supplemental Table 2A, with details
etation in Supplemental Table 5. B. MetaDome analysis of the ANKRD11 missense variants. Overview of the ANKRD
37407 4) tolerance landscape visualized via the MetaDome web server version 1 0 1 The green and blue peaks correspond Figure 1
Missense variants cluster in the intrinsic repressor domain 2 in the C-terminus of the ANKRD11 protein. A. Schematic
representation of ANKRD11 (UniProt: Q6UB99, having 100% sequence identity with NP_037407.4) indicating the location of variants
included in this study. De novo variants are indicated by black disks, inherited variants by gray disks, variants with unknown inheritance by
white disks, and variants marked with # are included in functional assays. The ANKRD11 protein sequence (2663 amino acids) contains an
ankyrin-repeat domain (ANK; purple; amino acids 133-296), 4 PEST sequences (gray; amino acids 286-324, 1796-1806, 2146-2212, and
2222-2297), 4 bipartite nuclear localization signals (NLS; black; amino acids 1184-1200, 1208-1236, 1358-1374, 1640-1656), 2 intrinsic
repressor domains (RD) (RD1; blue; amino acids 318-611 and RD2; green; amino acids 2369-2663), and an activator domain (AD1; pink;
amino acids 2076-2145). An overview with variant details per subject is provided in Supplemental Table 2A, with details on variant
interpretation in Supplemental Table 5. B. MetaDome analysis of the ANKRD11 missense variants. Overview of the ANKRD11 protein
(NP_037407.4) tolerance landscape visualized via the MetaDome web server version 1.0.1. The green and blue peaks correspond to regions
more tolerant to missense variation and the red valleys indicate intolerant regions. C. Sequence alignment of the region containing part of
ANKRD11 amino acid sequence in human (UniProt: Q6UB99), chimpanzee (A0A2I3TR65), mouse (E9Q4F7), chicken (A0A3Q2UE98),
African clawed frog (A0A1L8GEN1), and zebrafish (E7F5R3). Residues in which missense variants were found are highlighted in gray. ANKRD11 missense variants are predicted
deleterious and cluster at C-terminal RD2 We
found
20
unique
missense
variants
(Figure
1;
Supplemental Table 5) significantly clustering at the highly
intolerant C-terminal RD2 (P = 9.99e-9), with recurrence of
p.(Arg2512Gln),
p.(Glu2522Lys),
p.(Arg2579His),
p.(Arg2585Cys), and p.(Leu2605Arg). In addition, the
arginine residues at p.2512, p.2536, and p.2579 were
affected by 2 different missense variants, and p.(Glu2522-
Lys) is equivalent to the orthologous Ankrd11 p.(Glu2502-
Lys) in the Yoda mouse (Supplemental Figure 2). Most of
observed variants are predicted deleterious and affect
conserved and intolerant residues (Figure 1B and C), with
no
predicted
effects
on
premessenger
RNA
splicing
(Supplemental Table 5). Two variants, p.(Glu2522Lys) and
p.(Arg2523Trp), affect a ProViz32 predicted (low consensus
similarity) D-box (Supplemental Table 6; Supplemental
Figure
3)
and
2
variants,
p.(Arg2512Leu)
and
p.(Arg2512Gln), are located at an additional RxxL-motif,
marked as D-box in literature.13 We observed a signifi-
cantly greater number of missense variants affecting argi-
nine residues within RD2 (12/17 variants [70.6%]) than
would be expected by chance (P = 1.00e-4), whereas such
enrichment was not observed outside RD2 (0/8 variants;
0%; P = .37), visualized in Supplemental Figure 4. We
therefore hypothesized that generally, ANKRD11 missense We next compared the group of individuals with
missense variants with the collective KBG cohorts2-5 and
found that frequencies of most KBG-associated features
observed in the missense cohort lie within the range of
frequencies of these features seen in the group of individuals
with PTVs or microdeletions (Table 2). We therefore hy-
pothesized that KBG syndrome resulting from missense
variants is indistinguishable from KBG syndrome caused by
ANKRD11 PTVs or microdeletions. To
quantitatively
investigate
this
hypothesis,
we
compared standardized clinical data (Supplemental JSON21)
of 29 individuals with missense variants and 35 individuals
with KBG syndrome caused by ANKRD11 PTVs or
microdeletions affecting ANKRD11 only. Applying a Par-
titioning Around Medoids clustering algorithm33 on 68
features derived from HPO-data resulted in correct classifi-
cation of 40 of 64 individuals as either belonging to the PTV
or missense variant group (P = .04396; Figure 2D;
Supplemental Table 3A-C), indicating that the algorithm
recognizes a difference between the group with PTVs or
microdeletions and the group with missense variants. This
challenges
the
clinical
observation
that
pathogenic
ANKRD11 missense variants and PTVs or microdeletions
cause the same clinical entity. 2055 E. de Boer et al. 1
Missense variants cluster in the intrinsic repressor domain 2 in the C-terminus of the ANKRD11 protein. A. ANKRD11 missense variants are predicted
deleterious and cluster at C-terminal RD2 D-boxes, destruction box motifs. E. de Boer et al. 2056 2056 Table 1
Summary of observed clinical features Table 1
Summary of observed clinical features
Features Associated With KBG Syndrome
%
Present/Total
Assessed
Macrodontia upper central incisors2
54.2
13/24
Additional dental abnormalities2
66.7
14/21
Fitting the KBG characteristic facial
appearance2
79.3
23/29
Hand abnormalities2
80
20/25
Postnatal short stature2 (<–2 SD)
53.6
15/28
Delayed bone age2
57.1
8/14
Costovertebral anomalies2
7.1
1/14
ID2
81.5
22/27
Normal IQ
11.1
3/27
ID of unknown severity
11.1
3/27
Borderline
7.4
2/27
Mild
55.6
15/27
Moderate
14.8
4/27
Developmental delay2
92.9
26/28
Seizures2
22.2
6/27
Behavioral abnormalities2
89.3
25/28
ADHD/hyperactive behavior
69.2
18/26
Autism spectrum disorder
36
9/25
Anxiety
37.5
9/24
Cryptorchidism2
20
3/15
Congenital heart defect2
32
8/25
Palate defect2
7.1
2/28
Hearing loss2
39.3
11/28
First degree relative with KBG syndrome2
19.2
5/26
Large fontanelle at birth4
18.2
2/11
Feeding difficulties4
33.3
9/27
Precocious puberty3
5.9
1/17
Meeting diagnostic criteria of KBG
syndrome4
75.9
22/29
Other features
Motor delay
74.1
20/27
Speech delay
88.5
23/26
Hypotonia
41.7
10/24
Hypertonia/spasticity
12
3/25
Sleep disturbances
30.8
8/26
Abnormal brain MRI
20
3/15
Abnormalities during pregnancy
45.8
11/24
Abnormalities during delivery
57.7
15/26
Macrocephaly (>2 SD)
3.7
1/27
Microcephaly (<–2 SD)
22.2
6/27
Vision abnormalities
50
13/26
Gastrointestinal abnormalities
32
8/25
Endocrine/metabolic abnormalities
25.9
7/27
Immunologic abnormalities
18.5
5/27
Skin/hair/nail abnormalities
57.7
15/26
ADHD,
attention
deficit
hyperactivity
disorder;
ID,
intellectual
disability; IQ, intelligence quoteint; MRI, magnetic resonance imaging. transiently transfected with mutant ANKRD11. To obtain a
comprehensive insight of the spectrum of variants, we
examined variants spread across ANKRD11, including
p.(Leu509Pro)
located
in
RD1;
p.(Thr1721Met),
p.(Ala2017Thr), and p.(Asp2178Tyr) outside known protein
domains;
and
several
variants
(p.(Arg2512Gln),
p.(Arg2523Trp),
p.(Arg2536Trp),
p.(Arg2536Pro),
p.(Arg2579His), and p.(Arg2585Cys)) in RD2. transiently transfected with mutant ANKRD11. To obtain a
comprehensive insight of the spectrum of variants, we
examined variants spread across ANKRD11, including
p.(Leu509Pro)
located
in
RD1;
p.(Thr1721Met),
p.(Ala2017Thr), and p.(Asp2178Tyr) outside known protein
domains;
and
several
variants
(p.(Arg2512Gln),
p.(Arg2523Trp),
p.(Arg2536Trp),
p.(Arg2536Pro),
p.(Arg2579His), and p.(Arg2585Cys)) in RD2. We first assessed the effect of missense variants on
subcellular localization of ANKRD11. ANKRD11 missense variants are predicted
deleterious and cluster at C-terminal RD2 When transiently
expressed as EGFP-fusion proteins in HEK239T/17 cells,
wild-type ANKRD11 localized to the nucleus in a homo-
geneous speckle-like pattern, consistent with previous
findings in non-neuronal cell lines16 and mouse neocortical
neurons overexpressing wild-type Ankrd11.7 None of the
tested missense variants affected nuclear localization of
ANKRD11 (Figure 3A, Supplemental Figure 5), contrasting
with the nucleolar mislocalization of mutant Ankrd11 re-
ported in Yoda mice.13 On the basis of spatial clustering at the C-terminus,
which is critical for ANKRD11 degradation,13 we hypoth-
esized that missense variants might alter ANKRD11 sta-
bility,
possibly
via
altered
proteasome
degradation. Moreover, 4 variants are located at putative destruction
motifs (Supplemental Table 6, Supplemental Figure 3). To
assess ANKRD11 protein stability, we treated HEK239T/17
cells expressing EGFP-tagged ANKRD11 with cyclohexi-
mide to inhibit translation and measured relative fluores-
cence intensity over 24 hours. We found that all variants in
RD2 except p.(Arg2585Cys) showed reduced protein sta-
bility compared with wild type, whereas among variants
outside the RD2, only p.(Ala2017Thr) was less stable
(Figure 3B, Supplemental Figure 6 and 7). All other variants
outside RD2 showed stability similar to wild type. We next
examined the effect of missense variants on proteasome-
mediated
degradation
after
treating
EGFP-ANKRD11
expressing cells with proteasome inhibitor MG132. Only 2
variants, both located in RD2, affected proteasome degra-
dation, showing opposite directions of effect; whereas
p.(Arg2523Trp) displayed increased proteasome degrada-
tion, p.(Arg2585Cys) showed decreased proteasome degra-
dation (Figure 3B). To study the effects of missense variation on transcrip-
tional activity of ANKRD11, we performed luciferase re-
porter assays with the CDKN1A/P21 promoter, a known
downstream
target. Of
all
tested
variants,
only
p.(Arg2585Cys) affected transcriptional activity, leading to
a loss of transcriptional repression on CDKN1A/P21
(Figure 3C and D). Taken together, our cell-based assays indicate that most
missense variants yield a loss-of-function effect, either
resulting from a reduced dosage of ANKRD11 due to
decreased protein stability with or without increased pro-
teasome degradation or through a loss of transrepressive
activity. Three variants, p.(Leu509Pro), p.(Thr1721Met),
and p.(Asp2178Tyr), did not result in aberrations in any of
the tested protein functions and are therefore classified as of ANKRD11 missense variants act via 2 distinct loss-
of-function mechanisms We continued by studying the functional consequences on
protein localization, protein stability, proteasome degrada-
tion, and transcriptional activity of a subset of the observed
ANKRD11 missense variants using HEK293T/17 cells 2057 E. de Boer et al. 2
Clinical evaluation of individuals with ANKRD11 missense variants. A. Facial photographs of affected individuals
t individuals fit the characteristic facial gestalt of KBG syndrome (individuals 1, 2, 8, 10, 15, 17, 23, 24, 28). Individuals
lar face (individuals 4, 10, 15, 23, 24, 28), with full eyebrows and synophrys (individuals 1, 23, 28). Both downslant (indi
d upslant (individuals 1, 2, 8, 15, 21, 23, 28) of palpebral fissures can be observed. Individuals 2, 4, 8, 10, 15, 21, and 23 exh
ridge, with an upturned nasal tip and triangular-shaped nasal tip in individuals 4, 8, 10, 15, 17, 23, and 24. In individuals 1
d 23 a smooth philtrum is observed, with a thin upper lip in individuals 1, 2, 4, 15, 23, and 24. From the side, low-set, si
duals 2, 4, 23, 24, 28) and a flat facial profile (individuals 1, 2, 23, 24) can be observed. B. Photographs of teeth, showing ma
ral permanent incisors in individuals 8, 10, and 21, with teeth discolorization in individuals 21 and 22, and widely space
uals 4, 8, and 23. C. Photographs of hands, showing short palms (individuals 1, 2, 3, 17, 21, 22, 23, 28) and short fingers (i
24, 28) with tapering (individual 2, 21, 22, 23, 24) and clinodactyly of the fifth digit (individual 1, 2, 3, 22, 24, 28). D. Visua
Ph
t
O t l
b
d l
t i
l
i
i di
ti
th t th
l
t i
l
ith
i
diff
b t
i
Boer et al. Figure 2
Clinical evaluation of individuals with ANKRD11 missense variants. A. Facial photographs of affected individuals. Features
of most individuals fit the characteristic facial gestalt of KBG syndrome (individuals 1, 2, 8, 10, 15, 17, 23, 24, 28). Individuals display a
triangular face (individuals 4, 10, 15, 23, 24, 28), with full eyebrows and synophrys (individuals 1, 23, 28). Both downslant (individuals 3,
10) and upslant (individuals 1, 2, 8, 15, 21, 23, 28) of palpebral fissures can be observed. ANKRD11 missense variants act via 2 distinct loss-
of-function mechanisms Individuals 2, 4, 8, 10, 15, 21, and 23 exhibit a low
nasal bridge, with an upturned nasal tip and triangular-shaped nasal tip in individuals 4, 8, 10, 15, 17, 23, and 24. In individuals 1, 2, 4, 10,
15, and 23 a smooth philtrum is observed, with a thin upper lip in individuals 1, 2, 4, 15, 23, and 24. From the side, low-set, simple ears
(individuals 2, 4, 23, 24, 28) and a flat facial profile (individuals 1, 2, 23, 24) can be observed. B. Photographs of teeth, showing macrodontia
of central permanent incisors in individuals 8, 10, and 21, with teeth discolorization in individuals 21 and 22, and widely spaced teeth in
individuals 4, 8, and 23. C. Photographs of hands, showing short palms (individuals 1, 2, 3, 17, 21, 22, 23, 28) and short fingers (individuals
21, 22, 24, 28) with tapering (individual 2, 21, 22, 23, 24) and clinodactyly of the fifth digit (individual 1, 2, 3, 22, 24, 28). D. Visualization of
Human Phenotype Ontology-based clustering analysis, indicating that the clustering algorithm recognizes a difference between individuals
with missense variants and individuals with PTVs or 16q24.3 microdeletions. PC, principal component; PTV, protein truncating variant. Discussion Although KBG syndrome has been clinically recognized for
almost 50 years1 and PTVs and microdeletions affecting
ANKRD11 have been robustly implicated in its etiology
since 2011,7 the role of rare missense variation in ANKRD11
remained unclear. We characterized clinical, molecular, and
functional spectra of ANKRD11 missense variants by col-
lecting information for 29 individuals and assessing effects
of missense variation on ANKRD11 functions. We show
that almost all individuals carrying rare heterozygous pre-
dicted damaging ANKRD11 missense variants fit well within
the
clinical
spectrum
described
for
KBG
syndrome. Missense variants mainly affect the C-terminal RD2 with an
overrepresentation of mutated arginine residues. Based on
cellular assays, missense variants result in loss-of-function
of ANKRD11, either by impaired protein stability or
reduced transcriptional activity, consistent with ANKRD11
haploinsufficiency causing KBG syndrome through PTVs
and microdeletions. p (
g),
p
y)
ANKRD11 shows significant regional differences in
missense depletion in the general population, with 3 distinct
regions of missense tolerance: p.1-p.415 with modest
regional missense depletion (o/e = 0.51), p.416-p.2276
tolerating missense variation (o/e = 1.1), and p.2277-p.2664
showing high missense depletion (o/e = 0.11) (unpublished
data, Samocha KE, Kosmicki JA, Karczewski KJ, et al. 2017. https://doi.org/10.1101/148353). Consistently, in our
cohort, we observed that variants significantly clustered in
the highly depleted C-terminal region, which was previously
suggested to be implicated in the mechanism underlying
KBG syndrome,13 although we also observed missense
variants in the tolerant middle and N-terminal depleted re-
gions. The proportion of independently mutated arginine
residues is remarkable (total cohort 12/25 [48%]; RD2, 12/
17 [70.6]) and more pronounced than the overrepresentation
of mutated arginine residues seen for pathogenic variants
underlying genetic disorders in general (15%-20%35,36). Arginine is also the most frequently mutated residue in all
secondary structures when considering pathogenic vari-
ants.36 Therefore, we hypothesize that the molecular un-
derpinnings of the observed overrepresentation of mutated
arginine residues lies in the 3-dimensional structure of
ANKRD11, which could not be taken into account, because
the crystal structure of ANKRD11 is largely uncharacterized
and ab initio models are unreliable, despite recent advances
in the field.37 However, on the basis of our in silico studies,
we argue that if missense variants in ANKRD11 affect an
arginine residue in the C-terminal RD2, it is suggestive for
pathogenicity. ANKRD11 missense variants act via 2 distinct loss-
of-function mechanisms uals with ANKRD11 missense variants and previously reported KBG cohorts
et al4
Goldenberg et al3
Gnazzo et al5
Cumulative Frequencies in
Published Cohorts2-5
This Publication
TVs
PTVs + Microdeletions
PTVs + Microdeletions
PTVs + Microdeletions
Missense
Variants
esent/Total
Assessed
%
Present/Total
Assessed
%
Present/Total
Assessed
%
Present/Total
Assessed
Frequency
Range (%)
%
Present/Total
Assessed
23/27
69.2
18/26
76.7
23/30
76.7
89/116
69.2-85.2
54.2
13/24
NA
NA
NA
NA
NA
48.5
16/33
48.5
66.7
14/21
13/32
100
39/39
100
31/31
84.4
114/135
40.6-100
79.3
23/29
15/32
69.7
23/33
67.7
21/31
66.7
86/129
46.9-81.1
80
20/25
9/32
40.5
15/37
58.1
18/31
45.1
60/133
28.1-58.1
53.6
15/28
3/5
66.7
8/12
NA
NA
32
16/50
15.2-66.7
57.1
8/14
NA
NA
NA
NA
NA
30.3
10/33
30.3
7.1
1/14
NA
60
9/15
59.1
13/22
60
42/70
59.1-60.6
81.5
22/27
32/32
94.3
33/35
96.8
30/31
96.2
126/131
93.9-100
92.9
26/28
14/32
31.6
12/38
16.1
5/31
29.9
40/134
16.1-43.8
22.2
6/27
30/30
51.4
19/37
NA
NA
75
75/100
51.4-100
89.3
25/28
5/16
18.8
3/16
13.3
2/15
26.9
18/67
13.3-40
20
3/15
4/32
25.6
10/39
35.5
11/31
23
31/135
12.5-35.5
32
8/25
4/32
5.1
2/39
0
0/31
9.6
13/135
0-21.2
7.1
2/28
8/32
30.6
11/36
19.4
6/31
25
33/132
19.4-30.6
39.3
11/28
7/32
20.5
8/39
6.5
2/31
22.3
27/121
6.5-52.6
19.2
5/26
7/32
NA
NA
NA
NA
21.9
7/32
21.9
18.2
2/11
10/32
NA
NA
35.5
11/31
33.3
21/63
31.3-35.3
33.3
9/27
NA
15.6
5/32
5.6
1/18
12
6/50
5.6-15.6
5.9
1/17
ating variant. 2058
E. de Boer et al. 58
E. de Boer et al E. de Boer et al E. de Boer et al. 2059 uncertain significance, although the individuals carrying
p.(Thr1721Met) and p.(Asp2178Tyr) exhibit typical KBG
features (individual 6 and 8, respectively). All evidence per
observed missense variant including classification on the
basis of American College of Medical Genetics and
Genomics/Association for Molecular Pathology criteria are
provided in Supplemental Table 5. neurobehavioral abnormalities, a submucous cleft palate,
moderate hearing loss, mild growth hormone deficiency,
and microcephaly. The family with 5 affected individuals
also is a key example, in whom 2 presented with macro-
dontia (individual 21 and 24) and 3 exhibited the charac-
teristic facial gestalt (individuals 20, 23, and 24). We
therefore argue not to rule out pathogenicity for individual
ANKRD11 missense variants on inheritance or clinical
grounds only. ANKRD11 missense variants act via 2 distinct loss-
of-function mechanisms Also for the 2 individuals (individual 3 and
29) not clearly exhibiting symptoms of KBG syndrome, the
variants are classified as of uncertain significance and
pathogenic when applying American College of Medical
Genetics and Genomics/Association for Molecular Pathol-
ogy criteria (Supplemental Table 5; p.(Leu509Pro) and
p.(Leu2605Arg), respectively).34 uncertain significance, although the individuals carrying
p.(Thr1721Met) and p.(Asp2178Tyr) exhibit typical KBG
features (individual 6 and 8, respectively). All evidence per
observed missense variant including classification on the
basis of American College of Medical Genetics and
Genomics/Association for Molecular Pathology criteria are
provided in Supplemental Table 5. Discussion Most individuals presented with characteristics fitting the
KBG-associated phenotypic spectrum and, from a clinical
perspective, individuals with KBG syndrome caused by
ANKRD11 missense variants or by PTVs or microdeletions
are indistinguishable. However, unexpectedly, HPO-based
clustering
analysis
showed
a
difference
between
the
groups. Possibly, ascertainment bias influenced this anal-
ysis, because recognizing pathogenicity for missense vari-
ants is more challenging than that for PTVs. In addition, of
the 7 individuals with missense variants who did not meet
the KBG diagnostic criteria, 6 were correctly assigned to the
missense cluster (Supplemental Table 3B), potentially (in
part) driving the observed difference. Finally, the cohort of
cases with PTVs was obtained from 1 expert health care
center, whereas the missense cohort represents an interna-
tional collaboration. Larger cohorts are needed to assess
whether there are indeed phenotypic differences or whether
these results can be explained by cohort effects. Regarding functional impact, most tested missense vari-
ants resulted in reduced protein stability, but only for
p.(Arg2523Trp), it could be explained by increased pro-
teasome degradation. We hypothesize that variants reducing
protein stability without impairment of proteasome degra-
dation affect other mechanisms implicated in protein ho-
meostasis that could be activated by ubiquitination (eg,
autophagy). Of note, p.(Arg2523Trp) is located at a putative
D-box possibly affecting ANKRD11 ubiquitination and
subsequent proteasome degradation. However, the other
tested variant at a D-box, p.(Arg2512Gln), showed no
impairment of proteasome degradation, which challenges p
y
The clinical variability of KBG syndrome is noteworthy,
showing
considerable
phenotypic
differences
between
affected individuals within the same family or between
unrelated individuals with the same variant. This variability
is best shown by comparing individuals 13 and 14, carrying
de novo p.(Glu2522Lys). Although both presented with
macrodontia and the characteristic facial appearance, indi-
vidual 13 exhibited moderate ID, behavioral disturbances,
hypotonia, a duplex kidney, strabismus, and normal growth,
whereas
individual
14
had
normal
intelligence,
no E. de Boer et al. 2060 Figure 3
ANKRD11 variants in RD2 result in reduced protein stability and impaired proteasome degradation or loss of CDKN1A/
P21 transcriptional repression. A. Direct fluorescence imaging of cells expressing EGFP-tagged variants of the ANKRD11 protein using
confocal microscopy. Wild type and all variants showed a speckle-like pattern in the nucleus. Nuclei are stained with Hoechst 33342 (blue). Protein domains in which variants are located are indicated. Results are representative of 3 independent experiments. Scale bar = 5 μm. B. Discussion Relative fluorescence intensity of EGFP-tagged ANKRD11 variants overexpressed in HEK293T/17 cells treated with translation inhibitor
cycloheximide (CHX; 50 μg/mL) shown in upper panels and with proteasome inhibitor MG132 (5 μg/mL) in the lower panels. Equal volume
of dimethyl sulfoxide was used as a vehicle control. Fluorescence intensity was measured for 24 hours with 3-hour intervals. Values are
expressed relative to t = 0 hour and represent the mean ± SD of 3 independent experiments, each preformed in triplicate (*P < .05,
**P < .01, ***P < .001, ****P < .0001; 2-way analysis of variance and a post hoc Dunnett’s test). C-D. Results of luciferase assay with
constructs containing WT and ANKRD11 variants and 2 firefly luciferase reporter constructs with a CDKN1A/P21 promoter. Values are
expressed relative to the control condition that used a EGFP-C2 construct without ANKRD11 and represent the mean ± SD of 4 (C) or 5 (D)
independent experiments, each performed in triplicate (*P < .05, **P < .01, ****P < .0001 vs WT; 1-way analysis of variance and a post
h
D
) RD
d
i
WT
ild Figure 3
ANKRD11 variants in RD2 result in reduced protein stability and impaired proteasome de Figure 3
ANKRD11 variants in RD2 result in reduced protein stability and impaired proteasome degradation or loss of CDKN1A/
P21 transcriptional repression. A. Direct fluorescence imaging of cells expressing EGFP-tagged variants of the ANKRD11 protein using
confocal microscopy. Wild type and all variants showed a speckle-like pattern in the nucleus. Nuclei are stained with Hoechst 33342 (blue). Protein domains in which variants are located are indicated. Results are representative of 3 independent experiments. Scale bar = 5 μm. B. Relative fluorescence intensity of EGFP-tagged ANKRD11 variants overexpressed in HEK293T/17 cells treated with translation inhibitor
cycloheximide (CHX; 50 μg/mL) shown in upper panels and with proteasome inhibitor MG132 (5 μg/mL) in the lower panels. Equal volume
of dimethyl sulfoxide was used as a vehicle control. Fluorescence intensity was measured for 24 hours with 3-hour intervals. Values are
expressed relative to t = 0 hour and represent the mean ± SD of 3 independent experiments, each preformed in triplicate (*P < .05,
**P < .01, ***P < .001, ****P < .0001; 2-way analysis of variance and a post hoc Dunnett’s test). C-D. Results of luciferase assay with
constructs containing WT and ANKRD11 variants and 2 firefly luciferase reporter constructs with a CDKN1A/P21 promoter. Acknowledgments We are very grateful to all individuals and their families for
their participation in this study. This work was financially
supported by Aspasia grants of the Dutch Research Council
(015.014.036 to T.K. and 015.014.066 to L.E.L.M.V.),
Netherlands Organization for Health Research and Devel-
opment (91718310 to T.K.), and the Max Planck Society
(M.M.K.W., S.E.F.). Individual 4 was sequenced at the
Scottish Genomes Partnership. The Scottish Genomes
Partnership was funded by the Chief Scientist Office of the
Scottish Government Health Directorates (SGP/1) and the
Medical Research Council Whole Genome Sequencing for
Health and Wealth Initiative (MC/PC/15080). The Deci-
phering Developmental Disorders study presents indepen-
dent research commissioned by the Health Innovation
Challenge Fund (grant number HICF-1009-003). This study
makes use of Database of Chromosomal Imbalance and
Phenotype in Humans using Ensembl Resources (https://
www.deciphergenomics.org/), which is funded by Well-
come. See Deciphering Developmental Disorders study8
or
https://www.ddduk.org/access.html
for
full
acknowledgment. The 3 variants not showing aberrations in any of our assays
are all located outside RD2 and classified as variants of un-
certain significance (Supplemental Table 5). Of the 4 tested
variants located outside RD2, only p.(Ala2017Thr) affected
the assessed protein functions, whereas all tested variants
inside RD2 did affect protein function. It is therefore possible
that variants outside RD2 exert effects on ANKRD11 func-
tions not captured by our studies. Alternatively, they might
alter premessenger RNA splicing (Supplemental Figure 8)38
despite low SpliceAI scores.39 On the basis of the role of
ANKRD11 in chromatin remodeling, evaluating tran-
scriptomic and epigenetic profiles of individuals or cell-
models could increase understanding of the effects of
missense variants in the various domains. In conclusion, our study showed that (de novo) path-
ogenic missense variants in ANKRD11 cause the clinically
recognizable KBG syndrome with a similar phenotypic
spectrum as previously observed for PTVs and micro-
deletions affecting ANKRD11. We showed that loss of
transrepression capacity and reduced protein stability are
independent molecular mechanisms by which missense
variants cause a functional loss of ANKRD11. These
findings add to the mechanistic complexity underlying
ANKRD11 haploinsufficiency, already comprising deletion
of the locus,3 putative null alleles,12 and PTVs escaping
the NMD pathway,7,13 although effects of the latter on
protein stability and function have not been elucidated. Because inheritance of pathogenic variants in ANKRD11 is
regularly observed owing to the variability of the associ-
ated phenotype, missense variants pose diagnostic chal-
lenges,
warranting
stringent
variant
classification
and
careful phenotyping. Data Availability Code used for spatial clustering is shared at https://github. com/laurensvdwiel/SpatialClustering. Code used for per-
mutation
testing
is
available
at
https://github.com/
jhampstead/ANKRD11-simulations. All available pheno-
typic information in Human Phenotype Ontology terminol-
ogy is shared as a supplement (Supplemental JSON). Model
and code for Human Phenotype Ontology-based clustering
analysis are available at https://github.com/ldingemans/
HPO_clustering_Wang. Discussion Values are
expressed relative to the control condition that used a EGFP-C2 construct without ANKRD11 and represent the mean ± SD of 4 (C) or 5 (D)
independent experiments, each performed in triplicate (*P < .05, **P < .01, ****P < .0001 vs WT; 1-way analysis of variance and a post
hoc Dunnett’s test). RD, repressor domain; WT, wild type. E. de Boer et al. 2061 the previous suggestion that disruption of the C-terminal D-
boxes is the pathophysiological mechanism underlying
KBG syndrome.13 In contrast, the only other variant with
altered proteasome degradation, p.(Arg2585Cys), showed
reduced proteasome degradation and might slightly increase
protein stability, although the latter was not statistically
significant. The p.(Arg2585Cys) variant is also the only
tested variant resulting in reduced transcriptional repression
on CDKN1A/P21, consistent with previous observations
for
p.(Leu2143Val).14
These
findings
suggest
that
p.(Arg2585Cys) results in loss-of-function, despite a po-
tential accumulation of mutant ANKRD11 that contrasts
with the dosage reduction seen for the other tested variants. This is further supported by phenotypes of the 2 individuals
carrying p.(Arg2585Cys), which are characteristic for KBG
syndrome, without apparent differences from phenotypes of
individuals with missense variants impairing ANKRD11
stability. Finally, we did not observe changes in ANKRD11
subcellular localization for the assessed missense variants,
contrary to what has been reported for Yoda mice with
p.(Glu2502Lys).13 Acknowledgments However, because KBG syndrome is
a
relatively
common
cause
of
genetic
NDDs,
the
involvement of ANKRD11 missense variants in cohorts of
undiagnosed
individuals
with
NDD
should
be
considerable. In addition, the collaborations in this study were facilitated
by ERN ITHACA, one of the 24 European Reference Net-
works (ERNs) approved by the ERN Board of Member States,
cofunded by European Commission. The aims of this study
contribute to the Solve-RD project (E.d.B., A.S.D.P., L.F.,
C.G., T.K., A.V., L.E.L.M.V.), which has received funding
from the European Union’s Horizon 2020 research and
innovation
programme
under
grant
agreement
number 779257. E. de Boer et al. M.M.K.W., C.W.O., L.E.L.M.V., S.E.F., T.K.; Investiga-
tion: R.A.K., E.d.B, M.M.K.W., C.W.O., T.K.; Software:
L.L., A.J.M.D., J.E.H., C.G.; Writing-original draft: E.d.B.,
M.M.K.W.,
C.W.O.,
T.K.;
Supervision:
C.W.O.,
M.M.K.W., T.K.; All authors contributed to the final version
of the manuscript. Ruth McGowan28, Kristin G. Monaghan17, Sylvie Odent29,
Rolph Pfundt1, Audrey Putoux30,31, Jeroen van Reeuwijk1,2,
Gijs W.E. Santen32, Erina Sasaki25, Arthur Sorlin11,18,
Peter J. van der Spek23, Alexander P.A. Stegmann1,33,
Sigrid M.A. Swagemakers23, Irene Valenzuela26,
El´eonore Viora-Dupont18, Antonio Vitobello11,12,
Stephanie M. Ware20,34, Mathys W´eber18,
Christian Gilissen1,4, Karen J. Low35, Simon E. Fisher2,3,
Lisenka E.L.M. Vissers1,2, Maggie M.K. Wong3,
Tjitske Kleefstra1,2,36 Affiliations 1Department of Human Genetics, Radboudumc, Nijmegen,
The Netherlands; 2Donders Institute for Brain, Cognition
and
Behaviour,
Radboud
University,
Nijmegen,
The
Netherlands;
3Language and Genetics Department, Max
Planck
Institute
for
Psycholinguistics,
Nijmegen,
The
Netherlands; 4Radboud Institute for Molecular Life Sci-
ences, Radboudumc, Nijmegen, The Netherlands; 5Depart-
ment of Clinical Genetics, Great Ormond Street Hospital for
Children NHS Foundation Trust, London, United Kingdom;
6Clinical Genetics, Guy’s and St Thomas’ NHS Foundation
Trust, London, United Kingdom;
7Advocate Children's
Hospital, Park Ridge, IL; 8Genetic Laboratory of Rare and
Autoinflammatory Diseases, Department of Medical Ge-
netics, Rare Diseases and Personalized Medicine, Centre
Hospitalier
Universitaire
de
Montpellier,
Montpellier,
France; 9Service de G´en´etique, Hospices Civils de Lyon,
Bron, France; 10Institut NeuroMyoGene, CNRS UMR5310,
INSERM U1217, Universit´e Claude Bernard Lyon 1, Lyon,
France;
11G´en´etique des Anomalies du D´eveloppement,
Universit´e
de
Bourgogne
Franche-Comt´e,
UMR1231-
Inserm, Dijon, France; 12Laboratoire de G´en´etique Chro-
mosomique et Mol´eculaire, UF6254 Innovation en Diag-
nostic G´enomique des Maladies Rares, Centre Hospitalier
Universitaire de Dijon, Dijon, France;
13Department of
Neurology, NYU Grossman School of Medicine, NYU
Langone Health, New York, NY; 14Service de G´en´etique
Mol´eculaire et G´enomique M´edicale, CHU de Rennes,
Rennes, France; 15University of Rennes, CNRS, IGDR,
UMR
6290,
Rennes,
France;
16South
West
Thames
Regional Clinical Genetics Service, St George's Hospital,
University of London, London, United Kingdom; 17Gen-
eDx, Gaithersburg, MD; 18Centre de G´en´etique et Centre de
R´ef´erence Anomalies du D´eveloppement et Syndromes
Malformatifs de l'Interr´egion Est, Centre Hospitalier Uni-
versitaire Dijon, Dijon, France;
19F´ed´eration Hospitalo-
Universitaire M´edecine Translationnelle et Anomalies du
D´eveloppement (TRANSLAD), Centre Hospitalier Uni-
versitaire Dijon, Dijon, France; 20Department of Medical
and Molecular Genetics, Indiana University School of
Medicine, Indiana University, Indianapolis, IN; 21Medical
Genetic Department, Rare Diseases and Personalized Med-
icine,
Montpellier
University,
Inserm
U1183,
CHU Ethics Declaration We obtained informed consent to publish unidentifiable data
for all individuals reported in this study. Specific consent
was obtained for publication of clinical photographs. Con-
sent procedures were in accordance with the Declaration of
Helsinki and local ethical guidelines of the participating
centers. The institutional review board 'Commissie Mens-
gebonden Onderzoek Regio Arnhem-Nijmegen' approved
this study under number 2011/188 and 2022-13611. Num-
ber
2011/188 refers
to performing diagnostic
exome
sequencing. Discovery of novel syndromes and description
of clinical cohorts from this series can be taken as such. Number 2022-13611 refers to publishing Human Phenotype
Ontology data for individuals in Biobank Genetics and Rare
Diseases and Biobank Intellectual Disability of the Rad-
boudumc. All the appropriate institutional forms have been
archived locally. Conflict of Interest H.Z.E. and K.G.M. are employees of GeneDx, Inc. All other
authors declare no conflicts of interest. Additional Information The online version of this article (https://doi.org/10.1016/j. gim.2022.06.007) contains supplementary material, which
is available to authorized users. Author Information Conceptualization: C.W.O., M.M.K.W., T.K.; Data Cura-
tion: E.d.B., C.W.O., D.R., T.A., R.B., M.B.-H., B.A., N.C.,
A.-S.D.-P., O.D., C.D., F.E., H.Z.E., L.F., S.F.B., D.G.,
J.A.C.G., B.M.H., U.K., A.L.-A., G.L., S.A.L., I.M.J.M.,
R.M.G., K.G.M., S.O., R.P., A.P., J.v.R., G.W.E.S., E.S.,
A.S., P.J.v.d.S., A.P.A.S., S.M.A.S., I.V., E.V.-D., A.V.,
S.M.W., M.W., K.J.L., T.K.; Formal Analysis: E.d.B., 2062 E. de Boer et al. E. de Boer et al. Montpellier, Montpellier, France; 22Department of Plastic
and Reconstructive Surgery and Hand Surgery, Dutch
Craniofacial Center, Erasmus MC, University Medical
Center Rotterdam, Rotterdam, The Netherlands; 23Depart-
ment of Bioinformatics, Erasmus MC, University Medical
Center Rotterdam, Rotterdam, The Netherlands; 24Depart-
ment of Epidemiology, Richard M. Fairbanks School of
Public Health, Indiana University, Indianapolis, IN; 25Ox-
ford Centre for Genomic Medicine, Oxford University
Hospitals NHS Foundation Trust, Oxford, United Kingdom;
26Department of Clinical and Molecular Genetics, Vall
d'Hebron
University
Hospital
and
Medicine
Genetics
Group, Vall d'Hebron Research Institute, Barcelona, Spain;
27Department of Clinical Genetics, Children's Health Ireland
at Crumlin and Temple Street, Dublin, Ireland; 28West of
Scotland Centre for Genomic Medicine, Queen Elizabeth
University Hospital, Scottish Genomes Partnership, Glas-
gow,
United
Kingdom;
29CHU
Rennes,
Service
de
G´en´etique Clinique, Centre de R´ef´erence Maladies Rares
CLAD-Ouest, ERN ITHACA, Hˆopital Sud, Rennes, France;
30Service de G´en´etique - Centre de R´ef´erence Anomalies du
D´eveloppement, Hospices Civils de Lyon, Bron, France;
31 ´Equipe GENDEV, Centre de Recherche en Neurosciences
de Lyon, INSERM U1028 CNRS UMR5292, Universit´e
Claude Bernard Lyon 1, Lyon, France; 32Department of
Clinical Genetics, Leiden University Medical Center, Lei-
den, The Netherlands; 33Department of Clinical Genetics,
Maastricht University Medical Center+, Maastricht Uni-
versity, Maastricht, The Netherlands; 34Department of Pe-
diatrics,
Indiana
University
School
of
Medicine,
Indianapolis, IN; 35Department of Clinical Genetics, Uni-
versity Hospital Bristol and Weston NHS Foundation Trust,
Bristol, United Kingdom;
36Center of Excellence for
Neuropsychiatry, Vincent van Gogh Institute for Psychiatry,
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, Charlotte W. Ockeloen1,*,
Rosalie A. Kampen3, Juliet E. Hampstead1,4,
Alexander J.M. Dingemans1,2, Dmitrijs Rots1,2,
Lukas L¨utje3, Tazeen Ashraf5,6, Rachel Baker7,
Mouna Barat-Houari8, Brad Angle7, Nicolas Chatron9,10,
Anne-Sophie Denomm´e-Pichon11,12, Orrin Devinsky13,
Christ`ele Dubourg14,15, Frances Elmslie16,
Houda Zghal Elloumi17, Laurence Faivre11,18,19,
Sarah Fitzgerald-Butt20, David Genevi`eve21,
Jacqueline A.C. Goos22,23, Benjamin M. Helm20,24,
Usha Kini25, Amaia Lasa-Aranzasti26, Gaetan Lesca9,10,
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NGF and proNGF Regulate Functionally Distinct mRNAs in PC12 Cells: An Early Gene Expression Profiling
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PloS one
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cc-by
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Abstract The biological activities of NGF and of its precursor proNGF are quite distinct, due to different receptor binding profiles, but
little is known about how proNGF regulates gene expression. Whether proNGF is a purely pro-apoptotic molecule and/or
simply a ‘‘less potent NGF’’ is still a matter of debate. We performed experiments to address this question, by verifying
whether a proNGF specific transcriptional signature, distinct from that of NGF, could be identified. To this aim, we studied
gene expression regulation by proNGF and NGF in PC12 cells incubated for 1 and 4 hours with recombinant NGF and
proNGF, in its wild-type or in a furin-cleavage resistant form. mRNA expression profiles were analyzed by whole genome
microarrays at early time points, in order to identify specific profiles of NGF and proNGF. Clear differences between the
mRNA profiles modulated by the three neurotrophin forms were identified. NGF and proNGF modulate remarkably distinct
mRNA expression patterns, with the gene expression profile regulated by NGF being significantly more complex than that
by proNGF, both in terms of the total number of differentially expressed mRNAs and of the gene families involved. Moreover, while the total number of genes modulated by NGF increases dramatically with time, that by proNGFs is
unchanged or reduced. We identified a subset of regulated genes that could be ascribed to a ‘‘pure proNGF’’ signalling,
distinct from the ‘‘pure NGF’’ one. We also conclude that the composition of mixed NGF and proNGF samples, when the two
proteins coexist, influences the profile of gene expression. Based on this comparison of the gene expression profiles
regulated by NGF and its proNGF precursor, we conclude that the two proteins activate largely distinct transcriptional
programs and that the ratio of NGF to proNGF in vivo can profoundly influence the pattern of regulated mRNAs. Citation: D’Onofrio M, Paoletti F, Arisi I, Brandi R, Malerba F, et al. (2011) NGF and proNGF Regulate Functionally Distinct mRNAs in PC12 Cells: An Early Gene
Expression Profiling. PLoS ONE 6(6): e20839. doi:10.1371/journal.pone.0020839
Editor: Alfred Lewin, University of Florida, United States of America Received January 18, 2011; Accepted May 12, 2011; Published June 3, 2011 pyright: 2011 D’Onofrio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L
restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: The work was funded by grant PNR RBIP063ANC from the Italian Ministry of Higher Education and Scientific Research and IIT Grant from the Italian
Institute of Technology. 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: antonino.cattaneo@sns.it Competing Interests: The authors have declared that no competing interests exist. * E-mail: antonino.cattaneo@sns.it . These authors contributed equally to this work. biological activities, different from those of its mature NGF
counterpart, mediated by distinct, and somewhat complementary,
receptor binding properties [13,14]. In transfected cells and
cultured neurons, proNGF was shown to induce p75NTR-
dependent apoptosis [8,15,16], but also TrkA dependent neuronal
survival, although less effectively than mature NGF [17]. proNGF
is the predominant form of NGF in normal brain and its levels
increase in the brain of patients affected by Alzheimer’s Disease
(AD) [18]. Mara D’Onofrio1,2., Francesca Paoletti1., Ivan Arisi1,2, Rossella Brandi1,2, Francesca Malerba1,3, Luisa
Fasulo1, Antonino Cattaneo1,3* Mara D’Onofrio1,2., Francesca Paoletti1., Ivan Arisi1,2, Rossella Brandi1,2, Francesca Malerba1,3, Luisa
Fasulo1, Antonino Cattaneo1,3* 1 Neurotrophic Factors and Neurodegenerative Diseases Unit, EBRI-European Brain Research Institute, Rome, Italy, 2 Neurogenomics IIT Unit, EBRI-European Brain
Research Institute, Rome, Italy, 3 Scuola Normale Superiore, Pisa, Italy PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 Introduction Then, the medium was taken and 1 mg of recombinant proNGF or proNGF-KR or NGF was spiked into 50 mL of conditioned medium. Spiking controls in fresh medium and PBS were also carried out. The spiked medium was incubated at 37uC for 1 h or 4 h. The red arrow marks the
band corresponding to the full length proNGF, the blue arrow marks the band corresponding to mature NGF. In the figure, WT stands for proNGF-WT
and KR stands for proNGF-KR. Panel C: Densitometric analysis performed on the Western Blot of the spiking experiments representing the percentage of
proNGF proteolysis. The bands corresponding to proNGF and NGF in the Western blot challenged with the anti-NGF antibody were quantified. The
resulting intensities, normalized against the areas of the bands, were reported in the histogram. For each lane, corresponding to the different
proNGF-WT or -KR treatments, the band intensities of proNGF and NGF, derived from proNGF proteolysis, were measured and the sum of the two
bands intensities was assigned to a value of 100%. Among this total intensity, the intensity of the bands corresponding to proNGF and mature NGF
was evaluated and expressed as %. The histogram is the result of the average of four independent experiments. doi:10.1371/journal.pone.0020839.g001 Figure 1. Description of proNGF and its stability. Panel A: Schematic representation of the short form of proNGF. The arrows mark the cleavage
sites for furin, the double headed arrows represent the C-terminal processing site (post translational modification) and hexagons the potential N-
glycosylation sites. In red, the di-basic amino acids that are important in the processing of the protein. In green, the consensus site for the cleavage
by furin. In the present study, we obtained furin resistant mutants in this site (mutant proNGF-KR: RSKR to RSAA [52]). Panel B: Western blot to test the
stability of the proNGF samples in PC12 cells. PC12 cells were plated and treated for 1 h or 4 h with 20 ng/mL of proNGF-WT or proNGF- KR or 10 ng/
mL of NGF. Then, the medium was taken and 1 mg of recombinant proNGF or proNGF-KR or NGF was spiked into 50 mL of conditioned medium. Spiking controls in fresh medium and PBS were also carried out. The spiked medium was incubated at 37uC for 1 h or 4 h. Introduction NGF (Nerve Growth Factor), the prototype member of the
neurotrophin protein family [1], is involved in the maintenance
and growth of specific neuronal populations, both in the central
and peripheral nervous system, through the interaction with two
receptors: TrkA, a member of the Tyrosine Kinase receptors
superfamily [2], and p75NTR, belonging to the Tumor Necrosis
Factor (TNF) receptor superfamily [3]. As all neurotrophins, NGF is translated as a pre-pro-protein [4]. In the case of NGF, two alternative translation initiation sites have
been identified, leading to the formation of two precursor proteins,
a long and a short form, respectively (Figure 1A), that are
glycosylated in vivo [5]. The signal peptide is cleaved upon
translocation into the endoplasmic reticulum, and the protein is
further processed by furin protease in the trans-Golgi network
[6,7] or by extracellular proteases [8,9], to give rise to mature
NGF. Sortilin, a member of the family of Vps10p-domain receptors,
was identified as a specific receptor for proNGF [13] and proposed
to control the sorting pathways of pro-neurotrophins [19–21]. The
activation of the death signal by proNGF requires its interaction
with both sortilin and p75NTR receptors [13,22–24]. The protein
levels of proNGF, TrkA, p75NTR and sortilin appear to be
differently modulated in AD brains [25,26]. The overall picture of neurotrophins activity, as precursors or as
mature proteins, is therefore more complicated than previously
thought. Whether proNGF is a true apoptotic molecule or a sort of
‘‘less potent NGF’’ is still a matter of debate [8,17] and different
biological outcomes of NGF and proNGF signalling have been Besides its suggested roles as a precursor to mature NGF in the
regulation of neurotrophin secretion [10] and as an intramolecular
chaperone [11,12], proNGF was found to display independent June 2011 | Volume 6 | Issue 6 | e20839 1 June 2011 | Volume 6 | Issue 6 | e20839 Figure 1. Description of proNGF and its stability. Panel A: Schematic representation of the short form of proNGF. The arrows mark the cleavage
sites for furin, the double headed arrows represent the C-terminal processing site (post translational modification) and hexagons the potential N-
glycosylation sites. In red, the di-basic amino acids that are important in the processing of the protein. In green, the consensus site for the cleavage
by furin. In the present study, we obtained furin resistant mutants in this site (mutant proNGF-KR: RSKR to RSAA [52]). PLoS ONE | www.plosone.org Introduction Panel B: Western blot to test the
stability of the proNGF samples in PC12 cells. PC12 cells were plated and treated for 1 h or 4 h with 20 ng/mL of proNGF-WT or proNGF- KR or 10 ng/
mL of NGF. Then, the medium was taken and 1 mg of recombinant proNGF or proNGF-KR or NGF was spiked into 50 mL of conditioned medium. Spiking controls in fresh medium and PBS were also carried out. The spiked medium was incubated at 37uC for 1 h or 4 h. The red arrow marks the
band corresponding to the full length proNGF, the blue arrow marks the band corresponding to mature NGF. In the figure, WT stands for proNGF-WT
and KR stands for proNGF-KR. Panel C: Densitometric analysis performed on the Western Blot of the spiking experiments representing the percentage of
proNGF proteolysis. The bands corresponding to proNGF and NGF in the Western blot challenged with the anti-NGF antibody were quantified. The
resulting intensities, normalized against the areas of the bands, were reported in the histogram. For each lane, corresponding to the different
proNGF-WT or -KR treatments, the band intensities of proNGF and NGF, derived from proNGF proteolysis, were measured and the sum of the two
bands intensities was assigned to a value of 100%. Among this total intensity, the intensity of the bands corresponding to proNGF and mature NGF
l
t d
d
d
% Th
hi t
i th
lt
f th
f f
i d
d
t
i
t
Transcriptional Signature of proNGF in PC12 Cells Transcriptional Signature of proNGF in PC12 Cells Transcriptional Signature of proNGF in PC12 Cells Figure 1. Description of proNGF and its stability. Panel A: Schematic representation of the short form of proNGF. The arrows mark the cleavage
sites for furin, the double headed arrows represent the C-terminal processing site (post translational modification) and hexagons the potential N-
glycosylation sites. In red, the di-basic amino acids that are important in the processing of the protein. In green, the consensus site for the cleavage
by furin. In the present study, we obtained furin resistant mutants in this site (mutant proNGF-KR: RSKR to RSAA [52]). Panel B: Western blot to test the
stability of the proNGF samples in PC12 cells. PC12 cells were plated and treated for 1 h or 4 h with 20 ng/mL of proNGF-WT or proNGF- KR or 10 ng/
mL of NGF. 1. proNGF- versus NGF-regulated gene expression in
PC12 cells: experimental design and validation studies We first verified through a standard PC12 cells bioassay (50 ng/
mL of NGF and 100 ng/mL of the short form of proNGF, both
WT and KR) that the recombinant proteins used for the
experiments
are
able
to
induce
an
equivalent
extent
of
morphological differentiation after 72 hours exposure (Figure
S2), and are therefore active, in the frame of our experiment. PC12 cells treated with proNGF -WT and –KR had a normal
morphology and not apparent sign of apoptotic cells was observed. Indeed, it has been reported that after long time exposure, both
NGF and proNGF induce neurite sprouting in PC12 cells [14,33]. The final goal of the present work was to specifically evaluate
cellular responses to NGF and proNGF separately, in terms of
early transcriptional expression profiles. Therefore, we focussed on
a short-term exposure of naı¨ve PC12 cells to NGF or proNGF (1 h
and 4 h). Sub-saturating concentrations of the proteins were used
(namely 10 ng/mL for NGF and 20 ng/mL for the short form of
proNGF, both WT and KR), in order to isolate the specific early
cellular responses. We first verified through a standard PC12 cells bioassay (50 ng/
mL of NGF and 100 ng/mL of the short form of proNGF, both
WT and KR) that the recombinant proteins used for the
experiments
are
able
to
induce
an
equivalent
extent
of
morphological differentiation after 72 hours exposure (Figure
S2), and are therefore active, in the frame of our experiment. PC12 cells treated with proNGF -WT and –KR had a normal
morphology and not apparent sign of apoptotic cells was observed. Indeed, it has been reported that after long time exposure, both
NGF and proNGF induce neurite sprouting in PC12 cells [14,33]. The transcriptional profile regulated by NGF was analyzed first,
in order to set a comparison with published data [34–48],
confirming the well established activation of immediate early gene
after NGF treatment of PC12 cells [37,47,49,50]. Figure 3 reports
the analysis for immediate early genes and for other genes known
to be induced early by NGF. Many of these genes encode possible
transcription factors, and thus may play roles in the initiation and
regulation of subsequent responses to NGF (for review, [38]). The final goal of the present work was to specifically evaluate
cellular responses to NGF and proNGF separately, in terms of
early transcriptional expression profiles. 1. proNGF- versus NGF-regulated gene expression in
PC12 cells: experimental design and validation studies Therefore, we focussed on
a short-term exposure of naı¨ve PC12 cells to NGF or proNGF (1 h
and 4 h). Sub-saturating concentrations of the proteins were used
(namely 10 ng/mL for NGF and 20 ng/mL for the short form of
proNGF, both WT and KR), in order to isolate the specific early
cellular responses. It is noteworthy that none of these immediate early genes is
activated to any significant extent by either proNGF-WT or
proNGF-KR (Figure 3), suggesting already from the analysis of
this first set of genes that NGF and proNGF may activate distinct
transcriptional programs. We assessed the morphology of the PC12 cells in response to the
NGF and proNGF 1 h and 4 h treatments. A macroscopic
evaluation of the cells upon treatment with the neurotrophins
(Figure 2) does not show any significant difference at the early time
points used for the microarray study. Specifically, we cannot
identify any difference at 1 h, while at 4 h NGF-treated cultures
start showing a frequent incipient differentiated morphology,
which only in rare cells is observed in both proNGF treated
cultures. Transcriptional Signature of proNGF in PC12 Cells proNGF, wild-type, or furin-cleavage resistant (hereon simply
called proNGF-WT or proNGF-KR). The gene expression
changes in response to the different treatments were investigated
by microarray analysis. nick end labeling) using the ApopTag detection Kit. We could not
find any significant difference in the percentage of apoptosis
induced by either proNGF-WT or –KR, when compared to the
NGF treatment or the control (data not shown). The stability of the recombinant proNGF proteins, over the
same time scale of the microarray experiment, was assessed in the
culture conditions of the neurotrophin treatment of PC12 cells, in
order to compare the extent of processing of the wild-type and
furin-resistant proNGF proteins. The results show unequivocally that, at this relatively short time
scale, NGF and proNGF regulate the expression of significantly
different sets of mRNAs. Results A known (and measurable) amount of NGF, proNGF-WT and -
KR was spiked into PC12 conditioned medium and incubated in
the same conditions of temperature and time used for the cells’
treatment (see Materials and Methods). The extent of proNGF
degradation (the ratio between intact proNGF and the NGF
produced by proNGF processing) was evaluated by Western blot
analysis (Figure 1B) and densitometric analysis of the bands
corresponding to those of proNGF and NGF originating from the
proNGF proteolysis (Figure 1C). 1. proNGF- versus NGF-regulated gene expression in
PC12 cells: experimental design and validation studies 1. proNGF- versus NGF-regulated gene expression in
PC12 cells: experimental design and validation studies Introduction The red arrow marks the
band corresponding to the full length proNGF, the blue arrow marks the band corresponding to mature NGF. In the figure, WT stands for proNGF-WT
and KR stands for proNGF-KR. Panel C: Densitometric analysis performed on the Western Blot of the spiking experiments representing the percentage of
proNGF proteolysis. The bands corresponding to proNGF and NGF in the Western blot challenged with the anti-NGF antibody were quantified. The
resulting intensities, normalized against the areas of the bands, were reported in the histogram. For each lane, corresponding to the different
proNGF-WT or -KR treatments, the band intensities of proNGF and NGF, derived from proNGF proteolysis, were measured and the sum of the two
bands intensities was assigned to a value of 100%. Among this total intensity, the intensity of the bands corresponding to proNGF and mature NGF
was evaluated and expressed as %. The histogram is the result of the average of four independent experiments. doi:10.1371/journal.pone.0020839.g001 suggested to depend on the ratio between NGF and proNGF
[27,28], on the presence of different kinds of proteases [9,21,29]
and on the expression levels of the receptors [30]. suggested to depend on the ratio between NGF and proNGF
[27,28], on the presence of different kinds of proteases [9,21,29]
and on the expression levels of the receptors [30]. To this aim, in this paper we have undertaken a gene expression
profiling study, aimed at analyzing to what extent proNGF and
NGF activate different transcriptional programs in the NGF
responsive cell line PC12, which expresses the full complement of
NGF and proNGF receptors. PC12 cells were cultured for short
times with equimolar amounts of recombinant mouse NGF
(hereon simply called NGF) or two forms of recombinant mouse It is therefore crucial to address the question of whether, and to
what extent, NGF and proNGF have distinct signalling properties,
and whether the reported differences in their activities are
qualitative (i.e. highly distinct), or purely quantitative. PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 June 2011 | Volume 6 | Issue 6 | e20839 2 Transcriptional Signature of proNGF in PC12 Cells PLoS ONE | www.plosone.org 1. proNGF- versus NGF-regulated gene expression in
PC12 cells: experimental design and validation studies The experiment was designed to verify to what extent NGF and
proNGF activate different signalling pathways, thereby regulating
distinct sets of mRNAs, in the NGF responsive PC12 cells, an
extensively used model to study NGF-induced differentiation [31]. These cells express the full complement of NGF and proNGF
receptors (TrkA, p75NTR [32] and sortilin - Figure S1). The
initial signalling cascades triggered by NGF stimulation and the
patterns of NGF regulated mRNAs have been well characterized
in PC12 cells. On the other hand, nothing is known, so far, about
how proNGF influences gene expression, in this or in other
cellular systems. As shown in Figure 1B and 1C, the proNGF samples are not
cleaved upon incubation in PBS buffer, nor in fresh culture medium
upon the longer incubation (4 h). As expected, incubation of
proNGF samples for 1 h and 4 h in the corresponding PC12
conditioned medium yields to their partial degradation (Figure 1B). There are no substantial differences in the amount of NGF released
from both proteins, at the two time points, as seen in Figure 1C. The bioactivity of the recombinant NGF and proNGF proteins
was verified by incubating naı¨ve PC12 cells with equimolar
amounts of NGF and the short form of proNGF, both in its wild-
type (proNGF-WT) and in its furin-resistant form (proNGF-KR). The proNGF-WT and proNGF-KR proteins (Figure 1A) were
expressed in E.coli and purified, as described in the Methods
Section. The mature NGF was derived from proNGF-WT by
controlled trypsin proteolysis in vitro [14]. Therefore, we conclude that while the KR mutation does not
completely impair proNGF processing, due to extracellular
proteases,
besides
intracellular
furin,
present
in the
PC12
conditioned medium [8,9], the cleavage of proNGF-KR could
be different than that of wild-type proNGF. Indeed, the kinetic of
processing of the WT and the KR mutant are not easily
measurable. Therefore, we cannot exclude that there might be
different cleavage kinetics of the two proteins in vivo. The kinetics
would account for a difference in the NGF/proNGF ratio in the
system in the two cases, during the proteolysis progression, at
different time points. 2. Distinct mRNA expression patterns are regulated by
NGF and proNGF The differential activation of immediate early genes by NGF
versus proNGF (Figure 3) suggests that their transcriptional
responses may be significantly different. Therefore, the overall
statistics of the whole dataset of differentially expressed genes were
evaluated. This analysis showed that NGF and proNGF regulate
distinct mRNA sets in PC12 cells (Table S1), over the time scale
investigated. The ability of this PC12 cells clone to undergo apoptosis upon
treatment with proNGF was tested. In particular, cells were
treated with the described amounts of neurotrophins (namely
10 ng/mL for NGF and 20 ng/mL for proNGF, both WT and
KR) and apoptosis was evaluated at both 1 h and 4 h by means of
TUNEL method (Terminal deoxynucleotidyl transferase dUTP Figure 4A illustrates the overall counts of the differential genes. Among the different intersection areas of the Venn diagram, we
focussed our analysis on the coloured regions shown in Figure 4B,
highlighting the specific proNGF fingerprinting compared to the
NGF one. Genes activated exclusively by NGF are found in the PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 3 June 2011 | Volume 6 | Issue 6 | e20839 Transcriptional Signature of proNGF in PC12 Cells Figure 2. PC12 cells treated for 1 h or 4 h with equimolar amounts of the recombinant neurotrophins. Phase contrast pictures (upper
two rows, 206magnification) of PC12 cells in culture and confocal images of triple immunofluorescence of PC12 cells (last four rows) respectively for
betaIII-tubulin (green), actin (red) and DNA (blue) (636magnification) and immunofluorescence for actin cytoskeleton (1006magnification). After 1 h
or 4 h cells were fixed and stained with anti-betaIII tubulin antibody, Alexa 594 phalloidin to visualize filamentous actin and DAPI for nuclear staining. - first column: control cells with no addition. - second column: 10 ng/mL of NGF. - third column: 20 ng/mL of proNGF WT. - fourth column: 20 ng/mL
of proNGF KR. doi:10.1371/journal.pone.0020839.g002 Figure 2. PC12 cells treated for 1 h or 4 h with equimolar amounts of the recombinant neurotrophins. Phase contrast pictures (upper
two rows, 206magnification) of PC12 cells in culture and confocal images of triple immunofluorescence of PC12 cells (last four rows) respectively for
betaIII-tubulin (green), actin (red) and DNA (blue) (636magnification) and immunofluorescence for actin cytoskeleton (1006magnification). June 2011 | Volume 6 | Issue 6 | e20839 Transcriptional Signature of proNGF in PC12 Cells differentially expressed genes and about specific mechanisms or
pathways significantly modulated in the different experimental
groups. To this aim, differentially expressed genes for each
treatment
and
time
point
were
grouped
into
functional
categories. To this aim, 208 differentially expressed genes for
each treatment and time point were grouped into 209 functional
categories, by the Panther Ontology tool. As a first step, using the
Panther onthology tool, 8 disjoint lists of statistically significant
Panther Biological Processes were obtained (Table S2), one for
each treatment (NGF, proNGF-WT, proNGF-KR and the
intersection) and time point (1 and 4 hours), using the whole
filtered normalized transcriptomic dataset (see Materials and
Methods). Starting from these Panther categories, a more detailed
functional analysis was performed, by investigating, out of the
whole normalized dataset, only the differentially expressed genes. Therefore, as a second step, 8 sets of differentially expressed
genes were compiled, showing fold-change values larger than 1.2
or lower than 1/1.2 in the linear scale (see Materials and
Methods): these genes were further filtered by selecting those
belonging to statistically significant Panther Biological Processes,
and eventually classified into the categories summarized in
Table 1. In the case of NGF treated cells, the increase in the amount of
differential genes from 1 h to 4 h time is a result of the well known
early activation of transcription factors following NGF exposure. proNGF does not share this property of NGF, showing a more
restricted response at 4 hours. p
The differential regulation of gene expression in PC12 by NGF
and proNGF was further analyzed by comparing the correspond-
ing distributions of relative frequencies of ‘‘fold variation’’ ratio
(NGF or proNGF treated PC12/naı¨ve PC12) for differentially
expressed mRNAs (Figure 5). As shown in the Figure 5, the overall
cumulative distribution for NGF regulated genes is shifted towards
higher fold variation values than the overall distribution for
proNGF-WT and proNGF-KR regulated genes. The fold change
values at 90% of the cumulative distribution at 1 h for NGF,
proNGF-WT and proNGF-KR are respectively 1.40, 1.32 and
1.30, while at 4 h they are 1.74, 1.29 and 1.32 respectively. The
fold
change distributions
were compared
using
the
2-tails
Kolmogorov-Smirnov test for two samples and were found to be
significantly different. Thus, NGF exerts a much more potent and
widespread regulation of mRNA expression than proNGF, both in
terms of number of regulated genes and of fold variation. Transcriptional Signature of proNGF in PC12 Cells Transcriptional Signature of proNGF in PC12 Cells Figure 3. Relative expression data of typical early NGF-responsive genes, 1 hour after NGF, proNGF-WT, proNGF-KR treatments. The gene symbols refer to the following gene names: Egr1 (also known as Tis8) = early growth response 1; Egr2 = early growth response 2;
Egr4 = early growth response 4; Btg2 (also known as Pc3) = B-cell translocation gene 2, anti-proliferative; Fos = FBJ murine osteosarcoma viral
oncogene homolog; Ier2 = immediate early response 2; Nr4a1 (also known as Tis1) = nuclear receptor subfamily 4, group A, member 1; Jun = Jun
oncogene; Atf3 = activating transcription factor 3; Junb = Jun-B oncogene; Arc = activity-regulated cytoskeleton-associated protein; Fosl1 = fos-like
antigen 1; Klf4 = Kruppel-like factor 4 (gut); Ier3 = immediate early response 3; Cited2 = Cbp/p300-interacting transactivator, with Glu/Asp-rich
carboxy-terminal domain, 2; Id1 = Inhibitor of DNA binding 1, helix-loop-helix protein (splice variation); zfp36 (also known as Tis11) = zinc finger
protein 36; Ifrd1 (also known as Pc4) = interferon-related developmental regulator 1; Id3 = inhibitor of DNA binding 3; Vgf = VGF nerve growth factor
inducible. doi:10.1371/journal.pone.0020839.g003
Transcriptional Signature of proNGF in PC12 Cells Figure 3. Relative expression data of typical early NGF-responsive genes, 1 hour after NGF, proNGF-WT, proNGF-KR treatments. The gene symbols refer to the following gene names: Egr1 (also known as Tis8) = early growth response 1; Egr2 = early growth response 2;
Egr4 = early growth response 4; Btg2 (also known as Pc3) = B-cell translocation gene 2, anti-proliferative; Fos = FBJ murine osteosarcoma viral
oncogene homolog; Ier2 = immediate early response 2; Nr4a1 (also known as Tis1) = nuclear receptor subfamily 4, group A, member 1; Jun = Jun
oncogene; Atf3 = activating transcription factor 3; Junb = Jun-B oncogene; Arc = activity-regulated cytoskeleton-associated protein; Fosl1 = fos-like
antigen 1; Klf4 = Kruppel-like factor 4 (gut); Ier3 = immediate early response 3; Cited2 = Cbp/p300-interacting transactivator, with Glu/Asp-rich
carboxy-terminal domain, 2; Id1 = Inhibitor of DNA binding 1, helix-loop-helix protein (splice variation); zfp36 (also known as Tis11) = zinc finger
protein 36; Ifrd1 (also known as Pc4) = interferon-related developmental regulator 1; Id3 = inhibitor of DNA binding 3; Vgf = VGF nerve growth factor
inducible. d i 10 1371/j
l
0020839 003 doi:10.1371/journal.pone.0020839.g003 Figure 4B). The number of the differentially genes of the ‘‘pure
proNGF’’ genes is unchanged at 1 and 4 hours. Transcriptional Signature of proNGF in PC12 Cells Among all the functional categories listed in Table 1, the
following ones were analyzed in details: the transcription factor
family, well represented only in the NGF treatment dataset, the
lipid metabolism one, specific for the ‘‘pure proNGF’’ set, and the
cell cycle and DNA repair families, that are common between
NGF and proNGF specific sets. PLoS ONE | www.plosone.org 2. Distinct mRNA expression patterns are regulated by
NGF and proNGF After 1 h
or 4 h cells were fixed and stained with anti-betaIII tubulin antibody, Alexa 594 phalloidin to visualize filamentous actin and DAPI for nuclear staining. - first column: control cells with no addition. - second column: 10 ng/mL of NGF. - third column: 20 ng/mL of proNGF WT. - fourth column: 20 ng/mL
of proNGF KR. doi:10 1371/journal pone 0020839 g002 doi:10.1371/journal.pone.0020839.g002 At 1 h, the total number of mRNAs exclusively regulated by
NGF (N = 436, ‘‘pure NGF’’ cyan colour in Figure 4B) is very
similar to the total number of mRNAs regulated by proNGF-WT
(N = 444 orange colour in Figure 4B), while proNGF-KR regulates
a lower number of mRNAs (N = 204, blue colour in Figure 4B). The picture changes completely at 4 hours, when the total number
of differentially expressed genes is very different for the proNGF or
NGF treated cultures, showing that the number of genes up- or
down-regulated by proNGF-WT (N = 178, colour orange in
Figure 4B) or the pro-NGF-KR (N = 224, colour blue in
Figure 4B) is about ten times lower than the number of mRNAs
differentially regulated by NGF (N = 2231, colour cyan in cyan region; genes activated by proNGF WT or by proNGF KR
are represented in orange and blue, respectively; the brown region
represents the overlap of the genes activated by proNGF WT and
KR, but not by NGF. We refer to the brown region as the ‘‘pure
proNGF’’ activated genes set. The ‘‘pure proNGF’’ set identifies,
therefore, the genes most likely to be affected exclusively by the
proNGF component of the neurotrophin mixture, and provides
therefore a fingerprint of proNGF activity on a given cell. It is
interesting to notice that in this ‘‘pure proNGF’’ intersection set,
all the genes have the same trend in both the proNGF-WT and -
KR preparation, that is either both up-regulated or both down-
regulated (Table S1). PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 PLoS ONE | www.plosone.org 4 3. Functional analysis of NGF and proNGF regulated
genes doi:10 1371/journal pone 0020839 g004 different at 4 h, being much lower in the proNGF group, pointing
out a specific fingerprinting of the proNGF. The cell cycle category is well represented, both in the NGF and in
the proNGF treatments. Analyzing in detail the genes included in this
category for the NGF specific set and the proNGFs intersection set,
after 1 h treatment, two distinct trends can be highlighted (Figure 6). We observe indeed that, after 1 h treatment, while NGF activates the
expression of a mitotic and anti-apoptotic outcome mRNAs,
proNGFs activates pro-apoptotic genes, including tumour suppressor
mRNAs. It is noteworthy that the ‘‘proliferative signature’’ is
conserved for NGF, also at 4 hours of treatment, while the
proapoptotic signature is not maintained for proNGFs transcrip-
tional response at 4 hours. This could be due to a contribution of the
fraction of mature NGF derived from the cleavage of the precursor, a
contribution that increases with incubation time. Table 3 describes the analysis of the main functional categories
of genes whose expression levels correlate with those of the
transcription factors modulated by the NGF or proNGF. This kind
of analysis reconstructs a snapshot of the general status of the cell. ProNGF signalling indicates an early involvement of mitochon-
drial and metabolic genes, while NGF confirms the induction of
the transcription response, gene expression and angiogenesis
related genes. The picture of the cell treated with NGF is
characterised by an increased metabolism of the cell preparing
itself to progress toward the cell cycle division, while proNGF
treated cell could be less active. The differential regulation of genes coding for transcription
factors represents another major distinction signature of NGF
versus proNGF in PC12 cells. A summary count of the
transcription factors differentially expressed in this system by
NGF and proNGF is reported in Table 2, that shows a reduced
number of transcription factor genes represented in the proNGF
intersection set, compared to the NGF treatment set at 1 hour. The number of the differentially expressed transcription factor
genes in the ‘‘pure proNGF’’ or NGF treated cells is strikingly Other differences between NGF and proNGF signalling reside
in DNA replication and chromatin remodelling gene families,
where several genes involved in DNA repair are differentially
expressed after exposure to either NGF or proNGF (-WT or -KR),
but in opposite directions. 3. Functional analysis of NGF and proNGF regulated
genes The
microarray
datasets
were
functionally
analyzed
by
bioinformatics
tools,
to
learn
more
about
the
nature
of June 2011 | Volume 6 | Issue 6 | e20839 June 2011 | Volume 6 | Issue 6 | e20839 PLoS ONE | www.plosone.org 5 Transcriptional Signature of proNGF in PC12 Cells Figure 4. Overall statistics of differentially expressed genes. We selected genes with a fold change larger than 1.2 or lower than 1/1.2 in the
linear scale. Only genes with a gene symbol annotation were considered. Differential genes counts are shown for each of the three different
treatments, either 10 ng/ml NGF or 20 ng/ml of proNGF-WT or -KR, at the two selected time points (1 h and 4 h). (A) Counts of differential genes in
the different regions of the Venn diagram referring to the three treatments; all the seven gene sets are disjoint, at each time point, with no element
in common. (B) Counts of differential genes in four highlighted gene sets: color of bars correspond to the regions in the Venn Diagrams on the
right. doi:10.1371/journal.pone.0020839.g004 Figure 4. Overall statistics of differentially expressed genes. We selected genes with a fold change larger than 1.2 or lower than 1/1.2 in th
linear scale. Only genes with a gene symbol annotation were considered. Differential genes counts are shown for each of the three differen
treatments, either 10 ng/ml NGF or 20 ng/ml of proNGF-WT or -KR, at the two selected time points (1 h and 4 h). (A) Counts of differential genes in
h diff
i
f h V
di
f
i
h
h
ll h
di j i
h i
i
i h
l Figure 4. Overall statistics of differentially expressed genes. We selected genes with a fold change larger than 1.2 or lower than 1/1.2 in the
linear scale. Only genes with a gene symbol annotation were considered. Differential genes counts are shown for each of the three different
treatments, either 10 ng/ml NGF or 20 ng/ml of proNGF-WT or -KR, at the two selected time points (1 h and 4 h). (A) Counts of differential genes in
the different regions of the Venn diagram referring to the three treatments; all the seven gene sets are disjoint, at each time point, with no element
in common. (B) Counts of differential genes in four highlighted gene sets: color of bars correspond to the regions in the Venn Diagrams on the
right. PLoS ONE | www.plosone.org 3. Functional analysis of NGF and proNGF regulated
genes Main functional categories of differentially expressed genes belonging to the four analyzed set regions shown in the
Venn diagram of Figure 4B. NGF 1 h
NGF 4 h
proNGF-WT 1 h
proNGF-WT 4 h
proNGF-KR 1 h
proNGF-KR 4 h
proNGF 1 h
proNGF 4 h
cell cycle
cell cycle
cell cycle
cell cycle
cell cycle
cell cycle
cell cycle
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
DNA repair
DNA repair
DNA repair
DNA repair
DNA repair
DNA repair
ionic trasport
ionic trasport
ion transport
ion transport
ion transport
stress response
stress response
stress response
kinases and
phosphatases
kinases and
phosphatases
carbohydrate
metabolism
carbohydrate
metabolism
transcription factor transcription factor
transcription factor
development
development
lipid metabolism
lipid metabolism
DNA replication
RNA processing
Embryonic
development
structural proteins
cell adhesion
cell migration
chaperones
receptors
Main functional categories of differentially expressed genes belonging to the four analyzed set regions shown in the Venn diagram of Figure 4B: NGF\{proNGF-WT U
proNGF-KR} (light blue in Figure 4B, NGF in Table 1), proNGF-WT\{NGF U proNGF-KR} (orange in Figure 4B, proNGF-WT in Table 1), proNGF-KR\{NGF U proNGF-WT} (blue
in Figure 4B, proNGF-KR in Table 1), {proNGF-KR > proNGF-WT}\NGF (brown in Figure 4B, proNGF in Table 1). doi:10.1371/journal.pone.0020839.t001 Table 1. Main functional categories of differentially expressed genes belonging to the four analyzed set regions shown in the
Venn diagram of Figure 4B. Table 1. Main functional categories of differentially expressed genes belonging to the four analyzed set regions shown in the
Venn diagram of Figure 4B. al categories of differentially expressed genes belonging to the four analyzed set regions shown in the
4B g
y
p
g
g g
y
g
Venn diagram of Figure 4B. 3. Functional analysis of NGF and proNGF regulated
genes NGF at 4 h mainly down-regulates
DNA repair genes, such as DNA polymerase subtype, primarily
involved in repair and other related enzymes. Instead, 1 h of
treatment with proNGF (either -WT or -KR) up-regulates DNA
repair genes, both specific DNA polymerase involved in repair June 2011 | Volume 6 | Issue 6 | e20839 June 2011 | Volume 6 | Issue 6 | e20839 6 Transcriptional Signature of proNGF in PC12 Cells Figure 5. Distribution of directional fold changes. Cumulative distribution (normalized to 100%) of directional fold changes values (linear scale)
for differentially expressed genes, in the three disjoint sets NGF, proNGF-WT, proNGF-KR (see Figure 4B), at 1 hour (A) and 4 hour time (B). Genes
were selected by one-way ANOVA (p,0.05), assuming not equal variance. The fold change values (fc) for down-regulated probes, by definition ,1.0,
were converted into values .1.0 as 1/fc. The distributions of fold change, NGF vs proNGF-WT and NGF vs proNGF-KR, were clearly distinct and were
compared using the 2-side Kolmogorov-Smirnov test for two samples. The fold change values at 50% of the distribution at 1 h for (NGF, proNGF-WT,
proNGF-KR) are respectively (1.25, 1.23, 1.23) while at 4 h they are (1.34, 1.22, 1.23). The foldchange values at 90% of the distribution at 1 h for (NGF,
proNGF-WT, proNGF-KR) are respectively (1.40, 1.32, 1.30) while at 4 h they are (1.74, 1.29, 1.32). doi:10.1371/journal.pone.0020839.g005
Transcriptional Signature of proNGF in PC12 Cells Figure 5. Distribution of directional fold changes. Cumulative distribution (normalized to 100%) of directional fold changes values (linear scale)
for differentially expressed genes, in the three disjoint sets NGF, proNGF-WT, proNGF-KR (see Figure 4B), at 1 hour (A) and 4 hour time (B). Genes
were selected by one-way ANOVA (p,0.05), assuming not equal variance. The fold change values (fc) for down-regulated probes, by definition ,1.0,
were converted into values .1.0 as 1/fc. The distributions of fold change, NGF vs proNGF-WT and NGF vs proNGF-KR, were clearly distinct and were
compared using the 2-side Kolmogorov-Smirnov test for two samples. The fold change values at 50% of the distribution at 1 h for (NGF, proNGF-WT,
proNGF-KR) are respectively (1.25, 1.23, 1.23) while at 4 h they are (1.34, 1.22, 1.23). The foldchange values at 90% of the distribution at 1 h for (NGF,
proNGF-WT, proNGF-KR) are respectively (1.40, 1.32, 1.30) while at 4 h they are (1.74, 1.29, 1.32). doi:10.1371/journal.pone.0020839.g005 Table 1. 3. Functional analysis of NGF and proNGF regulated
genes NGF 1 h
NGF 4 h
proNGF-WT 1 h
proNGF-WT 4 h
proNGF-KR 1 h
proNGF-KR 4 h
proNGF 1 h
proNGF 4 h
cell cycle
cell cycle
cell cycle
cell cycle
cell cycle
cell cycle
cell cycle
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
intracellular
trafficking /
synaptic activity
DNA repair
DNA repair
DNA repair
DNA repair
DNA repair
DNA repair
ionic trasport
ionic trasport
ion transport
ion transport
ion transport
stress response
stress response
stress response
kinases and
phosphatases
kinases and
phosphatases
carbohydrate
metabolism
carbohydrate
metabolism
transcription factor transcription factor
transcription factor
development
development
lipid metabolism
lipid metabolism
DNA replication
RNA processing
Embryonic
development
structural proteins
cell adhesion
cell migration
chaperones
receptors
Main functional categories of differentially expressed genes belonging to the four analyzed set regions shown in the Venn diagram of Figure 4B: NGF\{proNGF-WT U
proNGF-KR} (light blue in Figure 4B, NGF in Table 1), proNGF-WT\{NGF U proNGF-KR} (orange in Figure 4B, proNGF-WT in Table 1), proNGF-KR\{NGF U proNGF-WT} (blue
in Figure 4B, proNGF-KR in Table 1), {proNGF-KR > proNGF-WT}\NGF (brown in Figure 4B, proNGF in Table 1). doi:10.1371/journal.pone.0020839.t001 Main functional categories of differentially expressed genes belonging to the four analyzed set regions shown in the Venn diagram of Figure 4B: NGF\{proNGF-WT U
proNGF-KR} (light blue in Figure 4B, NGF in Table 1), proNGF-WT\{NGF U proNGF-KR} (orange in Figure 4B, proNGF-WT in Table 1), proNGF-KR\{NGF U proNGF-WT} (blue
in Figure 4B, proNGF-KR in Table 1), {proNGF-KR > proNGF-WT}\NGF (brown in Figure 4B, proNGF in Table 1). doi:10.1371/journal.pone.0020839.t001 June 2011 | Volume 6 | Issue 6 | e20839 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org 7 Transcriptional Signature of proNGF in PC12 Cells Transcriptional Signature of proNGF in PC12 Cells Transcriptional Signature of proNGF in PC12 Cells Figure 6. Summary of regulated cell-cycle genes. Summary counts of up-regulated and down-regulated cell cycle genes with a clear
involvement in apoptotic and proliferative processes, for the NGF treatment and for the intersection between proNGF-WT and –KR at 1 h treatments. In the tables on the right, the names of the counted genes with the corresponding up-regulated (+) or down-regulated (2) trend. doi:10.1371/journal.pone.0020839.g006 Figure 6. Summary of regulated cell-cycle genes. June 2011 | Volume 6 | Issue 6 | e20839 3. Functional analysis of NGF and proNGF regulated
genes NGF\{proNGF-WT U
proNGF-KR}
(proNGF-WT >
proNGF-KR)\NGF
Up q
Down Q
Up q
Down Q
1 h
30
0
13
0
4 h
114
13
0
0
Summary counts of up- and down-regulated transcription factors genes with a
fold change larger than 1.2 or lower than 1/1.2 in the linear scale, for the NGF
treatment and for the intersection between proNGF-WT and -KR. The headers
correspond to the regions in the Venn diagram of Figure 4B and the gene sets, at
each time point, are disjoint. The transcription factors belong to the sets in
Figure 4B {NGF\{proNGF-WT U proNGF-KR}} (left side – corresponding to the blue
in Figure 4B) and {{proNGF-KR > proNGF-WT}\NGF} (right side – corresponding to
the brown in Figure 4B). doi:10.1371/journal.pone.0020839.t002 Table 2. Summary counts of the main up- and down-
regulated transcription factors genes. Table 2. Summary counts of the main up- and down-
regulated transcription factors genes. Table 2. Summary counts of the main up- and down-
regulated transcription factors genes. NGF\{proNGF-WT U
proNGF-KR}
(proNGF-WT >
proNGF-KR)\NGF
Up q
Down Q
Up q
Down Q
1 h
30
0
13
0
4 h
114
13
0
0
Summary counts of up- and down-regulated transcription factors genes with a
fold change larger than 1.2 or lower than 1/1.2 in the linear scale, for the NGF
treatment and for the intersection between proNGF-WT and -KR. The headers
correspond to the regions in the Venn diagram of Figure 4B and the gene sets, at
each time point, are disjoint. The transcription factors belong to the sets in
Figure 4B {NGF\{proNGF-WT U proNGF-KR}} (left side – corresponding to the blue
in Figure 4B) and {{proNGF-KR > proNGF-WT}\NGF} (right side – corresponding to
the brown in Figure 4B). doi:10.1371/journal.pone.0020839.t002 Finally, the expression trend of specific genes, known to be
linked to NGF and proNGF activity, were sought and analyzed in
the different datasets. We could identify furin to be down-regulated in ‘‘pure
proNGF’’ set, suggesting a feedback regulation loop possibly
fine-tuning and reinforcing proNGF activity, by reducing its
metabolism. Significantly, in the proNGF-WT at 1 h, the TrkA
receptor gene is down regulated, further suggesting a feedback
effect of proNGF-WT, leading to a reduced efficacy of signal
transduction mediated by TrkA receptor. 3. Functional analysis of NGF and proNGF regulated
genes Summary counts of up-regulated and down-regulated cell cycle genes with a clear
involvement in apoptotic and proliferative processes, for the NGF treatment and for the intersection between proNGF-WT and –KR at 1 h treatments. In the tables on the right, the names of the counted genes with the corresponding up-regulated (+) or down-regulated (2) trend. doi:10.1371/journal.pone.0020839.g006 mechanisms and other single-strand and double-strand binding
proteins that repair DNA breaks by recombination. differently. Since the two proNGF –WT and –KR proteins have
been widely used interchangeably [8,52] and functional differ-
ences among them have not been reported, we ascribe the
transcriptional differences highlighted in this analysis to their
differential processing during the incubation with PC12 cells, and
hence to a distinct contribution by mature NGF in the two
conditions. As for the ‘‘pure proNGF’’ set, many genes involved in
carbohydrate- as well as in lipid-metabolism were found to be
significantly down-regulated. Interestingly, it has been recently
shown [51] that proNGF is modified by non-enzymatic glycation
and lipidation in AD (Table 1), although we cannot directly
compare these modifications with the regulation by proNGF of
carbohydrate and lipid post-translationally modifying enzymes,
highlighted in PC12 cells. The results underscore the importance of the relative amount of
NGF versus proNGF in the biological outcome. It appears therefore
critical, when taking into account the signalling fingerprinting of
proNGF, to consider both the amount of mature and precursor
protein, particularly for in vivo situations. We then analyzed the genes specific for proNGF-WT and
proNGF-KR, represented in the Venn diagrams by the orange
and blue colours, respectively. The two proNGF-WT and -KR
specific datasets contain different number of genes and different
gene classes, suggesting that these two proteins behave somewhat One gene family significantly modulated in the two treatments
at 4 h is linked to synaptic functions and activity (genes involved in
vescicular transport, ion channels, protein trafficking), although in
the case of proNGF-WT, these genes are all down-regulated, while
in the case of proNGF-KR they do not have a homogeneous
trend, being partly up- and partly down-regulated. Table 2. Summary counts of the main up- and down-
regulated transcription factors genes. Transcriptional Signature of proNGF in PC12 Cells Table 3. Functional analysis of genes induced by the
transcription factors modulated by NGF and proNGF after 1 h
of treatment. Functional cluster
Geometric mean of cluster
terms p-values
NGF
regulation of gene expression
2.02E-06
regulation of transcription
1.05E-05
regulation of angiogenesis
1.58E-03
proNGF
mitochondrial membrane
3.09E-04
RNA transport
1.90E-02
regulation of metabolic process
2.61E-02
Main DAVID functional clusters of gene lists obtained by computing the
standard correlation of transcription factors after 1 h, listed in Table 2, with the
expression values of the whole set of data and selecting only the genes with
absolute correlation value .0.90. The transcription factors belong to the sets in
Figure 4B {NGF\{proNGF-WT U proNGF-KR}} (light blue in Figure 4B, NGF in
Table 3) and {{proNGF-KR > proNGF-WT}\NGF} (brown in Figure 4B, proNGF in
Table 3). doi:10.1371/journal.pone.0020839.t003 Table 3. Functional analysis of genes induced by the
transcription factors modulated by NGF and proNGF after 1 h
of treatment. observed that transcription factors and gene expression related
processes are heavily induced by NGF. We then analyzed the system by taking into account certain
subsets of differentially expressed genes. In particular, we focussed
on the intersection set genes induced both by proNGF-WT and
proNGF-KR, that we called the ‘‘pure proNGF’’ subset. We
compared this identified group of genes, with those activated by
NGF and with those activated by either proNGF-WT or proNGF-
KR selectively. In general, we observed in the proNGF transcriptional activity
the absence of certain gene families heavily activated by NGF. We
could identify certain gene families mainly activated by the ‘‘pure
proNGF’’. Most significantly, proNGF was shown to induce genes
connected to carbohydrate and lipid metabolism. y
p
Although in a different context, it has been recently shown [51]
that proNGF is modified by non-enzymatic glycation and
lipidation in AD, therefore this kind of modifications could be
interpreted as a specific signature of the protein. It is remarkable
that the modulation of the lipid metabolism, and of genes of the
cholesterol biosynthesis among these, is a specific signature for the
proNGF treatment. Indeed, it has been shown that cholesterol
biosynthesis is connected on one side to the p75NTR-mediated
signalling and apoptosis [55–57], and on the other side to the
progression of AD [58,59]. Discussion A further discriminating category between NGF and proNGF is
the cell cycle family, encompassing mainly pro-proliferative genes
in the case of NGF and pro-apoptotic genes in the case of proNGF
at 1 h of treatment. Other mRNA families distinctly regulated
involve DNA replication and chromatin remodelling, which are
differentially expressed after exposure to either NGF or proNGF,
but usually in opposite directions, which leads to suggest a
differential effect of the two neurotrophin forms even on common
pathways. The cellular response to NGF has been extensively studied in
the PC12 cell line, both in terms of the cellular phenotype, of
signalling, and, more recently, of the transcriptional profiles
[45,54]. In light of recent studies pointing to an independent and
distinct biological role for the NGF precursor protein proNGF,
particularly relevant in neurodegeneration, we investigated the
properties of proNGF signalling by gene expression microarray,
since very little is known in this respect, for proNGF. Particularly notable is the difference in the regulation of
mRNAs coding for transcription factors. In particular, proNGF
was found to modulate a smaller number of transcription factor
genes compared to NGF and the treatment of PC12 cells with
NGF or proNGF appears to have a completely different effect on
the cellular response. While in the case of NGF, the modulated
transcription factors are connected with a regenerative/differen-
tiative trend, those modulated by proNGF are more connected
with a less proliferative cell. The aim of the experiment was to exploit transcriptional
regulation as a ‘‘signalling signature’’ to address the question
whether NGF and proNGF show only quantitative or also
qualitative differences in their respective transcriptional activation
programs. The present is therefore the first study, aimed at an overall
comparison of the genes induced in PC12 cells upon treatment
with mature NGF or its precursor. In order to isolate as much as possible the effects of a ‘‘pure’’
proNGF system, we treated the cells either with proNGF-WT or
with the furin resistant mutant proNGF-KR. A limited partial
processing of the proteins by other extracellular protease still
occurs, as also demonstrated in the literature [8,9]. Therefore we
concentrated on the early response in the system (1 and 4 hours),
when the processing of the proNGF proteins is lower. Using this
approach, we were able to conclude that NGF and proNGF
activate distinct transcriptional programs and to identify a specific
proNGF transcription signature, distinct from NGF. Transcriptional Signature of proNGF in PC12 Cells Given the proposed role of proNGF in
p75NTR-mediated
apoptosis
[8] and
the
unbalance
of the
proNGF/NGF ratio in AD [18,27,60], further analysis will be
required to evaluate the importance of this pathway in the specific
biological outcome of proNGF in cellular systems and in vivo. doi:10.1371/journal.pone.0020839.t003 Another gene recently discovered to be regulated by proNGF in
rat CNS neurons, the phosphatase and tensin homolog deleted on
chromosome 10 gene pTen [53], is not differentially expressed in
our system. PLoS ONE | www.plosone.org 3. Functional analysis of NGF and proNGF regulated
genes Summary counts of up- and down-regulated transcription factors genes with a
fold change larger than 1.2 or lower than 1/1.2 in the linear scale, for the NGF
treatment and for the intersection between proNGF-WT and -KR. The headers
correspond to the regions in the Venn diagram of Figure 4B and the gene sets, at
each time point, are disjoint. The transcription factors belong to the sets in
Figure 4B {NGF\{proNGF-WT U proNGF-KR}} (left side – corresponding to the blue
in Figure 4B) and {{proNGF-KR > proNGF-WT}\NGF} (right side – corresponding to
the brown in Figure 4B). doi:10 1371/journal pone 0020839 t002 Summary counts of up- and down-regulated transcription factors genes with a
fold change larger than 1.2 or lower than 1/1.2 in the linear scale, for the NGF
treatment and for the intersection between proNGF-WT and -KR. The headers
correspond to the regions in the Venn diagram of Figure 4B and the gene sets, at
each time point, are disjoint. The transcription factors belong to the sets in
Figure 4B {NGF\{proNGF-WT U proNGF-KR}} (left side – corresponding to the blue
in Figure 4B) and {{proNGF-KR > proNGF-WT}\NGF} (right side – corresponding to
the brown in Figure 4B). doi:10.1371/journal.pone.0020839.t002 Despite the evidence of the cross-talk between the p75NTR and
sortilin in the cell death induced by proNGF, we could not find a
modulation in these receptors’ genes in the proNGF treatments. However, this agrees well with the finding that the protein levels of
p75NTR and sortilin are unaffected in neurodegeneration states
[25,26]. June 2011 | Volume 6 | Issue 6 | e20839 PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 8 Transcriptional Signature of proNGF in PC12 Cells PC12 cells treatment and differentiation bioassay PC12 cells treatment and differentiation bioassay Rat pheochromocytoma PC12 cells [31] (PC12 SB subclone,
kindly provided by Maurizia Caruso, Consiglio Nazionale delle
Ricerche, INMM, Rome, Italy) were maintained with RPMI 1640
Medium (Invitrogen) and grown as monolayer cultures on Falcon
dishes, supplemented with 10% Horse Serum (Invitrogen) and 5%
Foetal Calf Serum (Invitrogen), in a humidified atmosphere at
37uC and 5% CO2. For differentiation, PC12 cells were plated at a
concentration of 106/dish (100 mm plates, BD Falcon) and kept in
culture for 12 hours. Cells were treated with 10 ng/ml NGF,
20 ng/ml of wild type (WT) proNGF or 20 ng/ml of furin-
cleavage resistant proNGF-KR. These results fit well with data in the literature [30], showing
how the biological outcome of proNGF is finely tuned by the
amount of protein, as well as by the relative ratios of the involved
receptors. Our results help also explaining why proNGF is able to
induce distinct biological effects in different cellular systems and
different biological conditions [8,17,29,61]. A strong unbalance
towards ‘‘pure’’ NGF is unlikely to be found in vivo, where the co-
existence of the mature and precursor neurotrophins has been
widely observed. Moreover, in pathological conditions, such as
brain injury or Alzheimer’s disease, the equilibrium between
synthesis and cleavage of proNGF was demonstrated to be in
favour of the precursor form [18,52]. Therefore, the life/death
effects induced by NGF and proNGF appear to be strictly related
to their relative ratio in vivo. Indeed, being the two neurotrophin
forms able to activate different transcriptional programs, we
suggest that the biological effects exerted in vivo are a result of a
complex balance between their specific signalling and transcrip-
tional programs. After 72 hours, the medium was changed and free medium+-
proteins was supplied. Following different incubation times, light
microscope pictures were taken of the living differentiating cells
and neurite extension was evaluated. For microarray mRNA expression analysis, half volume of
medium was replaced with fresh medium and the naı¨ve PC12 cells
were treated for 1 h or 4 h with 20 ng/mL of proNGF or
proNGF-KR or 10 ng/mL NGF. A negative control with no
neurotrophin addition was performed. In conclusion, we report herein the first characterization of the
differential transcriptional signature of proNGF versus NGF, in
PC12 cells. From our data, we confirm that the mature and the
precursor proteins are biologically different, and show a different
transcriptional
signature. PC12 cells treatment and differentiation bioassay These
results
open
the
path
for
subsequent more detailed studies of the distinct transcriptional
pathways activated by NGF and proNGF, in order to better
characterize their different biological activity in vivo. In order to evaluate cells morphology in the same experimental
conditions used for RNA extraction, a triple immunofluorescence
was performed on PC12 cells exposed to analogous treatments
(20 ng/mL of proNGF or proNGF-KR or 10 ng/mL NGF). After
1 h or 4 h cells were fixed [62] and stained with anti-beta III
tubulin antibody (Covance, 1:250 dilution), Alexa 594 phalloidin
(Invitrogen, 1:40 dilution), to visualize filamentous actin, and
DAPI for nuclear staining. Coverslips were mounted using
Vectashield (Vector) mounting medium. Stained cells were
analyzed by confocal laser scanning microscopy on Leika
microscope. Materials and Methods Cloning and expression of mouse short proNGF (proNGF)
The cDNA sequence of the short form of proNGF, corresponds
to bp 348 to 1010 (NCBI entry M35075); from aminoacid 2103 to
aminoacid +118 (Figure 1A). proNGF was cloned and expressed in
E. coli according to the protocols described in [14]. p
The apoptosis in PC12 cells was evaluated with the TUNEL
method (Terminal deoxynucleotidyl transferase dUTP nick end
labeling) using the ApopTag detection Kit (Merck Millipore),
according to manufacturer’s instructions. The mutant protein proNGF-KR carries a change from
aminoacids KR to AA at position 22, 21 (Figure 1A), which
destroys the cleavage site by furin protease [52]. The mutant was
obtained by site-directed mutagenesis by Stratagene Kit. The
correct cDNAs were subcloned into the pET11a vector (Novagen). The protein was expressed following the same protocol described
for wild-type proNGF [14]. NGF was obtained from proNGF, by
‘‘in vitro’’ proteolytic cleavage with trypsin [14]. Determination of proNGF stability The processing of the proNGF proteins in the PC12 cells
culture was evaluated by a spiking experiment. The amount of
neurotrophins used in the PC12 cells treatment for the microarray
experiments is too low to be detected in a direct Western Blot
analysis, and does not allow to quantify the precise percentage of
NGF cleaved from the two forms of the precursor during the
incubation. To this aim, a sufficient amount of the recombinant
proteins was spiked into the conditioned medium (CM) of treated
PC12 cells, in the same conditions of temperature and time used
for the microarray experiment. In details, 1 mg of recombinant
proNGF-WT, proNGF-KR or NGF was spiked into 50 mL of 1 h
the conditioned medium (CM) or 4 h CM of treated PC12 cells, in
the same conditions of temperature and time used for the
microarray experiment. Discussion p
From our analysis, we suggest that the relative ratio of NGF
versus proNGF is critical for the downstream transcriptional
signalling. In fact, we observe that there is a significant number
of genes selectively modulated by proNGF-WT or proNGF-KR,
and that for each of the two subsystems, the genes overlapping
with those of NGF in the two cases are also different. This
observation well fit with our hypothesis that the kinetic of
interconversion of proNGF-WT and –KR into NGF is likely to
be different, due to the removal of one dibasic aminoacid site in
the proNGF-KR. The consequence of a different proteolytic
kinetic is that the PC12 cells system is exposed to a progressively
different NGF/proNGF protein ratio, in the time windows
considered. This could account for the difference between the
two datasets. The relative ratio of NGF/proNGF is surely
important for the biological response of the cellular treatment
with the neurotrophins. Our results clearly show that NGF and proNGF signalling
mediate distinct mRNA expression patterns, not only in terms of
total number of modulated genes (a higher number for NGF than
for the proNGFs – See Figure 4), but also in terms of gene families
(see Table 2). The functional analysis of NGF-induced transcriptional data
allowed us, at first, to confirm previously published studies on
NGF-induced microarray profiles in PC12 cells. Indeed, we The regulation of the NGF/proNGF ratio in vivo might
therefore have profound consequences, further underscoring the June 2011 | Volume 6 | Issue 6 | e20839 June 2011 | Volume 6 | Issue 6 | e20839 9 Transcriptional Signature of proNGF in PC12 Cells Transcriptional Signature of proNGF in PC12 Cells crucial importance of determining this parameter in different in
vivo situations. RNA isolation RNA was isolated from four different PC12 cell cultures, PC12
cells treated with NGF, wild type proNGF (proNGF-WT) or furin
resistant proNGF (proNGF-KR) or untreated cells. Neurotrophin
treated cell cultures were sampled at two different incubation times
(1 and 4 hours). Two biological replicates were used for each time
point and treatment. PC12 cell cultures were scraped and lysed with Trizol
(Invitrogen) and DNAse treated by Qiagen columns. RNA
quantity was determined on a NanoDrop UV-VIS. Only samples
with an absorbance ratio of 1.8,OD260/OD280,2.0 were
processed further. Each sample was then quality checked for
integrity using the Agilent BioAnalyzer 2100 (Agilent G2938C,
RNA 6000 nano kit): samples with a RNA Integrity Number
(RIN) index lower than 8.0 were discarded. 500 ng of RNA were
used for each reaction. cRNA was synthesised from double-
stranded cDNA during in vitro transcription with T7 RNA
polymerase, labelled using Cyanine 3-CTP or Cyanine 5-CTP
(Perkin Elmer) and purified (Qiagen’s RNeasy mini spin columns). PC12 cell cultures were scraped and lysed with Trizol
(Invitrogen) and DNAse treated by Qiagen columns. RNA
quantity was determined on a NanoDrop UV-VIS. Only samples
with an absorbance ratio of 1.8,OD260/OD280,2.0 were
processed further. Each sample was then quality checked for
integrity using the Agilent BioAnalyzer 2100 (Agilent G2938C,
RNA 6000 nano kit): samples with a RNA Integrity Number
(RIN) index lower than 8.0 were discarded. 500 ng of RNA were
used for each reaction. cRNA was synthesised from double-
stranded cDNA during in vitro transcription with T7 RNA
polymerase, labelled using Cyanine 3-CTP or Cyanine 5-CTP
(Perkin Elmer) and purified (Qiagen’s RNeasy mini spin columns). As a result, 4 lists of statistically significant Panther categories
were obtained, one for each treatment (NGF, proNGF-WT,
proNGF-KR) and one for the intersection between proNGF-WT
and proNGF-KR treatments, including in each list only disjoint
category terms. Each of these lists was analyzed at two time points
(1 and 4 hours). Four disjoint sets of differentially expressed genes, showing
directional fold change larger than 1.2 or lower than 1/1.2 in the
linear scale, were compiled: one for each treatment (NGF,
proNGF-WT, proNGF-KR) and one for the intersection between
proNGF-WT and proNGF-KR treatments, at the two time points
(1 and 4 h) (see the Venn diagram in Figure 4). Hybridization of oligonucleotide Rat microarrays The gene expression profiling was performed using the two-
color Agilent protocol (http://www.chem.agilent.com/en-US/
Products/Instruments/dnamicroarrays/Pages/default.aspx). cRNA samples from the treated cells were labelled by Cyanine-
5, while the control samples (from untreated cells) were labelled by
Cyanine-3. Labelled cRNA samples (825 ng each sample) were
hybridized to Agilent 4644 k whole Rat genome oligonucleotide
microarrays (G4131F) at 65uC for 17 hours using Agilent’s Gene
Expression Hybridization Kit. The hybridized microarrays were
disassembled at room temperature in Agilent Gene Expression
Wash Buffer 1. After the disassembly, the microarrays were
washed in Gene expression Buffer 1 for one minute at room
temperature, followed by washing with Gene Expression Wash
Buffer 2 for one minute at 37uC. The microarrays were then
treated with acetonitrile for one minute at room temperature. Transcription factors were identified by the DAVID tool [63]
from the sets of differentially expressed genes; DAVID was also
used for annotation of individual genes. Supporting Information Figure S1
PC12 cells treated with the recombinant
neurotrophins – 72 h after treatment. Panel A – 50 ng/mL
of NGF. Panel B – 100 ng/mL of proNGF-WT. Panel C – 100 ng/
mL of proNGF-KR. Panel D – Control without addition of
neurotrophins. Transcriptional Signature of proNGF in PC12 Cells values. The PANTHER Classification System is a freely available
web-based resource that classifies genes by their functions, using
both published experimental evidence and evolutionary relation-
ships for predictions. Genes and proteins are classified by expert
curators according to: Gene families and subfamilies, Gene
Ontology classes (molecular function, biological process, cellular
component), PANTHER Protein Classes, Pathways. The gene lists
used for this Panther analysis were obtained from the whole set of
filtered and normalized data, with the following two additional
criteria: genes without an official Gene Symbol were excluded
from the analysis; in the case of more than one mRNA probe
referring to the same gene, the most significant value (based on the
largest absolute log2 value of fold change) was chosen. RNA isolation Only the genes
with a match in the corresponding sets of statistically significant
Panther terms, based on the gene annotation in the Panther
database, were eventually selected. Scanning, feature extraction and analysis Post-hybridization image acquisition was accomplished using
the Agilent scanner G2564B, equipped with two lasers (532 nm
and 635 nm) and a 48 slide auto-sampler carousel. The ‘‘extended
range’’ scanning protocol was used, where the output of two
following scannings at 10% and 100% of laser power are
numerically combined. Data extraction from the images was
accomplished by Agilent Feature Extraction 9.1 software, using
the standard Agilent two-color gene expression extraction protocol
(GE2-v4_91). Receptors characterization in the PC12 SB subclone Rat pheochromocytoma PC12 cells [31] (PC12 SB subclone)
were maintained with RPMI 1640 Medium (Invitrogen) and
grown as monolayer cultures on Falcon dishes, supplemented
with 10% Horse Serum (Invitrogen) and 5% Foetal Calf
Serum (Invitrogen), in a humidified atmosphere at 37uC and 5%
CO2. The spiked medium was incubated at 37uC for 1 h or 4 h and
then 20 mL/sample were run on SDS-PAGE for Western blotting
analysis with an anti-NGF antibody (Jackson Lab). Cell dishes were washed with PBS, incubated on ice with lysis
buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100,
0.1% SDS, 10 mM EDTA), then scraped and centrifuged. Protein
concentration of the supernatant was evaluated by Bradford Assay
(Sigma). 60 mg of the total protein lysate was loaded onto a
CriterionTM XT Bis-Tris Gel, 4–12% (BioRad) to perform
Western Blotting. The membrane was cut and challenged with
anti TrkA (Chemicon), anti Sortilin (R&D System) and anti
p75NTR (Alomone Labs), according by the manufacturer’s
protocol, and then with the proper secondary antibodies, HRP
conjugated, at 1:7000 (all by Jackson Lab). PC12 cells were washed, fresh medium was added and kept for
1 h or 4 h (1 h CM or 4 h CM). Primary antibody: anti-NGF
M13 (Santa Cruz), at a 1:200 concentration, 16 hours at 4uC. Secondary antibody: Goat Anti-Rabbit, HRP conjugated (Jackson
Lab), 1:7000, 1 hour at room temperature. The ratio between the intact proNGF and the NGF produced
by processing from input proNGF (degradation ratio) was
determined by a densitometric analysis of the bands in each lane,
performed with the Kodak digital imager. PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 10 PLoS ONE | www.plosone.org (TIF) Figure S2
Receptors characterization in the PC12 SB
subclone. PC12 cells lysate was subjected to Western blotting. The antibodies for the three receptors (TrkA, p75NTR and
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21. Hempstead BL (2006) Dissecting the diverse actions of pro- and mature
neurotrophins. References 27. Capsoni S, Cattaneo A (2006) On the molecular basis linking Nerve Growth
Factor (NGF) to Alzheimer’s disease. Cell Mol Neurobiol 26: 619–633. 1. Levi-Montalcini R (1987) The nerve growth factor 35 years later. Science 237:
1154–1162. 2. Hempstead BL, Martin-Zanca D, Kaplan DR, Parada LF, Chao MV (1991)
High-affinity NGF binding requires coexpression of the trk proto-oncogene and
the low-affinity NGF receptor. Nature 350: 678–683. 28. Chao MV, Rajagopal R, Lee FS (2006) Neurotrophin signalling in health and
disease. Clin Sci (Lond) 110: 167–173. 29. Pang PT, Teng HK, Zaitsev E, Woo NT, Sakata K, et al. (2004) Cleavage of
proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306: 487–491. 3. Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, et al. (1986)
Gene transfer and molecular cloning of the human NGF receptor. Science 232:
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Biological activity of nerve growth factor precursor is dependent upon relative
levels of its receptors. J Biol Chem 284: 18424–18433. 4. Shooter EM (2001) Early days of the nerve growth factor proteins. Annu Rev
Neurosci 24: 601–629. 5. Fahnestock M, Yu G, Coughlin MD (2004) ProNGF: a neurotrophic or an
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rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc
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role of basic amino acid clusters in the pro-region. Biochem Biophys Res 6. Mouri A, Nomoto H, Furukawa S (2007) Processing of nerve growth factor: The
role of basic amino acid clusters in the pro-region. Biochem Biophys Res
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ligand-induced conversion from low- to high-affinity states. Proc Natl Acad
Sci U S A 77: 4751–4755. 7. Nomoto H, Takaiwa M, Mouri A, Furukawa S (2007) Pro-region of
neurotrophins determines the processing efficiency. Biochem Biophys Res
Commun 356: 919–924. 33. Sobottka B, Reinhardt D, Brockhaus M, Jacobsen H, Metzger F (2008) ProNGF
inhibits NGF-mediated TrkA activation in PC12 cells. J Neurochem 107:
1294–1303. 8. Author Contributions Conceived and designed the experiments: MD FP AC. Performed the
experiments: MD FP IA RB FM LF. Analyzed the data: MD FP IA RB
FM LF AC. Contributed reagents/materials/analysis tools: FP RB FM LF. Wrote the paper: MD FP IA RB FM LF AC. (TIF) The processes specific to each treatment
and time point are highlighted in bold while the processes common Differentially modulated gene families were identified bioinfor-
matically from the Panther (Protein ANalysis THrough Evolu-
tionary Relationships) database of Biological Processes (http://
www.pantherdb.org/tools/genexAnalysis.jsp), using the Wilcoxon
Rank-Sum Test (p-value,0.05) on two-column tables including
the list of differentially expressed genes and the related fold change PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 11 Transcriptional Signature of proNGF in PC12 Cells Transcriptional Signature of proNGF in PC12 Cells to both the pro-NGF treatments (KR and wild type), separately at
1 h and 4 h, are highlighted in italic bold. (PDF) to both the pro-NGF treatments (KR and wild type), separately at
1 h and 4 h, are highlighted in italic bold. (PDF) Acknowledgments The authors wish to acknowledge the technical assistance of Alessandra Di
Mambro with microarray analysis, and Nina Krako and Paolo Capelli for
their kind help with confocal microscopy of PC12 cells. The authors wish to acknowledge the technical assistance of Alessandra Di
M
b
ith
i
l i
d Ni
K
k
d P
l C
lli f The authors wish to acknowledge the technical assistance of Alessandra Di
Mambro with microarray analysis, and Nina Krako and Paolo Capelli for
their kind help with confocal microscopy of PC12 cells. Mambro with microarray analysis, and Nina Krako and Paolo Capelli for
their kind help with confocal microscopy of PC12 cells. (TIF) Table S1
List of differentially expressed genes at the
two selected time points, 1 and 4 hours. Selected genes have
an absolute directional fold change larger than 1.2 and an official
gene symbol annotation. The colours on the top correspond to
the highlighted subsets in the Venn diagram of Figure 4B:
NGF\{proNGF-WT U proNGF-KR} in light blue, proNGF-
WT\{NGF U proNGF-KR} in orange, proNGF-KR\{NGF U
proNGF-WT} in blue, {proNGF-KR > proNGF-WT}\NGF in
brown. (XLS) Raw data filtering was performed in Microsoft Excel using any
of the following criteria to discard spots: spots with more than 5%
of saturated pixel in any of the two channels, spots flagged as ‘‘not
found’’ by the Feature Extraction software in any of the two
channels, spots with a Signal/Noise ratio smaller than 3 in any of
the two channels, where Signal = (median of the spot2median
spot background level) and Noise is the IQR (interQuantileRange)
of the median spot background. Data analysis was performed on
filtered data using Agilent GeneSpring GX 7.3 and Microsoft
Excel. Each array was normalized by the Lowess algorithm within
GeneSpring, using 20% of data as smoothing window. (XLS) Table S2
Differentially modulated processes for each of
the three different treatments, at 1 and 4 hours. The
items were selected from the Panther Database of Biological
Processes (http://www.pantherdb.org/tools/genexAnalysis.jsp) us-
ing the Wilcoxon Rank-Sum Test (p-value,0.05) on two-columns
tables including the list of differentially expressed genes and the
related fold change values. The processes specific to each treatment
and time point are highlighted in bold while the processes common Table S2
Differentially modulated processes for each of
the three different treatments, at 1 and 4 hours. The
items were selected from the Panther Database of Biological
Processes (http://www.pantherdb.org/tools/genexAnalysis.jsp) us-
ing the Wilcoxon Rank-Sum Test (p-value,0.05) on two-columns
tables including the list of differentially expressed genes and the
related fold change values. References Curr Alzheimer Res 3: 19–24. 46. Levi A, Eldridge JD, Paterson BM (1985) Molecular cloning of a gene sequence
regulated by nerve growth factor. Science 229: 393–395. 22. Massa SM, Xie Y, Yang T, Harrington AW, Kim ML, et al. (2006) Small,
nonpeptide p75NTR ligands induce survival signaling and inhibit proNGF-
induced death. J Neurosci 26: 5288–5300. 47. Wu BY, Fodor EJ, Edwards RH, Rutter WJ (1989) Nerve growth factor induces
the proto-oncogene c-jun in PC12 cells. J Biol Chem 264: 9000–9003. 23. Volosin M, Song W, Almeida RD, Kaplan DR, Hempstead BL, et al. (2006)
Interaction of survival and death signaling in basal forebrain neurons: roles of
neurotrophins and proneurotrophins. J Neurosci 26: 7756–7766. 48. Bartel DP, Sheng M, Lau LF, Greenberg ME (1989) Growth factors and
membrane depolarization activate distinct programs of early response gene
expression: dissociation of fos and jun induction. Genes Dev 3: 304–313. 24. Kim T, Hempstead BL (2009) NRH2 is a trafficking switch to regulate sortilin
localization and permit proneurotrophin-induced cell death. Embo J 28:
1612–1623. 49. Altin JG, Kujubu DA, Raffioni S, Eveleth DD, Herschman HR, et al. (1991)
Differential induction of primary-response (TIS) genes in PC12 pheochromo-
cytoma cells and the unresponsive variant PC12nnr5. J Biol Chem 266:
5401–5406. 25. Counts SE, Mufson EJ (2005) The role of nerve growth factor receptors in
cholinergic basal forebrain degeneration in prodromal Alzheimer disease. J Neuropathol Exp Neurol 64: 263–272. 50. Kujubu DA, Lim RW, Varnum BC, Herschman HR (1987) Induction of
transiently expressed genes in PC-12 pheochromocytoma cells. Oncogene 1:
257–262. 26. Mufson EJ, Wuu J, Counts SE, Nykjaer A (2010) Preservation of cortical sortilin
protein levels in MCI and Alzheimer’s disease. Neurosci Lett 471: 129–133. PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 June 2011 | Volume 6 | Issue 6 | e20839 12 Transcriptional Signature of proNGF in PC12 Cells 51. Kichev A, Ilieva EV, Pinol-Ripoll G, Podlesniy P, Ferrer I, et al. (2009) Cell
death and learning impairment in mice caused by in vitro modified pro-NGF
can be related to its increased oxidative modifications in Alzheimer disease. Am J Pathol 175: 2574–2585. 57. Korade Z, Kenchappa RS, Mirnics K, Carter BD (2009) NRIF is a regulator of
neuronal cholesterol biosynthesis genes. J Mol Neurosci 38: 152–158. 58. Wolozin B (2004) Cholesterol and the biology of Alzheimer’s disease. Neuron
41: 7–10. J
52. Beattie MS, Harrington AW, Lee R, Kim JY, Boyce SL, et al. (2002) ProNGF
induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron 36: 375–386. 59. Wollmer MA (2010) Cholesterol-related genes in Alzheimer’s disease. Biochim
Biophys Acta 1801: 762–773. 60. Capsoni S, Tiveron C, Vignone D, Amato G, Cattaneo A (2010) Dissecting the
involvement of tropomyosin-related kinase A and p75 neurotrophin receptor
signaling in NGF deficit-induced neurodegeneration. Proc Natl Acad Sci U S A
107: 12299–12304. 53. Song W, Volosin M, Cragnolini AB, Hempstead BL, Friedman WJ (2010)
ProNGF induces PTEN via p75NTR to suppress Trk-mediated survival
signaling in brain neurons. J Neurosci 30: 15608–15615. 54. Brynczka C, Merrick BA (2008) The p53 transcriptional target gene wnt7b
contributes to NGF-inducible neurite outgrowth in neuronal PC12 cells. Differentiation 76: 795–808. 61. Althaus HH, Kloppner S (2006) Mature pig oligodendrocytes rapidly process
human recombinant pro-nerve growth factor and do not undergo cell death. J Neurochem 98: 506–517. 55. Casademunt E, Carter BD, Benzel I, Frade JM, Dechant G, et al. (1999) The
zinc finger protein NRIF interacts with the neurotrophin receptor p75(NTR)
and participates in programmed cell death. Embo J 18: 6050–6061. 62. Sakane A, Honda K, Sasaki T (2010) Rab13 regulates neurite outgrowth in
PC12 cells through its effector protein, JRAB/MICAL-L2. Mol Cell Biol 30:
1077–1087. 56. Yan C, Mirnics ZK, Portugal CF, Liang Y, Nylander KD, et al. (2005)
Cholesterol biosynthesis and the pro-apoptotic effects of the p75 nerve growth
factor receptor in PC12 pheochromocytoma cells. Brain Res Mol Brain Res 139:
225–234. 63. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative
analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:
44–57. PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20839 13
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https://openalex.org/W2957461219
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https://eprints.soton.ac.uk/435031/1/fmicb_10_01729.pdf
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English
| null |
Origin of Short-Chain Organic Acids in Serpentinite Mud Volcanoes of the Mariana Convergent Margin
|
Frontiers in microbiology
| 2,019
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cc-by
| 19,446
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ORIGINAL RESEARCH
published: 26 July 2019
doi: 10.3389/fmicb.2019.01729 Origin of Short-Chain Organic Acids
in Serpentinite Mud Volcanoes of the
Mariana Convergent Margin Philip Eickenbusch 1*, Ken Takai 2, Olivier Sissman 3, Shino Suzuki 4, Catriona Menzies 5,6,
Sanae Sakai 2, Pierre Sansjofre 7, Eiji Tasumi 2, Stefano M. Bernasconi 8,
Clemens Glombitza 1,9, Bo Barker Jørgensen 10, Yuki Morono 4 and Mark Alexander Lever 1* 1 Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, Zurich, Switzerland, 2 SUGAR Program, Institute for
Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-star), Japan Agency for Marine-Earth Science
Technology, Yokosuka, Japan, 3 IFP Energies Nouvelles, Rueil-Malmaison, France, 4 Geomicrobiology Research Group, Kochi
Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology, Kochi, Japan, 5 Ocean and
Earth Science, National Oceanography Centre, University of Southampton, Southampton, United Kingdom, 6 Department of
Geology and Petroleum Geology, University of Aberdeen, Aberdeen, United Kingdom, 7 Laboratoire Géosciences Océan
UMR 6538, Université de Bretagne Occidentale, Brest, France, 8 Geological Institute, ETH Zürich, Zurich, Switzerland,
9 NASA Ames Research Center, Moffett Field, CA, United States, 10 Department of Bioscience, Center for Geomicrobiology,
Aarhus University, Aarhus, Denmark Keywords: limits of life, deep biosphere, serpentinization, abiotic synthesis, formate, acetate, methane,
International Ocean Discovery Program Edited by:
Dawn Cardace,
University of Rhode Island,
United States Serpentinitic systems are potential habitats for microbial life due to frequently high
concentrations of microbial energy substrates, such as hydrogen (H2), methane (CH4),
and short-chain organic acids (SCOAs). Yet, many serpentinitic systems are also
physiologically challenging environments due to highly alkaline conditions (pH > 10)
and elevated temperatures (>80◦C). To elucidate the possibility of microbial life in
deep serpentinitic crustal environments, International Ocean Discovery Program (IODP)
Expedition 366 drilled into the Yinazao, Fantangisña, and Asùt Tesoru serpentinite mud
volcanoes on the Mariana Forearc. These mud volcanoes differ in temperature (80,
150, 250◦C, respectively) of the underlying subducting slab, and in the porewater
pH (11.0, 11.2, 12.5, respectively) of the serpentinite mud. Increases in formate and
acetate concentrations across the three mud volcanoes, which are positively correlated
with temperature in the subducting slab and coincide with strong increases in H2
concentrations, indicate a serpentinization-related origin. Thermodynamic calculations
suggest that formate is produced by equilibrium reactions with dissolved inorganic
carbon (DIC) + H2, and that equilibration continues during fluid ascent at temperatures
below 80◦C. By contrast, the mechanism(s) of acetate production are not clear. Besides
formate, acetate, and H2 data, we present concentrations of other SCOAs, methane,
carbon monoxide, and sulfate, δ13C-data on bulk carbon pools, and microbial cell
counts. Even though calculations indicate a wide range of microbial catabolic reactions
to be thermodynamically favorable, concentration profiles of potential energy substrates,
and very low cell numbers suggest that microbial life is scarce or absent. We discuss the
potential roles of temperature, pH, pressure, and dispersal in limiting the occurrence of
microbial life in deep serpentinitic environments. Reviewed by:
Timothy Ferdelman,
Max Planck Institute for Marine
Microbiology (MPG), Germany
Craig Lee Moyer,
Western Washington University,
United States Reviewed by:
Timothy Ferdelman,
Max Planck Institute for Marine
Microbiology (MPG), Germany
Craig Lee Moyer,
Western Washington University,
United States *Correspondence:
Philip Eickenbusch
philipeickenbusch@gmail.com
Mark Alexander Lever
mark.lever@usys.ethz.ch *Correspondence:
Philip Eickenbusch
philipeickenbusch@gmail.com
Mark Alexander Lever
mark.lever@usys.ethz.ch Specialty section:
This article was submitted to
Extreme Microbiology,
a section of the journal
Frontiers in Microbiology Received: 01 May 2019
Accepted: 12 July 2019
Published: 26 July 2019 INTRODUCTION Importantly, while the production
of CH4 and hydrocarbons with ≥2 C atoms by FTT in the
presence of a gas phase is generally accepted, recent studies
have challenged earlier reports of CH4 production by FTT in
water-saturated serpentinitic systems at 200–300◦C (McDermott
et al., 2015; McCollom, 2016; Wang et al., 2018). Instead stable
and clumped isotopic compositions point toward synthesis of
these hydrocarbons at ≥400◦C from magmatic volatiles that
are trapped within fluid-vapor inclusions. Alteration of mantle
rocks by serpentinization then releases these compounds into
circulating fluids. In
subseafloor
sediments,
most
microorganisms
are
chemoorganotrophic
and
rely
on
the
breakdown
of
photosynthetically fixed organic matter (OM) and in situ-
produced microbial necromass as power sources (Canfield et al.,
2005; Lomstein et al., 2012). Under anaerobic conditions,
SCOAs,
such
as
formate,
acetate,
propionate,
butyrate,
and lactate, in addition to H2, are important metabolic
intermediates, being the end products of microbial fermentation
and acetogenesis reactions (Stams, 1994; Wellsbury et al.,
2002; Worm et al., 2010). In addition, SCOAs are key energy
substrates for microorganisms involved in terminal oxidation
reactions to carbon dioxide (CO2) and CH4 involving nitrate,
manganese(IV), iron(III), sulfate, and CO2 as electron acceptors
(Froelich et al., 1979; Sørensen et al., 1981; Canfield et al.,
1993; Finke and Jørgensen, 2008). Microbial production and
turnover of SCOAs has been reported from 0 (Finke et al., 2007)
to 80◦C (Wellsbury et al., 1997; Parkes et al., 2007), and pH
values of <4 (Goodwin and Zeikus, 1987; Koschorreck, 2008)
to >12 (Yu et al., 2013), and to sediment depths of ∼800 mbsf
(Wellsbury et al., 2002). A typical indicator of serpentinization reactions is the
presence of fluids with pH >10 and high formate concentrations
(Mottl et al., 2003; McCollom and Bach, 2009; Schrenk et al.,
2013). In some places, large macrofaunal populations are present
where serpentinite-influenced fluids mix with seawater at the
seafloor (Kelley et al., 2005; Fryer, 2012; Ohara et al., 2012; Joseph,
2017). These macrofauna feed on abundant chemotrophic
microbiota which are sustained by abiotically produced H2, CH4,
and SCOAs as energy donors and seawater-derived O2 and
sulfate as electron acceptors. It has been suggested that the same
abiotically produced compounds might also support microbial
communities in the upper mantle and in subseafloor basaltic
ocean crust (Früh-Green et al., 2004; Lever et al., 2013; Bach,
2016; Smith et al., 2019). INTRODUCTION sulfate reduction (e.g., Mottl et al., 1979; Kiyosu and Krouse,
1990; Worden et al., 2000; Cross et al., 2004; Truche et al., 2009). Since the 1950s, advances in sampling techniques have extended
explorations of subseafloor life from ∼8 meters below seafloor
(mbsf) (Morita and Zobell, 1955) to ∼2,500 mbsf (Inagaki et al.,
2015). Today, the subseafloor microbial biosphere is estimated
to account for 0.18–3.6% of total living biomass on Earth
(Kallmeyer et al., 2012), and to persist in many places despite low
energy supply and harsh environmental conditions, such as high
temperature, pressure, salinity, and/or pH (Takai, 2011; Hoehler
and Jørgensen, 2013; Lever et al., 2015). These variables affect
the power requirements of microbial life, e.g., by increasing rates
of biomolecule damage, and consequently also raise the power
required by cells to maintain and repair essential biomolecules
(Lever et al., 2015). As a result, the presence and abundance of
microorganisms within subseafloor habitats varies greatly with
location as a result of stark differences in cell-specific power
supply and cell-specific power demand (Inagaki et al., 2015; Lever
et al., 2015; Møller et al., 2018; Heuer et al., 2019). In addition to the breakdown of photosynthetically fixed
and necromass bound OM, SCOAs can be synthesized via the
abiotic reduction of inorganic carbon with electron donors
released by serpentinization reactions (Holm and Andersson,
1998; McCollom and Seewald, 2007; Schrenk et al., 2013; Früh-
Green et al., 2014; Preiner et al., 2018). Serpentinization reactions
include chemical reactions whereby water reacts with ultramafic
rocks rich in iron(II)-containing minerals, such as olivine and
pyroxene, to release H2 (McCollom and Bach, 2009; Preiner et al.,
2018). This H2 can then reduce inorganic carbon to formate,
with which it forms a metastable equilibrium between 175 and
300◦(McCollom and Seewald, 2001, 2003a). Mineral surface-
catalyzed Sabatier-type and Fischer-Tropsch type reactions can
furthermore cause H2 to react abiotically with CO or CO2 to
form methanol, CH4, and SCOAs and hydrocarbons with ≥2 C
atoms (Sabatier and Senderens, 1899; Fischer and Tropsch, 1926;
McCollom and Seewald, 2003a, 2007; Holm and Neubeck, 2009). Rates of serpentinization and FTT reactions strongly depend on
temperature, availability of suitable catalysts, and H2 and CO
partial pressures (Van Der Laan and Beenackers, 1999), with
H2 release peaking at ∼300◦C and CH4 production peaking
at 320◦C in batch experiments ranging from 200 to 320◦C
(McCollom et al., 2016). Citation: Eickenbusch P, Takai K, Sissman O,
Suzuki S, Menzies C, Sakai S,
Sansjofre P, Tasumi E,
Bernasconi SM, Glombitza C,
Jørgensen BB, Morono Y and
Lever MA (2019) Origin of Short-Chain
Organic Acids in Serpentinite Mud
Volcanoes of the Mariana Convergent
Margin. Front. Microbiol. 10:1729. doi: 10.3389/fmicb.2019.01729 July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 1 Organic Acids in Serpentinite Mud Eickenbusch et al. Frontiers in Microbiology | www.frontiersin.org Site Description p
IODP Expedition 366 (8 December 2016 to 7 February 2017)
onboard the R/V JOIDES Resolution drilled into the Yinazao
(previously known as Blue Moon), Fantangisña (previously
known as Celestial), and Asùt Tesoru (previously known as
Big Blue) serpentinite mud volcanoes on the Mariana forearc
(Fryer et al., 2018b). These mud volcanoes are located to the
west of the Mariana Trench and differ in distance to the trench
and temperature of the underlying subducting slab (Figure 1). The three mud volcanoes also differ in geographic location, i.e.,
Fantagisña is located ∼90 km north of Yinazao, and ∼170 km
south of Asùt Tesoru (Fryer et al., 2018b). Yet, distance to trench,
which only differs by 17 km, and associated changes in the slab
temperature, which varies from 80◦C to around 250◦C, are more
important drivers of deep geochemical processes (Hulme et al.,
2010). For this reason we schematically represent the three mud
volcanoes as a transect in Figure 1. Distinguishing microbial, thermogenic, and abiotic origins
of SCOAs remains a challenge in many locations because these
processes overlap in temperature, pressure, and redox ranges
and can, in theory, co-occur in the same environments (Holm
and Andersson, 1998; Schrenk et al., 2013). Furthermore,
as
mentioned
earlier,
microbially,
thermogenically,
and
abiotically produced SCOAs can also be removed by microbial,
thermogenic, and abiotic processes. Thus, accumulation of
SCOAs is not a reliable indicator of origin. Even if microbial,
thermogenic, and abiotic processes are spatially separated, fluids
may contain products of all three processes if they are mixtures
with multiple origins. Fortunately, in some cases SCOA origin
can be revealed by concentration, isotopic, and thermodynamic
analyses of natural educts and products (Heuer et al., 2009; Lang
et al., 2010, 2018; Lever et al., 2010). All three mud volcanoes are formed as fluids, liberated during
the subduction of both sediments and crustal rock from the
Pacific plate, hydrate the overlying plate’s mantle and drive
serpentinization, whereby mud is produced by rock-alteration
and breakdown, and the pH of fluids becomes highly alkaline
(Mottl et al., 2003; Fryer, 2012). Due to volume expansion and
corresponding density changes, this mud wells up via conduits,
which are likely related to fault intersections, to the seafloor
(Früh-Green et al., 2004), where it forms serpentinite mud
volcanoes with heights and diameters of multiple kilometers
(Fryer et al., 2018b). INTRODUCTION Lever, unpubl.) indicates the
presence of Lokiarchaeota with close relatives in methane
hydrates, methane seeps, and anoxic subseafloor sediments,
and of aerobic nitrifying Marine Group I Thaumarchaeota
with close relatives in diverse deep sea benthic surface habitats. Cultivation studies have resulted in the isolation of alkaliphilic
bacteria (Marinobacter alkaliphilus), which grow at a pH
of 6.5 to 10.8–11.4 using organic substrates as electron
donors, and O2, nitrate, or fumarate as electron acceptors
(Takai et al., 2005). the reactions that produce microbial energy substrates deep
within subduction zones. INTRODUCTION In
addition
to
being
degraded
by
microorganisms,
photosynthetically fixed OM, and microbial necromass can
be broken down at high temperature by thermochemical or
“thermogenic” reactions (Wellsbury et al., 1997; Egeberg and
Barth, 1998). Elevated temperatures in petroleum and gas
reservoirs, hydrothermal sediments, or deeply buried sediment
layers produce many of the same intermediates and end products
released during the microbial breakdown of OM, including
SCOAs, H2, and CH4 (Wellsbury et al., 1997; Parkes et al.,
2007). Field and laboratory experiments show that thermogenic
SCOA pools are typically dominated by acetate (e.g., Cooles
et al., 1987; Lundegard and Kharaka, 1990; Barth and Bjørlykke,
1993; Kharaka et al., 1993; Shebl and Surdam, 1996), and in
exceptional cases by propionate (Carothers and Kharaka, 1978). Furthermore, in thermogenic environments with temperatures
>100◦C in the presence of sulfate, significant fractions of
the SCOAs, H2, and hydrocarbons produced by thermogenic
breakdown of OM can be removed through thermochemical Well-known
sites
of
serpentinization
in
the
marine
environment include the Lost City Hydrothermal Field (LCHF;
Früh-Green et al., 2003, 2014; Kelley et al., 2005; Proskurowski
et al., 2008; Konn et al., 2009; Lang et al., 2010, 2018) and the
Mariana Forearc (Fryer et al., 2003, 2018b; Mottl et al., 2003;
Hulme et al., 2010; Fryer, 2012). The LCHF is located on the
Atlantis Massif, an oceanic core complex, 15 km west of the
Mid-Atlantic Ridge and is a model environment for rock-hosted July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 2 Eickenbusch et al. Organic Acids in Serpentinite Mud serpentinitic marine systems. In contrast, serpentinite mud
volcanoes of the Mariana forearc are located in a subduction
zone. Here, highly alkaline serpentinite muds, produced >10
kmbsf in the mantle wedge by alteration of mantle rock with
fluids liberated from the underlying subducting plate, are
transported to the seafloor (Hulme et al., 2010; Fryer, 2012). Geochemical profiles and membrane lipids in muds of the top
20 mbsf indicate the presence of metabolically active microbial
life that is dominated by Archaea (Mottl et al., 2003; Curtis
et al., 2013; Aoyama et al., 2018; Kawagucci et al., 2018). Comparing 16S rRNA gene sequences of Curtis et al. (2013)
with published sequences (M. Sampling
T bl
d Table 1 provides an overview of drilled sites included in
this study. Most samples were obtained using a Half-Length
Advanced Piston Corer. Site U1492C Core 1 was obtained
by a full-length Advanced Piston Corer. Site U1498 samples
were obtained using a Rotary Core Barrel. Based on estimated
temperature gradients for all flank and summit sites, in situ
temperatures of all cores were <10◦C. For further details, we refer
to Fryer et al. (2018a). Site Description Maximum vertical mud flow velocities
at Yinazao and Asùt Tesoru have been estimated to be 10.3
and 36.3 cm yr−1, respectively (Hulme et al., 2010; no data for
Fantangisña), which correspond to ascent times of ∼130,000
(Yinazao) and ∼50,000 years (Asùt Tesoru) from the décollement
to the seafloor. Detailed site data from IODP Expedition 366 are
publicly available on the IODP homepage (Fryer et al., 2018b). Here we investigate the origin of SCOAs in three serpentinite
mud volcanoes (Yinazao, Fantangisña, and Asùt Tesoru) of the
Mariana forearc system based on mud and extracted pore fluids
that were obtained during IODP Expedition 366 in 2016/2017. We compare depth-related trends in SCOA concentrations to
those observed for other microbial energy substrates (H2, CO,
CH4), and electron acceptors (SO2−
4 , DIC), to stable isotopic
compositions of bulk carbon pools [DIC, total organic carbon
(TOC), dissolved organic carbon (DOC)], and to cell counts. Furthermore, we present results of incubation experiments and
of thermodynamic calculations to elucidate likely SCOA sources. Despite high concentrations of SCOAs and other microbial
substrates and low temperatures of samples (<10◦C), microbial
activity and microbial populations are around the detection
limit. This (near-)absence of active microbial populations is
likely due to elevated temperature (∼80–250◦C) in the slab
and/or high pH (pH 11.0–12.5) of serpentinite mud, and due
to absence of significant microbial colonization of serpentinite
mud during ascent from the mantle wedge to the seafloor. Due
to the absence of a clear microbial imprint, the mud fluids
sampled during Expedition 366 provide a unique window into Frontiers in Microbiology | www.frontiersin.org DIC and DOC Concentrations DIC and DOC concentrations were measured onboard with
the OI Analytical Aurora 1030C TOC analyzer, consisting of a
syringe module, a sample-stripping manifold, and an infrared
CO2 analyzer. Porewater samples (1 mL per injection) were
acidified with 0.2 mL of 2 M HCl. The CO2 released during
this acid addition step was stripped and injected into the CO2
analyzer. Subsequently, any remaining carbon in the sample was
combusted, and the DOC was obtained by difference. The CO2
Beer-Lambert absorption law was integrated to determine the
total CO2 released from the sample (Fryer et al., 2018a). SCOA Quantification Porewater samples for SCOA quantification were stored at
−80◦C in baked vials (6 h at 450◦C) immediately after retrieval
and were quantified in the home laboratory using two-
dimensional ion chromatography as described in Glombitza et al. (2014). Porewater Interstitial water samples were extracted by squeezing water
out of inner parts of cores using Carver presses (Manheim,
1966) with filtering through prewashed 11 µm cellulose filters
(Whatman, Cat.-No. 1001090). Water samples were then filtered
through 0.45 µm PES syringe filters (GE Puradisc, Cat-No. 6780.2504) during splitting into aliquots. pH, and concentrations July 2019 | Volume 10 | Article 1729 3 Organic Acids in Serpentinite Mud Eickenbusch et al. FIGURE 1 | Schematic dissection of the Yinazao, Fantangisña, and Asùt Tesoru mud volcanoes in relation to the Mariana Trench, which is formed by subduction of
the Pacific Plate under the Philippine Plate. The Pacific Plate and its sediment cover are exposed to increasing temperatures as they are subducted. The Yinazao,
Fantangisña, and Asùt Tesoru mud volcanoes are shown in blue due to the distinct blue color of the dominant lithology, upwelling serpentinite mud. Investigating how
SCOA compositions, and origins (microbial, thermogenic, abiotic) change both horizonally and vertically between and within these mud volcanoes provides insights
into the controls on carbon transformation reactions in the deep subseafloor. FIGURE 1 | Schematic dissection of the Yinazao, Fantangisña, and Asùt Tesoru mud volcanoes in relation to the Mariana Trench, which is formed by subduction of
the Pacific Plate under the Philippine Plate. The Pacific Plate and its sediment cover are exposed to increasing temperatures as they are subducted. The Yinazao,
Fantangisña, and Asùt Tesoru mud volcanoes are shown in blue due to the distinct blue color of the dominant lithology, upwelling serpentinite mud. Investigating how
SCOA compositions, and origins (microbial, thermogenic, abiotic) change both horizonally and vertically between and within these mud volcanoes provides insights
into the controls on carbon transformation reactions in the deep subseafloor. Serpentinite Mud
Cell Counts To determine cell abundances, 2 cm3 of mud were subsampled
from central portions of cores using sterile cut-offsyringes in
an ultra clean air environment (KOACH T-500f, Koken, Ltd.,
Morono et al., 2018). Samples were immediately fixed with 2%
paraformaldehyde
in
3-(cyclohexylamino)-1-propanesulfonic
acid (CAPS) buffer solution adjusted to pH 11. The fixed samples
were stored at 4◦C until the analysis at the home laboratory,
whereby slurry samples were subjected to cell detachment and
separation steps at the super clean room in Kochi Core Center,
Japan (Morono et al., 2013, 2017). In brief, 1 mL of fixed mud
slurry was mixed with 1.4 mL of 2.5% NaCl, 300 µL of detergent
mix (100 mM ethylenediamine tetra-acetic acid [EDTA], 100 mM
sodium pyrophosphate, 1% [v/v] Tween-80), and 300 µL of
pure methanol, and homogenized on a Shake Master (Bio
Medical Science, Japan) at 500 rpm for 60 min. Samples were δ13C-DIC Values of [Mg(aq)], sulfate, DIC and DOC were determined onboard as
described in Fryer et al. (2018a). All data are available online
(Fryer et al., 2018b,c). Porewater samples for δ13C-DIC analyses were preserved
by adding HgCl2 and stored at 4◦C. Depending on the
concentration, 1–2 ml of sample were injected into He-flushed
exetainers containing 150 µl of 85% phosphoric acid to
lower the pH and convert all DIC into CO2. CO2 was
then measured after equilibration using isotope-ratio mass
spectrometry. Standardization was accomplished by measuring
Na-bicarbonate solutions of different concentrations prepared
from a Na-bicarbonate powder, for which the δ13C was
determined by digestion with phosporic acid, and by comparison
to calcium carbonate standards as described in Breitenbach and
Bernasconi (2011). Gas Analyses y
To quantify concentrations of H2, CH4, and CO, 1 cm3 of mud
was collected from cut ends of core sections immediately after
core arrival. Samples were placed in 20 cm3 glass vials with
3 mL of distilled water and a small amount of HgCl2 to prevent
microbial activity. Vials were sealed with Teflon-coated butyl
rubber septa and crimped aluminum caps and then placed in an
oven at 80◦C for 30 min. A 0.5 cm3 aliquot of the headspace was
sampled with a standard gas syringe and automatically injected
into a GL Science GC4000 GC equipped with a helium ionization
detector set at 250◦C. The column (2 mm inner diameter; 6.3 mm
outer diameter) was packed with carbosieve (Agilent/Hewlett
Packard). The GC oven program was set to 40◦C during the initial
5 min with a subsequent rise to 250◦C at 20◦C/min. A second 0.5
cm3 aliquot of the headspace was then automatically injected into
an Agilent/Hewlett Packard 6890 Series II gas chromatograph
(GC) equipped with a flame ionization detector set at 250◦C. The column (2 mm inner diameter; 6.3 mm outer diameter)
was packed with 80/100 mesh HayeSep (Restek). The GC oven
program was set to 80◦C for 8.25 min with a subsequent rise to
150◦C at 40◦C/min. All measurements were calibrated using two
different gas standards. All data from Fryer et al. (2018c), except distance to trench (from Hulme et al., 2010), and distance to slab data (Oakley, 2008; from Oakley et al., 2007, 2008). then sonicated at 160 W for 30 s for 10 cycles (Bioruptor UCD-
250HSA; Cosmo Bio, Japan), followed by loading onto density
layers composed of 30% Nycodenz (1.15 g/cm3), 50% Nycodenz
(1.25 g/cm3), 80% Nycodenz (1.42 g/cm3), and 67% sodium
polytungstate (2.08 g/cm3), and centrifugation at 10,000×g for
1 h at 25◦C with swinging rotors. The light density layer was
collected, whereas the heavy fraction was subjected to a second
round of separation after washing with 5 mL of 2.5% NaCl. The
recovered supernatants were then pooled and passed through a
0.22 µm polycarbonate membrane filter. Cells on the membrane
filter were stained with SYBR Green I staining solution (1/40
of SYBR Green I in Tris-EDTA [TE] buffer). The number of
SYBR Green I–stained cells was enumerated by automated
epifluorescence microscopic counting (Morono et al., 2009;
Inagaki et al., 2015). To quantify procedural contamination,
blank controls involving 1 mL of 2.5% NaCl solution were also
subjected to the above cell separation and staining procedures. The procedural contamination was on average 1.0 cells per
counted membrane (n = 10), which corresponds to a minimum
quantification limit of 24 cells/cm3 (average plus three times the
standard deviation of blank counts). TABLE 1 | Background information on drilled holes during IODP Expedition 366. Hole
Seamount
Location
Latitude
Longitude
Water depth
(mbsl)
Coring
Total
penetration
(m)
Distance to
trench (km)
Distance to
slab (km)
Temperature
at slab (◦C)
U1492C
Yinazao
Summit
15◦42.5590′N
147◦10.6001′E
3666.47
APC/HLAPC
139.1
55
13
∼80
U1493B
Asùt Tesoru
Flank
17◦59.1665′N
147◦06.0060′E
3358.92
HLAPC
32.6
72
18
∼250
U1494A
Asùt Tesoru
Flank
18◦3.0896′N
147◦6.0003′E
2199.80
HLAPC
39
72
18
∼250
U1495A
Asùt Tesoru
Flank
18◦05.6693′N
147◦06.0004′E
1405.81
HLAPC
10.7
72
18
∼250
U1495B
Asùt Tesoru
Flank
18◦05.6788′N
147◦05.9901′E
1401.89
HLAPC
10.8
72
18
∼250
U1496A
Asùt Tesoru
Summit
18◦6.5936′N
147◦6.0999′E
1243.38
HLAPC
44.8
72
18
∼250
U1496B
Asùt Tesoru
Summit
18◦6.6205′N
147◦6.0998′E
1240.18
HLAPC
36
72
18
∼250
U1497A
Fantangisña
Summit
16◦32.2536′N
147◦13.2642′E
2019.24
HLAPC
34.2
62
14
∼150
U1497B
Fantangisña
Summit
16◦32.2528′N
147◦13.2606′E
2018.22
HLAPC
23.8
62
14
∼150
U1498A
Fantangisña
Flank
16◦27.0898′N
147◦09.8502′E
3496.21
RCB
181.6
62
14
∼150
U1498B
Fantangisña
Flank
16◦27.3716′N
147◦10.1166′E
3284.70
RCB
260
62
14
∼150 δ13C-DOC Values Porewater samples for δ13C-DOC analyses were stored at −80◦C
in baked vials (6 h at 450◦C). Isotopic compositions were
measured after wet-chemical oxidation of DOC with persulfate
(1 h at 100◦C) on decarbonized subsamples (acidification to pH
< 3 with 85% H3PO4). Two to four ml of headspace were then
transferred into He flushed vials and analyzed using isotope-ratio
mass spectrometry as described in Lang et al. (2012). July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 4 Organic Acids in Serpentinite Mud Eickenbusch et al. Total Organic Carbon (TOC) and δ13C-TOC Total Organic Carbon (TOC) and δ
C TOC
Analyses were performed on solid residue samples after
porewater squeezing. TOC and δ13Corg were analyzed using
an elemental analyzer (EA, Flash 2000; Thermo Scientific)
coupled to a Delta V+ isotope ratio mass spectrometer
(Thermo Scientific) at the Pôle de Spectrométrie Océan
(PSO, Brest, France). Approximately 25 mg of decarbonated
samples were loaded into tin capsules and introduced into
an autosampler. Flash combustion was performed using an
8 s injection time of dioxygen at a flux of 240 mL min−1. Carbon isotope ratios were obtained against reference standards
(SED-IVA reference number: 33802151) and in-house standards July 2019 | Volume 10 | Article 1729 5 Organic Acids in Serpentinite Mud Eickenbusch et al. (Acetanilide: reference number 274462 from Thermo Fisher;
CAP (leaf litter) and LIPG (yeast) from Institut de Physique
du Globe de Paris, France). δ13C-TOC values are given as
the per mil (‰) difference from the PDB standard. TOC was
measured using the thermal conductivity detector of the Flash
2000 instrument. Routine replicate measurements had internal
deviations of 0.15‰ for δ13C-TOC and <5% for TOC. (Acetanilide: reference number 274462 from Thermo Fisher;
CAP (leaf litter) and LIPG (yeast) from Institut de Physique
du Globe de Paris, France). δ13C-TOC values are given as
the per mil (‰) difference from the PDB standard. TOC was
measured using the thermal conductivity detector of the Flash
2000 instrument. Routine replicate measurements had internal
deviations of 0.15‰ for δ13C-TOC and <5% for TOC. provide insights into the role of deep, tectonically driven
reactions in determining the sources and chemical compositions
of microbial energy substrates in mud volcanoes of the
Mariana forearc. Thermodynamic Calculations Calculations were done
for activities of aqueous species, which were calculated using the
activity coefficients γCO32−= 0.038 (Plummer and Sundquist,
1982), γCH4 = 1.24 (Millero, 2000), γSO42−= 0.104 (Millero
and Schreiber, 1982), and γHS−= 0.685 (Clegg and Whitfield,
1991). The activity coefficients of H2 and CO were approximated
with that of CH4, those of SCOAs were approximated with that
of HS−, and those of glucose were set to 1.0. All concentrations
were measured, except for glucose and HS−, which were assumed
to equal 1 nM and 1 mM, respectively. The ∆Gr was calculated
for temperature, pressure, and pH in sediment cores and for
temperature, pressure, and pH under slab conditions. The pH
under slab conditions was 11.2 for Yinazao, 11.0 for Fantangisña,
and 12.5 for Asùt Tesoru (based on Fryer et al., 2018b). Magnesium (Mg2+)concentrations are mostly below or close
to the detection limit of 0.1 mM in subsurface high-pH mud
fluids of all three mud volcanoes. In a few high-pH samples,
concentrations of up to ∼5 mM are reached, which correspond
to ∼10% of seawater values (∼54 mM). Drilling fluid (seawater)
contamination is a likely source of these elevated Mg2+ values
(Figure 2B) according to drilling fluid intrusion estimates based
on perfluorocarbon tracer compounds (Fryer et al., 2017; Lever
et al., in prep.). Mg2+ concentrations in high-pH fluids increase
steeply near the seafloor, in the same intervals where pH values
decrease, indicating significant fluid exchange, e.g., by diffusive
mixing with seawater, in the top meters of sediment. By contrast,
moderate-pH mud fluids show a gradual decrease but sustain
significantly higher Mg2+ concentrations throughout the cored
intervals. These Mg2+ concentrations cannot be explained with
measured drilling fluid contamination (Fryer et al., 2017; Lever
et al., in prep.) and indicate that moderate-pH pore fluids are
a mixture of seawater and/or shallower sedimentary pore fluids
and deeply-sourced serpentinitic fluids. Geochemical Setting Porewater pH values increase downward from the seafloor
(Figure 2A), where values approach those of seawater (pH 8.1). In samples with high fluid upflow, the pH stabilizes at values that
increase with distance to trench (Yinazao: ∼10.7; Fantangisña:
∼11.2; Asùt Tesoru: ∼12.4) and at different sediment depths
(Yinazao: ∼8 mbsf; Fantangisña: unclear but within top 66 mbsf;
Asùt Tesoru: ∼4 mbsf). Within Fantangisña and Asùt Tesoru,
fluids from flank sites or areas with less fluid upflow are clearly
distinguishable from fluids in areas of higher upflow based on pH
values closer to those in surface sediments (Mottl et al., 2003). For the sake of simplicity, we from now on refer to samples
from boreholes with high upflow and pH>10.0 as “high-pH
mud fluids,” and samples from boreholes on flank sites and sites
with less upflow and pH<10.0 as “moderate-pH mud fluids.”
We, moreover, distinguish between surface samples with a clear
seawater influence, and subsurface samples, where pH values are
constant at higher values. Thermodynamic Calculations Gibbs energy yields (∆Gr) of potential microbial, thermogenic,
and abiotic reactions were calculated based on the equation ∆Gr = ∆G0
r (T,p) + RT ln Qr
(1) (1) where ∆G0
r (T,p) is the Gibbs energy (kJ mol−1 of reaction) at
standard concentrations (1 M per each reactant and product, pH
7.0) corrected for in situ temperature T (K) and pressure p (bar),
R is the universal gas constant (0.008314 kJ mol−1 K−1), and
Qr the quotient of product and reactant activities. To obtain
∆G0
r (T,p), the standard Gibbs energies of the reaction ∆G0
r was
corrected to estimated in situ slab temperature and pressure as
outlined in Stumm and Morgan (1996). Standard Gibbs energies,
standard enthalpies, and standard molal volumes of formation
are shown in Supplementary Table 1. Calculations were done
for activities of aqueous species, which were calculated using the
activity coefficients γCO32−= 0.038 (Plummer and Sundquist,
1982), γCH4 = 1.24 (Millero, 2000), γSO42−= 0.104 (Millero
and Schreiber, 1982), and γHS−= 0.685 (Clegg and Whitfield,
1991). The activity coefficients of H2 and CO were approximated
with that of CH4, those of SCOAs were approximated with that
of HS−, and those of glucose were set to 1.0. All concentrations
were measured, except for glucose and HS−, which were assumed
to equal 1 nM and 1 mM, respectively. The ∆Gr was calculated
for temperature, pressure, and pH in sediment cores and for
temperature, pressure, and pH under slab conditions. The pH
under slab conditions was 11.2 for Yinazao, 11.0 for Fantangisña,
and 12.5 for Asùt Tesoru (based on Fryer et al., 2018b). where ∆G0
r (T,p) is the Gibbs energy (kJ mol−1 of reaction) at
standard concentrations (1 M per each reactant and product, pH
7.0) corrected for in situ temperature T (K) and pressure p (bar),
R is the universal gas constant (0.008314 kJ mol−1 K−1), and
Qr the quotient of product and reactant activities. To obtain
∆G0
r (T,p), the standard Gibbs energies of the reaction ∆G0
r was
corrected to estimated in situ slab temperature and pressure as
outlined in Stumm and Morgan (1996). Standard Gibbs energies,
standard enthalpies, and standard molal volumes of formation
are shown in Supplementary Table 1. RESULTS Blue marks on the x-axis indicate seawater concentrations from Mottl et al. (2003). Red symbols indicate high-pH holes, black symbols indicate moderate-pH holes
see text for definitions). FIGURE 3 | Depth profiles of (A) formate, (B) acetate, (C) propionate, (D) butyrate, (E) CH4, and (F) H2 concentrations in the Yinazao, Fantangisña, and Asùt Tesoru
mud volcanoes ordered in ascending distance to the Mariana Trench (top to bottom). Red symbols indicate high-pH holes with an average pH > 10, black symbols
ndicate moderate-pH holes with an average pH < 10. oles U1497A and B decreasing steeply with sediment depth,
hile values from U1498A stabilize at ∼25 mM, which is ∼5-
also vary between boreholes. Steep depth-related decreases to
∼14 mM occur in the top 12 mbsf at U1495A and B, while more FIGURE 2 | Depth profiles of (A) pH, (B) magnesium (Mg2+), (C) sulfate, (D) DIC, (E) DOC, (F) TOC, and (G) cell counts ordered in ascending distance to the
Mariana Trench (top to bottom). All pH data, and magnesium (Mg2+), sulfate, DIC, and DOC concentrations were measured shipboard and were obtained from Fryer
et al. (2018b). TOC (% sample dry weight) and cell abundances are from this study (gray area indicates counts that are below the quantification limit of 20 cells cm−3). Blue marks on the x-axis indicate seawater concentrations from Mottl et al. (2003). Red symbols indicate high-pH holes, black symbols indicate moderate-pH holes
(see text for definitions). FIGURE 2 | Depth profiles of (A) pH, (B) magnesium (Mg2+), (C) sulfate, (D) DIC, (E) DOC, (F) TOC, and (G) cell counts ordered in ascending distance to the
Mariana Trench (top to bottom). All pH data, and magnesium (Mg2+), sulfate, DIC, and DOC concentrations were measured shipboard and were obtained from Fryer
et al. (2018b). TOC (% sample dry weight) and cell abundances are from this study (gray area indicates counts that are below the quantification limit of 20 cells cm−3). Blue marks on the x-axis indicate seawater concentrations from Mottl et al. (2003). Red symbols indicate high-pH holes, black symbols indicate moderate-pH holes
(see text for definitions). FIGURE 3 | Depth profiles of (A) formate, (B) acetate, (C) propionate, (D) butyrate, (E) CH4, and (F) H2 concentrations in the Yinazao, Fantangisña, and Asùt Tesoru
mud volcanoes ordered in ascending distance to the Mariana Trench (top to bottom). RESULTS In the following sections we first compare geochemical
background data (pH; magnesium, sulfate, DIC, and DOC
concentrations; TOC contents) and cell counts on high-pH
subsurface serpentinite mud fluids (pH 10.5–12.5) with shallow
and adjacent samples, that are under stronger seawater or
sedimentary influence and have moderate pH values (pH
7.8–10;
Figure 2). We
then
examine
how
concentration
profiles of potential microbial energy substrates (SCOAs,
CH4, H2, CO; Figure 3) vary with pH within and across the
three volcanoes. In the final part, we investigate how the
relative contributions of different SCOAs to DOC change
between and within mud volcanoes (Figure 4), how the
concentrations
of
individual
SCOAs
change
in
high-pH
samples with distance to the trench and with increasing slab
temperature (Figures 5, 6), and how δ13C-isotopic values
of DIC, DOC, and TOC change between and within mud
volcanoes (Figure 7). Through this integrated analysis, we Sulfate concentrations in high-pH fluids (Figure 2C) show
similar patterns to pH and magnesium, and in relation to chloride
(Supplementary Figure S1B), i.e., changes toward seawater
values (∼28 mM) in the upper meters, and steady profiles below. Chloride concentrations below those of seawater in high-pH
fluids, moreover, indicate fluid freshening due to dewatering of
deep clay-bearing minerals (Supplementary Figure S1A). There
is no clear trend in sulfate concentrations with distance to
trench, as is evident from the fact that high-pH fluids from
Fantangisña are depleted in sulfate (∼4 mM), whereas those
of Yinazao and Asùt Tesoru are only slightly lower (∼27 mM)
or even higher (∼31 mM) than seawater values (28 mM; Mottl
et al., 2003). By contrast, sulfate concentrations in moderate-
pH fluids at Fantangisña show a striking variability, also in
relation to chloride (Supplementary Figure S1), with values in July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 6 Organic Acids in Serpentinite Mud Eickenbusch et al. FIGURE 2 | Depth profiles of (A) pH, (B) magnesium (Mg2+), (C) sulfate, (D) DIC, (E) DOC, (F) TOC, and (G) cell counts ordered in ascending distance to the
Mariana Trench (top to bottom). All pH data, and magnesium (Mg2+), sulfate, DIC, and DOC concentrations were measured shipboard and were obtained from Fryer
t al. (2018b). TOC (% sample dry weight) and cell abundances are from this study (gray area indicates counts that are below the quantification limit of 20 cells cm−3). Frontiers in Microbiology | www.frontiersin.org RESULTS Red symbols indicate high-pH holes with an average pH > 10, black symbols
indicate moderate-pH holes with an average pH < 10. FIGURE 3 | Depth profiles of (A) formate, (B) acetate, (C) propionate, (D) butyrate, (E) CH4, and (F) H2 concentrations in the Yinazao, Fantangisña, and Asùt Tesoru
mud volcanoes ordered in ascending distance to the Mariana Trench (top to bottom). Red symbols indicate high-pH holes with an average pH > 10, black symbols
indicate moderate-pH holes with an average pH < 10. holes U1497A and B decreasing steeply with sediment depth,
while values from U1498A stabilize at ∼25 mM, which is ∼5-
fold higher than in high-pH fluids of the same mud volcano. Sulfate concentration profiles on the flanks of Asùt Tesoru also vary between boreholes. Steep depth-related decreases to
∼14 mM occur in the top 12 mbsf at U1495A and B, while more
moderate decreases to ∼15 mM and ∼22 mM at ∼30 mbsf occur
at U1493B and U1494A, respectively. Unlike at Fantangisña, also vary between boreholes. Steep depth-related decreases to
∼14 mM occur in the top 12 mbsf at U1495A and B, while more
moderate decreases to ∼15 mM and ∼22 mM at ∼30 mbsf occur
at U1493B and U1494A, respectively. Unlike at Fantangisña, July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 7 Organic Acids in Serpentinite Mud Eickenbusch et al. FIGURE 4 | SCOA percent fractions of DOC vs. depth at the Yinazao, Fantangisña, and Asùt Tesoru mud volcanoes. Black labeling indicates moderate pH fluids with
an average pH < 10 (Left), red labeling indicates high pH fluids with an average pH > 10 (Right). FIGURE 4 | SCOA percent fractions of DOC vs. depth at the Yinazao, Fantangisña, and Asùt Tesoru mud volcanoes. Black labeling indicates moderate pH fluids with
an average pH < 10 (Left), red labeling indicates high pH fluids with an average pH > 10 (Right). sulfate concentrations at Asùt Tesoru are higher in high-pH than
in moderate-pH fluids. in the same range as high-pH subsurface fluids, moderate-pH
fluids at Asùt Tesoru consistently had 5–10-fold lower DIC
concentrations than high-pH fluids. DIC concentrations in high-pH fluids at Yinazao and
Fantangisña remain mostly within a narrow range of 0 to
1 mM and are lower than those in seawater (Mottl et al.,
2003; Figure 2D). Frontiers in Microbiology | www.frontiersin.org RESULTS By comparison, despite showing considerable
scatter, measured DIC concentrations of high-pH fluids at Asùt
Tesoru are higher (mostly 2–4 mM), in a range that is similar to
or above seawater values. While DIC concentrations at shallow
sediment depths and moderate-pH fluids at Fantangisña were DOC
concentrations
in
high-pH
fluids
show
straight
subsurface profiles, with an increase in average concentration
with distance to trench (Yinazao: 211 ± 51 µM; Fantangisña: 419
± 79 µM; Yinazao: 1,684 ± 234 µM; Figure 2E). In moderate-
pH fluids of Fantangisña and Asùt Tesoru, subsurface DOC
concentrations also show no clear depth-related trends and have
similar (Fantangisña; 317 ± 72 µM) or lower (Asùt Tesoru: 350 July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 8 Organic Acids in Serpentinite Mud Eickenbusch et al. FIGURE 5 | Formate-to-acetate ratios for three serpentinite mud volcanoes drilled during IODP Expedition 366. Black symbols indicate moderate pH fluids with an
average pH < 10 (A), and red symbols indicate high pH fluids with an average pH > 10 (B). Brown symbols indicate data on high pH fluids from Conical seamount
from ODP Leg 125 (Haggerty and Fisher, 1992). FIGURE 5 | Formate-to-acetate ratios for three serpentinite mud volcanoes drilled during IODP Expedition 366. Black symbols indicate moderate pH fluids with an
average pH < 10 (A), and red symbols indicate high pH fluids with an average pH > 10 (B). Brown symbols indicate data on high pH fluids from Conical seamount
from ODP Leg 125 (Haggerty and Fisher, 1992). FIGURE 6 | Formate, acetate and propionate concentrations in high pH fluids vs. distance to trench (A) and vs. slab temperatures (B). Regression includes data from
Conical seamount, which was drilled during Ocean Drilling Program Leg 125 (Haggerty and Fisher, 1992). FIGURE 6 | Formate, acetate and propionate concentrations in high pH fluids vs. distance to trench (A) and vs. slab temperatures (B). Regression includes data from
Conical seamount, which was drilled during Ocean Drilling Program Leg 125 (Haggerty and Fisher, 1992). ± 195 µM) concentrations than in respective high-pH fluids. At
Fantangisña, where measurements were also made in shallow
sediment layers, there is a clear decrease in DOC concentrations
toward the seafloor. TOC contents in serpentinite muds are generally low,
mostly scattering around ∼0.01% dry weight, and do not
show systematic differences between high- and moderate-pH
fluids or between mud volcanoes (Figure 2F). RESULTS Nonetheless, July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 9 Organic Acids in Serpentinite Mud Eickenbusch et al. FIGURE 7 | Box plots of δ13C-isotopic compositions of TOC, DOC, and DIC of moderate-pH and high-pH subsurface mud fluids. Boxes show the entire data range,
vertical lines within boxes indicate the mean value. FIGURE 7 | Box plots of δ13C-isotopic compositions of TOC, DOC, and DIC of moderate-pH and high-pH subsurface mud fluids. Boxes show the entire data range,
vertical lines within boxes indicate the mean value. FIGURE 7 | Box plots of δ13C-isotopic compositions of TOC, DOC, and DIC of moderate-pH and high-pH subsurface mud fluids
vertical lines within boxes indicate the mean value. Tesoru, however, moderate-pH, subsurface fluids have ∼40-fold
lower concentrations (2.7 ± 1.7 µM) than high-pH fluids. there are several outliers, including values with 2.1% (U1492C
1H2; Yinazao) and 3.3% dry weight (U1496B 8XCC; Asùt
Tesoru). Comparing porosity-corrected DOC to TOC contents
in subsurface fluids, there are clear differences between and
within mud volcanoes. DOC accounts for similar contributions
of TOC in high-pH subsurface fluids at Yinazao (1.5 ±
0.7%) and at high-pH (∼2.0%) and moderate-pH (1.6 ±
1.3%) fluids of Fantangisña. By contrast, at Asùt Tesoru,
the average DOC contribution to TOC is higher, i.e., 3.0
± 1.2% in moderate-pH, and 13.1 ± 8.0% in high-pH
subsurface fluids. Acetate concentrations (Figure 3B) show similar trends to
formate, except that the characteristic decrease toward the
seafloor is absent from shallow sediments of Fantangisña,
Moreover, the increase in high-pH fluids with distance to trench
is not as strong as for formate. Average acetate concentrations
range between 1 and 10 µM in high-pH (Yinazao: 2.4 ±
1.6 µM; Fantangisña: 6.2 ± 1.6 µM) and moderate-pH fluids
(Fantangisña: 4.9 ± 4.0 µM; Asùt Tesoru: 5.6 ± 4.4 µM), except
in high-pH subsurface fluids of Asùt Tesoru, where acetate
concentrations are clearly elevated (42.0 ± 4.7 µM). Cell counts reach values of ∼106 cells cm−3 in the upper tens
of meters, but are mostly below or within an order of magnitude
above the quantification limit of 20 cells cm−3 in deeper layers,
both in high-pH and moderate-pH muds (Figure 2G). RESULTS Propionate concentrations (Figure 3C) show no clear trends
related to depth below the seafloor, fluid pH, or distance to
trench, and range mostly from 1 to 10 µM both in high-pH
(Yinazao: 6.3 ± 1.7 µM; Fantangisña: 5.9 ± 1.2 µM; Asùt Tesoru:
4.4 ± 2.8 µM) and in moderate-pH fluids (Fantangisña: 5.8 ±
2.5 µM; Asùt Tesoru: 3.5 ± 4.0 µM). Propionate concentrations
are relatively uniform across boreholes at Fantangisña, but
vary significantly in moderate-pH fluids of Asùt Tesoru, where
U1493B and U1491A have 5–10 times lower concentrations than
U1495A and B. Similarly, there is an offset among high-pH
fluids of Asùt Tesoru (U1496), where hole B has ∼3 times lower
concentrations than hole A. Frontiers in Microbiology | www.frontiersin.org Microbial Activity Within Samples Based
on Incubation Experiments Other SCOAs were also detected in numerous samples
(Supplementary Figure S2). Pyruvate was mainly detected in
high-pH fluids of Asùt Tesoru (0.2 ± 0.1 µM). Valerate was
detected in most high-pH fluid samples from Yinazao and Asùt
Tesoru, with roughly 10-fold higher average concentrations in
Asùt Tesoru (0.4 ± 0.2 µM vs. 0.04 ± 0.03 µM at Yinazao). Low
(sub)micromolar concentrations of lactate were also present in
most samples. Notably, lactate was the only SCOA with higher
average concentrations in moderate-pH (1.5 ± 1.5 µM) than in
high-pH fluids (0.7 ± 1.2 µM) of Asùt Tesoru. To check for measurable microbial activity, we incubated high-
pH muds of all three mud volcanoes at in situ pH in the
laboratory using formate as an energy substrate and monitored
concentrations of the metabolites formate, H2, CH4, and DIC
for 6 weeks. None of the samples showed significant changes
in metabolite concentrations over time or between formate
incubations (100 µM 13C-formate), killed controls (100 µM 13C-
formate + sodium azide), and negative controls (no formate). Similar to formate, CH4 concentrations (Figure 3E) in
high-pH fluids show relatively straight downcore profiles
and increase dramatically with distance to trench (Yinazao:
2.4 ± 1.4 µM, Fantangisña: 10.4 ± 6.3 µM; Asùt Tesoru:
2,170
±
1,630 µM). CH4
concentrations
in
moderate-
pH subsurface fluids of Fantangisña and Asùt Tesoru are
10 to 100 times lower than in high-pH fluids from the
same depths. Concentration Profiles of Microbial Energy
Substrates Concentration profiles of potential microbial energy substrates
show patterns with respect to depth below the seafloor and
between high-pH and moderate-pH fluids that resemble those
observed for pH, DIC, sulfate, and DOC. Formate concentrations in high-pH fluids show straight
subsurface profiles and increase with distance to trench (Yinazao:
1.8 ± 1.2 µM, Fantangisña: 7.0 ± 2.7 µM; Asùt Tesoru: 104 ±
11 µM; Figure 3A). Moderate-pH fluids from subsurface layers
of Fantangisña are in a similar range (8.0 ± 3.7 µM) to high-
pH fluids, and show the same characteristic decrease toward the
seafloor that is also present in DOC concentrations. At Asùt Butyrate concentrations scatter in the submicromolar to
low micromolar range (Figure 3D), showing no systematic
relationship with depth below the seafloor, pH, or distance to
trench. Average values of high-pH fluids are 1.5 ± 0.5 µM, 0.5
± 0.2 µM, and 2.1 ± 0.3 µM at Yinazao, Fantangisña, and Asùt
Tesoru, respectively. Average values of moderate-pH fluids are July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 10 Organic Acids in Serpentinite Mud Eickenbusch et al. Asùt Tesoru are consistently dominated by formate and acetate,
which together account for 69–92% of the total SCOA-C. Asùt Tesoru are consistently dominated by formate and acetate,
which together account for 69–92% of the total SCOA-C. 0.6 ± 0.3 µM and 3.4 ± 3.6 µM at Fantangisña and Asùt Tesoru,
respectively. The only notable trend is a linear decrease to the
seafloor within the upper 10 mbsf at Fantangisña. Frontiers in Microbiology | www.frontiersin.org SCOA Concentrations in High-PH Fluids in
Relation to Distance to Trench and Slab
Temperature p
The very low cell numbers, the nearly constant concentration
profiles of SCOAs in high-pH fluids, and the absence of
measurable formate turnover in incubations raise the possibility
that microbial activity is absent from high-pH subsurface muds. If so, then this raises the possibility that SCOAs measured
in high-pH muds were thermogenically released from organic
matter in the subducting slab and its sediments and/or produced
by abiotic synthesis reactions linked to serpentinization of mantle
rock of the Philippine Plate, and subsequently preserved over
thousands of years during ascent. Under such a scenario, where
all SCOAs in high-pH muds would be thermogenic or abiotic
in origin, the changes in SCOA concentrations across mud
volcanoes would reflect different environmental conditions deep
within the subduction zone. Similar to formate and CH4, H2 concentrations (Figure 3F)
in high-pH fluids show a similar, strong increase with distance
to trench (Yinazao: 16 ± 40 µM, Fantangisña: 380 ± 250 µM,
Asùt Tesoru: 1,660 ± 1,560 µM). Compared to corresponding
depths at Fantangisña and Asùt Tesoru, these H2 concentrations
are approximately two orders of magnitude higher than those in
moderate-pH fluids (Fantangisña: 22.2 ± 32.4 µM; Asùt Tesoru:
225 ± 660 µM). Carbon monoxide (CO) concentrations were barely detectable
in high-pH fluids of Yinazao, but were clearly above detection
in high-pH fluids of the other two mud volcanoes (Fantangisña:
11 ± 3 µM; Asùt Tesoru: 7 ± 2 µM; Supplementary Figure S3). Compared to high-pH fluids, CO concentrations in moderate-
pH fluids of Fantangisña and Asùt Tesoru are in a similar
range or slightly higher (Fantangisña: 13 ± 9 µM; Asùt
Tesoru: 12 ± 3 µM). Our data are consistent with the possibility of SCOAs in high-
pH fluids having deep origins. Cross-plots, in which previously
measured formate and acetate concentrations from Conical
Seamount (Haggerty and Fisher, 1992), a mud volcano that is
located further away (86 km) from the Mariana Trench than
Asùt Tesoru, are included, show that measured concentrations
of formate and acetate in high-pH subsurface fluids are strongly
correlated (power relationship; Figure 5B). Such a relationship
is absent from moderate-pH samples (Figure 5A), or for
propionate or butyrate in high-pH samples (not shown). Stable Isotopic Compositions of Bulk
Carbon Pools microbial populations. Downward SCOA concentrations in
high-pH subsurface muds of all three mud volcanoes show
no clear changes, and neither do concentrations of other
potential metabolites, such as sulfate, DIC, and DOC. Though an
interpretation of the dissolved CH4 and H2 data is confounded
by potential outgassing during core retrieval, these gases also
show no clear depth-related production or consumption profiles. Microbial populations, quantified by microscopic counting, are
mainly around the minimum quantification limit of 20 cells
cm−3. Local cell population peaks in subsurface high-pH fluids
have to be interpreted with caution, given the local detection
of millimolar concentrations of seawater-derived Mg2+, and
evidence of significant contamination of sediment porewater by
drilling fluid (surface seawater) containing cell concentrations of
105-106 cells cm−3 (Lever et al., in prep.). Yet, even if the local
peaks in cell counts accurately reflect in situ cell populations,
these cells may not be metabolically active due to the highly
alkaline in situ pH. As with most other analytes, the subsurface δ13C-isotopic
values of TOC, DOC, or DIC show no clear depth-related
trends (Supplementary Figure S5), but instead indicate clear
differences related to mud volcanoes and fluid pH (Figure 7). Subsurface δ13C-TOC values fall into a narrow range
(Yinazao, high-pH: −27.5 ± 1.9‰; Fantangisña: moderate-pH:
−27.8 ± 1.5‰; high-pH: −27.6 ± 3.0‰; Asùt Tesoru: moderate-
pH: −28.7 ± 0.6‰; high-pH: −26.9 ± 2.0‰), and thus do not
differ significantly between mud volcanoes or between pH-muds
within mud volcanoes (Mann Whitney Test; p > 0.05). y
p
All subsurface δ13C-DOC data also fall within a narrow
range, with the exception of high-pH fluids from Asùt Tesoru,
where the average δ13C-DOC is 11.1–12.1‰ higher than in all
other mud volcano fluids (Yinazao, high-pH: −25.2 ± 1.2‰;
Fantangisña: moderate-pH: −25.4 ± 0.4‰; high-pH: −26.2 ±
1.7‰; Asùt Tesoru: moderate-pH: −25.5 ± 2.3‰; high-pH:
−14.1 ± 1.6‰). This difference in δ13C-DOC between Asùt
Tesoru high-pH fluids and all other mud volcano fluids is highly
significant (p < 0.01). If significant microbial activity is absent, then this would mean
that concentrations and compositions of potential microbial
electron donors, such as SCOAs, CH4, H2, and CO, and electron
acceptors, such as sulfate and DIC, in high-pH fluids could
provide useful insights into deep, non-biological processes in the
subducting slab and the overlying forearc mantle. Contribution of SCOAs to DOC SCOAs make up ∼10–30% of the C fraction of total DOC
(Figure 4). The contribution of different SCOA species to the
DOC pool varies with depth and pH. At Yinazao, the average
SCOA fraction in high-pH fluids increases from ∼15% at the
seafloor to ∼30% in the deepest samples. Propionate dominates
the contribution in high-pH fluids (60–95% of SCOA C-pool),
followed by acetate, butyrate, and then formate. Moderate pH-
fluids at Fantangisña have trends similar to high-pH fluids at
Yinazao, i.e., similar C contributions of SCOAs and propionate
clearly dominating followed by acetate, but here the formate
fraction is higher than the butyrate fraction. By comparison,
SCOAs in high-pH fluids at Fantangisña account for a lower
fraction of DOC, mainly due to a much lower C contribution of
propionate, which barely exceeds acetate. The difference between
moderate- and high-pH fluids is biggest at Asùt Tesoru. While
moderate-pH fluid compositions fluctuate, and are variably
dominated by acetate, propionate, and butyrate, high-pH fluids at Next, we examined potential drivers behind the observed
concentration trends in SCOAs in high-pH fluids across different
mud volcanoes. Specifically, we investigated the relationship
of formate, acetate, and propionate concentrations and mud
volcano distance to the Mariana Trench, as a proxy for
time since the initial subduction. Furthermore, we investigated
the relationship between formate, acetate, and propionate
concentrations and temperature in the subducting slab. The
concentrations of formate and acetate, but not propionate,
show a highly significant power relationship with distance
to trench (Figure 6, upper panel) and with modeled in situ
temperature in the underlying subducting slab (Figure 6, lower
panel). Similarly, the ratios of formate to acetate, show strong
power relationships with distance to trench and slab temperature
(Supplementary Figure S4). July 2019 | Volume 10 | Article 1729 11 Organic Acids in Serpentinite Mud Eickenbusch et al. Free Energies of Catabolic Reactions Free Energies of Catabolic Reactions
To determine whether catabolic reactions are thermodynamically
favorable, we calculated Gibbs energies for a range of reactions
at in situ temperature, pressure, and pH in cores, as well as
at estimated temperature, and pressure in the subducting slab
using the in situ pH in the cores (Table 2). We include three
respiration reaction types (methanogenesis, sulfate reduction,
acetogenesis) that are important in marine serpentinitic systems
(Brazelton et al., 2006; Quéméneur et al., 2014; Rempfert et al.,
2017; Ijiri et al., 2018; Lang et al., 2018). Besides SCOAs,
we include H2, CO, and methane as substrates of microbial
respiration, because of the ubiquitously high concentrations of
H2 and CH4 and the detectable concentrations of CO in some
of the high-pH muds. We also calculate free energy yields for
the fermentative breakdown of propionate and butyrate, and the
fermentative conversion of glucose to propionate and butyrate,
as fermentation reactions are also likely to occur in serpentinitic
environments (e.g., Kohl et al., 2016; Brazelton et al., 2017;
Rempfert et al., 2017). While the currently known temperature
limit of microbial life is 122◦C (Takai et al., 2008), and thus Frontiers in Microbiology | www.frontiersin.org Stable Isotopic Compositions of Bulk
Carbon Pools For these high-
pH fluids to be indeed informative, non-biological alterations
of electron donor and acceptor compositions during the tens
of thousands of years of mud fluid ascent would need to be
absent or sufficiently slow to not overprint original trends. In
the following sections we first investigate possible explanations
for the absence of detectable microbial activity in this subseafloor
environment, focusing on free energy yields of catabolic reactions
and the environmental variables pressure, temperature, pH, and
fluid mixing during ascent. Afterward we discuss possible deep
sources and production mechanisms of measured SCOAs, as
well as sulfate, methane, and bulk carbon pools, across the
Mariana forearc. Despite considerable scatter, subsurface δ13C-DIC data show
a clear division between Asùt Tesoru and the other two mud
volcanoes. At Asùt Tesoru, moderate-pH and high-pH fluids
have highly similar δ13C-DIC ranges (moderate pH: −7.4 ±
3.6‰; high-pH: −7.6 ± 2.6‰). These values are significantly
higher than in high-pH fluids of Yinazao (−16.3 ± 1.9‰) and
Fantangisña (−15.3 ± 1.7‰; note: no data from moderate-pH
fluids), which did not differ significantly from each other. Comparing subsurface δ13C-isotope data to each other reveals
several trends. Overall, the mean δ13C-DOC is consistently
higher than the mean δ13C-TOC. This difference is small (1.5–
2.3‰) in muds of Yinazao and Fantangisña, and moderate-
pH muds of Asùt Tesoru, but comparatively large (12.8‰)
in high-pH muds of Asùt Tesoru (−14.1 ± 1.6‰ vs. −26.9
± 2.0‰). Comparing δ13C-DIC to δ13C-TOC and δ13C-DOC
within each location and pH category shows that the δ13C-
DIC is consistently higher than the δ13C-TOC and δ13C -DOC. The average difference between δ13C-DIC and δ13C-TOC is
lower in high-pH muds of Yinazao (−11.2‰) and Fantangisña
(−12.3‰) compared to moderate-pH (−20.5‰) and high-pH
muds (−19.3‰) at Asùt Tesoru. The average differences between
δ13C-DIC and δ13C-DOC are in a similar range in high-pH muds
of Yinazao (8.9‰) and Fantangisña (10.9‰) and in moderate-
pH muds of Asùt Tesoru (18.1‰). The only exception is again
high-pH mud of Asùt Tesoru, where the average δ13C-DIC is only
6.6‰ higher than the average δ13C-DOC. DISCUSSION By contrast, methanogenesis
from H2 (+CO2−
3 ) is thermodynamically favorable under core
conditions but in the range of the BEQ or even endergonic
under slab conditions. Acetogenesis from formate, CO, and
CO+H2 is also always exergonic. Acetogenesis with H2 (+CO2−
3 )
or formate+H2 as substrates is furthermore exergonic at core
conditions, whereas the reverse reaction, acetate oxidation to
H2 (+CO2−
3 ) or formate+H2, is thermodynamically favorable
under slab conditions. Fermentation of glucose to propionate
or butyrate is highly favorable under all conditions, whereas
propionate or butyrate fermentations to acetate are only clearly
exergonic under slab conditions. The only reactions that are
always endergonic, whether under core or slab conditions, are
reactions involving the conversion of H2+CO2−
3
or formate to
CO (∆Gr > +50 kJ mol−1). On the other hand, the reverse
reaction is clearly exergonic, underscoring the potential for CO
oxidation as a catabolic pathway in serpentinitic fluids (Morrill
et al., 2014). Finally, the oxidation of formate to CO2−
3 +H2 is
close to thermodynamic equilibrium under core conditions, but
exergonic under slab conditions. Temperature
is
a
more
likely
limiting
factor. As
aforementioned, the slab temperatures at Fantangisña (150◦C)
and Asùt Tesoru (250◦C) are above the known temperature limit
of life (122◦C; Takai et al., 2008). Furthermore, even though
temperature in the subducting slab at Yinazao (80◦C) is below
this temperature limit, there is evidence that microbial life ceases
at or below 80◦C in energy-limited subsurface environments
(Head et al., 2003; Inagaki et al., 2015; Møller et al., 2018; Heuer
et al., 2019), despite heat-driven increases in the release of energy
substrates from thermogenic (e.g., Wellsbury et al., 1997; Parkes
et al., 2007) or abiotic reactions (e.g., McCollom and Seewald,
2001, 2003a). This lower temperature limit in the subsurface may
exist for the following reasons. Rates of biomolecule-damage
increase exponentially with temperature (e.g., Lindahl and
Nyberg, 1972; Wolfenden et al., 1998; Steen et al., 2013). Cells
may adjust their biomolecule compositions toward building
blocks with higher thermal stability, e.g., DNA with higher
GC content, and amino acids with lower racemization rates
at elevated temperature. Yet, the resulting enhanced thermal
stability is minor compared to the impact of temperature on
biomolecule damage rates. DISCUSSION Concentrations of potential microbial energy sources, such
as formate, acetate, CH4, H2, and DOC, in deeply sourced
high-pH fluids of Mariana forearc serpentinite mud volcanoes
increase systematically with distance to the Mariana Trench
and with underlying slab temperature. Yet, despite these
increases, there are no clear indications of metabolically active Frontiers in Microbiology | www.frontiersin.org July 2019 | Volume 10 | Article 1729 12 Organic Acids in Serpentinite Mud Eickenbusch et al. below the slab temperatures at Fantangisña and Asùt Tesoru,
we nonetheless include calculations at all slab temperatures. This is because it cannot be ruled out that deep life exists at
>122◦C. Furthermore, many of the reactions in Table 2 can also
operate thermochemically or abiotically at high temperature and
thus provide insights into potential non-biological sources and
production pathways discussed later. below the slab temperatures at Fantangisña and Asùt Tesoru,
we nonetheless include calculations at all slab temperatures. This is because it cannot be ruled out that deep life exists at
>122◦C. Furthermore, many of the reactions in Table 2 can also
operate thermochemically or abiotically at high temperature and
thus provide insights into potential non-biological sources and
production pathways discussed later. or any subseafloor cores that have been recovered to date,
laboratory experiments suggest that microbial life can remain
metabolically active at >10,000 bar (Sharma et al., 2002) and
survive even higher pressures (20,000–30,000 bar; Hazael et al.,
2016). Furthermore, experiments suggest that the ability to
survive high pressure (>10,000 bar) can evolve rapidly, even in
microbial strains that are not pre-adapted to elevated pressure,
such as Escherichia coli or Shewanella oneidensis (Vanlint et al.,
2011; Hazael et al., 2014). y
Our calculations indicate that the majority of reactions are
thermodynamically favorable in all three mud volcanoes under
both core and slab conditions (Table 2). Gibbs energies are
mostly more negative than the theoretical minimum amount
of energy that can be conserved per biochemical reaction (−20
to −10 kJ mol−1; Schink and Thauer, 1988; Hoehler et al.,
2001), also known as the “biological energy quantum” (BEQ;
Thauer and Morris, 1984). Sulfate reduction is exergonic from
all substrates, with reactions from SCOAs and CO in many
cases being highly exergonic (∆Gr < −100 kJ mol−1), especially
at slab temperatures. Methanogenesis from formate, acetate,
and CO is also always exergonic. DISCUSSION For instance, over a temperature
increase from 2 to 80◦C, increases in racemization rates may
vary by one order of magnitude between amino acids; yet this
difference is small considering that racemization rates of all
amino acids increase by ∼3–4 orders of magnitude over this
temperature interval (Steen et al., 2013; Lever et al., 2015). Due
to this dramatic increase in energy needed for biomolecule
repair, it has been proposed that the upper temperature limit of
microorganisms in energy-limited subsurface habitats is lower
than in energy-replete environments, e.g., laboratory growth
media or hydrothermal vent chimneys (Lever et al., 2015). Yet,
crucially, the temperature argument only applies if the mud
fluids remain free of microbial recolonization during ascent. If there is any significant microbial re-inoculation of cooled
but presumably sterile serpentinite mud fluids from sediments
or other fluids during ascent, then these microbial colonizers
could potentially thrive as a result of the high concentrations
of energy substrates and the high free energy yields of catabolic
reactions. Indeed, geochemical data from our study, and from a
study on a borehole observatory at the nearby South Chamorro
Seamount (IODP Site 1200C; Kawagucci et al., 2018), suggest
at most minimal recolonization of Mariana forearc serpentinite
muds during ascent from the subducting slab. In summary, given that many known catabolic reactions have
significant free energy yields under core and slab conditions, the
very low to absent microbial activity in high-pH fluids of the
three mud volcanoes cannot be explained with absence of suitable
substrates for energy-yielding catabolic reactions. Frontiers in Microbiology | www.frontiersin.org Potential Physiological Limits to Microbial
Life in Subseafloor Serpentinites Estimated pH values under slab
conditions were 11.2 for Yinazao, 11.0 for Fantangisña, and 12.5 for Asùt Tesoru. In situ pressures were calculated based on hydrostatic pressure assuming a seawater density of
1.013 g cm−3 and a mud density of 1.8 g cm−3 (based on Fryer et al., 2018b). Thermodynamically unfavorable reactions with positive Gibbs energies are shaded. Fermentation Fermentation H2 + CO2−
3
+ 2 H+ →CO + 2 H2O
HCOO−+ H+ →CO + H2O H2 Calculations were done at in situ temperature, pressure, and pH of cores during sampling (left column) and under slab conditions (right column). Estimated pH values under slab
conditions were 11.2 for Yinazao, 11.0 for Fantangisña, and 12.5 for Asùt Tesoru. In situ pressures were calculated based on hydrostatic pressure assuming a seawater density of
1.013 g cm−3 and a mud density of 1.8 g cm−3 (based on Fryer et al., 2018b). Thermodynamically unfavorable reactions with positive Gibbs energies are shaded. Chamorro, which has the same measured pH value as Asùt
Tesoru (12.5), has been estimated to be significantly higher (13.1;
Mottl, 2009), and thus significantly above the known pH limit
of microbial life. Furthermore, it is possible that high pH poses
an effective barrier to microbial recolonization from sediment or
other fluids during ascent, if colonizing cells are not adapted to
such high pH. (Takai et al., 2005; Suzuki et al., 2014; current record: pH
12.5, Takai et al., 2001; reviewed in Takai, 2019). Furthermore,
there have been several enrichments of microorganisms and
microbial DNA detections based on natural serpentinitic fluids
with a pH≥12 (e.g., Crespo-Medina et al., 2014; Morrill et al.,
2014; Kohl et al., 2016; Brazelton et al., 2017; Suzuki et al.,
2017) including mud and borehole fluids of the nearby South
Chamorro Seamount (Curtis et al., 2013; Kawagucci et al., 2018). Based on measured pH values, microbial life should therefore
be possible in high-pH muds of the three mud volcanoes. Nonetheless, it is possible that the in situ pH is considerably
higher than measured. The in situ pH in mud fluids at South In conclusion, elevated temperature (80–250◦C) and extreme
pH can explain the (near) absence of microbial life in ascending
serpentinite mud fluids of the Yinazao, Fantangisña, and Asùt
Tesoru mud volcanoes. Potential Physiological Limits to Microbial
Life in Subseafloor Serpentinites Next we will discuss the potential roles of pressure, temperature,
pH, and lack of fluid mixing as variables that limit the
proliferation of microbial life in serpentinite mud volcanoes of
the Mariana Forearc. Among these variables, pressure alone is perhaps the least
likely to be a strong limiting factor. We calculate in situ pressures
of 2,740, 2,670–2,820, and 3,300–3,500 bar for the subducting
slabs of Yinazao, Fantangisña, and Asùt Tesoru, respectively. While these values are higher than those in surface sediments
of the deepest part of the world’s oceans, the Mariana’s Trench, The other potentially important factor is pH. Porewater pH
values in high-pH fluids (Yinazao: ∼10.7; Fantangisña: ∼11.2;
Asùt Tesoru: ∼12.4) and for the underlying subducting slab
(Yinazao: ∼11.2; Fantangisña: ∼11.0; Asùt Tesoru: ∼12.5; Fryer
et al., 2018c) are in the growth range of microbial pure cultures July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 13 Organic Acids in Serpentinite Mud Eickenbusch et al. TABLE 2 | Mean Gibbs energies (∆Gr) (±standard deviation) of potential catabolic reactions in high-pH fluids of each mud volcano. Potential Physiological Limits to Microbial
Life in Subseafloor Serpentinites e−donors by
reaction type
Reaction
Yinazao
Fantangisña
Asùt Tesoru
2.6◦C
80◦C
3.5◦C
150◦C
2.2◦C
250◦C
Sulfate reduction
H2
4 H2 + SO2−
4
+ H+ →4 H2O + HS−
−95 ± 16
−57 ± 20
−127 ± 10
−77 ± 15
−140 ± 6
−63 ± 12
Formate
4 HCOO−+ SO2−
4 →HS−+ 4 CO2−
3
+ 3 H+
−111 ± 10
−149 ± 9
−129 ± 5
−165 ± 5
−163 ± 3
−253 ± 5
Acetate
CH3COO−+ SO2−
4
→HS−+ 2 CO2−
3
+ 2 H+
−69 ± 3
−114 ± 4
−73 ± 2
−134 ± 1
−87 ± 1
−207 ± 3
Propionate
2 CH3CH2COO−+ 3 SO2−
4
+ 2 H2O →3 HS−+ 6 CO2−
3
+ 2
H2 + 7 H+
−230 ± 12 −379 ± 15 +225 ± 10 −449 ± 11
−252 ± 4
−677 ± 9
Butyrate
2 CH3CH2CH2COO−+ 4 SO2−
4
+ 4 H2O →4 HS−+ 8 CO2−
3
+ 4 H2 + 10 H+
−310 ± 20 −533 ± 26 −277 ± 16 −604 ± 17
−322 ± 7
−947 ± 14
Carbon monoxide
4 CO + SO2−
4
+ 4 H2O →4 CO2−
3
+ HS−+ 7 H+
−322 ± 5
−397 ± 7
−370 ± 7
−444 ± 4
−399 ± 5
−552 ± 11
Methane
CH4 + SO2−
4
→HS−+ CO2−
3
+ H+ + H2O
−27 ± 4
−43 ± 6
−32 ± 2
−58 ± 2
−52 ± 2
−117 ± 3
Methanogenesis
H2
4 H2 + CO2−
3
+ 2 H+ →CH4 + 3 H2O
−68 ± 14
−10 ± 18
−95 ± 10
−15 ± 15
−88 ± 5
+59 ± 9
Formate
4 HCOO−+ H2O →CH4 + 3 CO2−
3
+ 2 H+
−84 ± 8
−102 ± 11
−98 ± 4
−103 ± 5
−112 ± 2
−130 ± 4
Acetate
CH3COO−+ H2O →CH4 + CO2−
3
+ H+
−42 ± 4
−66 ± 5
−41 ± 2
−72 ± 2
−36 ± 1
−84 ± 2
Carbon monoxide
4 CO + 5 H2O →CH4 + 3 CO2−
3
+ 6 H+
−295 ± 5
−350 ± 6
−339 ± 6
−382 ± 5
−346 ± 6
−430 ± 9
Acetogenesis
H2
4 H2 + 2 CO2−
3
+ 3 H+ →CH3COO−+ 4 H2O
−25 ± 16
+57 ± 20
−53 ± 11
+59 ± 16
−52 ± 6
+145 ± 11
Formate
4 HCOO−→CH3COO−+ 2 CO2−
3
+ H+
−42 ± 6
−35 ± 8
−56 ± 3
−29 ± 5
−76 ± 1
−45 ± 3
Formate + H2
2 HCOO−+ 2 H2 + H+ →CH3COO−+ 2 H2O
−33 ± 8
+12 ± 10
−55 ± 6
+15 ± 10
−64 ± 3
+50 ± 6
Carbon monoxide
4 CO + 4 H2O →CH3COO−+ 2 CO2−
3
+ 5 H+
−252 ± 4
−283 ± 5
−296 ± 5
−309 ± 4
−311 ± 4
−344 ± 9
Carbon monoxide
+ H2
2 CO + 2 H2 →CH3COO−+ H+
−135 ± 8
−92 ± 10
−172 ± 5
−104 ± 8
−181 ± 4
−74 ± 8
Fermentation
Propionate
CH3CH2COO−+ 3 H2O →CH3COO−+ CO2−
3
+ 3 H2 + 2 H+
+2 ± 12
−59 ± 16
+25 ± 8
−64 ± 12
+32 ± 4
−116 ± 9
Butyrate
CH3CH2CH2COO−+ 2 H2O →2 CH3COO−+ 2 H2 + H+
−16 ± 3
−60 ± 11
+7 ± 5
−55 ± 7
−14 ± 3
−89 ± 6
Glucose to
propionate
C6H12O6 + 5 H2O →CH3CH2COO−+ 3 CO2−
3
+ 5 H2 + 7 H+ −358 ± 21 −519 ± 26 −323 ± 15 −554 ± 20
−340 ± 7
−765 ± 14
Glucose to
butyrate
C6H12O6 + 2 H2O →CH3CH2CH2COO−+ 2 CO2−
3
+ 2 H2 +
5 H+
−365 ± 9
−467 ± 11
−361 ± 8
−512 ± 10
−375 ± 3
−659 ± 6
Carbon monoxide
production
H2
H2 + CO2−
3
+ 2 H+ →CO + 2 H2O
+57 ± 4
+85 ± 5
+61 ± 3
+92 ± 4
+65 ± 1
+122 ± 3
Formate
HCOO−+ H+ →CO + H2O
+53 ± 2
+62 ± 2
+60 ± 1
+70 ± 1
+59 ± 1
+75 ± 2
Formate oxidation
Formate
HCOO−+ H2O→CO2−
3
+ H2 + H+
−4 ± 5
−23 ± 6
−1 ± 2
−22 ± 3
−6 ± 1
−47 ± 3
Calculations were done at in situ temperature, pressure, and pH of cores during sampling (left column) and under slab conditions (right column). Frontiers in Microbiology | www.frontiersin.org Potential Sources and Production
Mechanisms of Deep SCOAs We document a strong increase in total SCOA concentrations
and systematic shift in the composition of SCOAs in high-pH
serpentinite muds that is correlated with distance to the Mariana
Trench and slab temperature (Figures 3–6). The shift in SCOA
compositions is mainly due to concentration increases of acetate
and especially formate (Figure 5). Concentrations of propionate,
which is the dominant SCOA at Yinazao, butyrate, or other
SCOAs do not change substantially with distance to trench or slab
temperature. Lactate (and CO) concentrations are even overall
higher in moderate-pH compared to high-pH fluids. High formate concentrations are a common feature of
serpentinizing systems (Mottl et al., 2003; McCollom and Bach,
2009; Schrenk et al., 2013), and are attributed to a metastable
equilibrium of formate with H2 and CO2−
3
(McCollom and
Seewald, 2001, 2003a). Hereby high formate concentrations result
from the chemical reaction of H2, produced during reactions
of iron(II)-rich minerals with water, with CO2−
3
and H+. This
reaction is equivalent to the reversal of biological formate
oxidation, which is shown in Table 2. While earlier studies
suggest that formate equilibration with H2+CO2−
3 +H+ takes
place at 175–300◦C (McCollom and Seewald, 2001, 2003a), recent
laboratory incubations of olivine-rich rocks indicate significant
formate production from H2+CO2 at only 100◦C (Miller et al.,
2017). These data are in line with our calculations, which
suggest that formate is at or near thermodynamic equilibrium
with H2+CO2−
3 +H+ in cores, but not under estimated slab
temperature, pressure, and pH (Table 2). The fact that this is even
true for Yinazao, suggests that equilibration between formate
and H2+CO2−
3 +H+ continues at temperatures significantly
below 80◦C. The factors that are responsible for the strong changes in
SCOA compositions are unclear. Distance to trench per se is an
unlikely driver, but distance to trench is correlated with time
since initial subduction and with pressure. SCOA compositions
could evolve as production rates of different SCOAs change as
a function of time and pressure. However, time and pressure
effects are unlikely to be the main drivers behind the observed
trends. Relative to Yinazao, distance to trench only changes
by ∼13, ∼31, and ∼56% at Fantangisña, Asùt Tesoru, and
Conical Seamount, respectively, and pressure also only increases
by at most ∼30% from Yinazao to Asùt Tesoru (no data
for Conical). Potential Physiological Limits to Microbial
Life in Subseafloor Serpentinites If temperature functions as a natural
sterilizing agent in the subducting slab, then this would indicate Frontiers in Microbiology | www.frontiersin.org July 2019 | Volume 10 | Article 1729 14 Organic Acids in Serpentinite Mud Eickenbusch et al. Kharaka, 1978). H2 concentrations (∼1–100 µM) are clearly
higher than in typical marine sediments (<1 µM; e.g., Hoehler
et al., 1998; Lin et al., 2012) and are consistent with a low-
temperature serpentinization fluid input (Mayhew et al., 2013;
Miller et al., 2017). Going from Yinazao (80◦C) to Fantangisña
(150◦C), the clear increases in formate (to ∼10 µM) and
especially H2 concentrations (to ∼100–1,000 µM) are consistent
with temperature-driven increases in serpentinization rates. By
contrast, a significant stimulation of thermogenic processes,
which would be expected to clearly increase concentrations of
others SCOAs, is not evident. minimal recolonization of fluids during their long ascent
(Yinazao: ∼50,000 years, Asùt Tesoru: ∼130,000 years), during
which their temperatures drop within the known limits of life
(Fryer et al., 2018b; Table 2). The absence of recolonization could
be due to recruitment limitation, e.g., no or only minimal mixing
with other fluids or sediments containing microbial life. This
notion is supported by the very low cell numbers and absence
of clear microbial activity in moderate-pH fluids of the same
three mud volcanoes. Alternatively, microbial life present in
other fluids or sediments that come into contact with high-pH
serpentinite fluids during ascent may not tolerate the high pH of
serpentinite fluids. A big shift in SCOA compositions occurs from Fantangisña
(150◦C) to Asùt Tesoru (250◦C), where formate and acetate
concentrations increase 15- and 7-fold, and methane and
H2 concentrations increase ∼200- and 4-fold, respectively
(Figure 3). The observed increases in formate and acetate
concentrations continue as temperatures increase further from
Asùt Tesoru to Conical Seamount (350◦C). For reasons, that
we discuss in the next paragraphs, we believe that these strong
increases in formate and acetate concentrations are driven
by serpentinization. Frontiers in Microbiology | www.frontiersin.org Geochemical Trends in Sulfate, Methane,
and Bulk Organic Carbon Pools While δ13C-values of sedimentary TOC in
the Mariana Trench are dominated by marine phytoplankton-
derived organic carbon with higher isotopic values (−19 to
−21‰), local layers with potentially significant terrestrial
contributions (−24 to −25‰) were noted previously (Luo et al.,
2017). Thus, it is possible that terrestrial TOC or certain 13C-
enriched marine TOC fractions are selectively enriched during
subduction, as a result of greater resistance to microbial and
heat-driven degradation processes. Alternatively, given that the
TOC contents and δ13C values are in the same range as those
from other oceanic basement rocks [mostly 0–0.02 wt. %, −25
to −30‰; compiled in (Delacour et al., 2008)], it is also possible
that most of the TOC is indigenous to the overlying plate. Heat-driven serpentinitic alteration would then release soluble
fractions of mantle rock-bound TOC into solution and drive
the increase in DOC concentrations in high-pH fluids from
Yinazao to Asùt Tesoru. At Asùt Tesoru, the 4-fold increase
in DOC concentrations compared to Fantangisña can, however,
only in part be explained by release of rock-bound indigenous
organic carbon. Here the strong isotopic offset (∼+10‰) of
DOC relative to TOC suggests that a major fraction of DOC
derives from a source that is significantly heavier than TOC. The
contributions of formate and acetate, which account for ∼10–
12% of the DOC and have 13C-isotopic values in the range of DIC
at Asùt Tesoru, can only explain an isotopic offset of ∼+2‰. Thus, the origin of a major component of the DOC pool in
high-pH fluids of Asùt Tesoru remains unknown. As mentioned earlier, average CH4 concentrations in high-
pH subsurface fluids increase ∼200-fold from Fantangisña to
Asùt Tesoru. Earlier data on high-pH fluids from Conical
Seamount suggest that CH4 concentrations are within the range
of Asùt Tesoru (Shipboard Scientific Party, 1990), which would
indicate no substantial further increases in CH4 release as
slab temperatures increase from 250 to 350◦C. The sources
of these very high CH4 concentrations are controversial, and
could in principle be abiotic or thermogenic. Although our
SCOA data suggest that thermogenic breakdown of OM is not
a dominant process of C cycling in high-pH fluids of Asùt Tesoru
or Conical Seamount, potential mechanisms of abiotic CH4
production are also unclear. Our thermodynamic calculations
indicate that abiotic CH4 formation from H2 (+CO2−
3 ) under
aqueous conditions in the slab is an endergonic process (Table 2). Potential Sources and Production
Mechanisms of Deep SCOAs By contrast, formate and acetate concentrations
increase exponentially by ∼3 and ∼2 orders of magnitude,
respectively, from Yinazao to Conical Seamount (Figures 5, 6). This exponential increase is consistent with the well-known
mathematical relationship between temperature and rates of
chemical reactions (Arrhenius equation), and suggests that
temperature is the main driver behind the observed differences
in formate and acetate concentrations. The elevated acetate concentrations at Asùt Tesoru and
Conical Seamount are more difficult to interpret than the
elevated formate concentrations, as acetate is produced by
serpentinization-related processes (Miller et al., 2017), and also
is the dominant SCOA produced by thermogenic breakdown of
OM (e.g., Kharaka et al., 1993; Shebl and Surdam, 1996; Dhillon
et al., 2005). Though thermogenic acetate could be produced
by thermal breakdown of OM from subducting sediment or
mantle rock during serpentinitic alteration (Kelley and Früh-
Green, 2001; McDermott et al., 2015), we consider abiotic
synthesis processes to be the most likely source. This is because
high rates of thermogenic acetate production are typically
accompanied by significant increases in propionate and/or
butyrate concentrations, which were not observed. Furthermore,
isotopic data from Asùt Tesoru (Sissmann et al., in preparation)
support an abiotic serpentinization-related production of acetate As slab temperatures increase, the sources of individual
SCOAs and/or the balance between production and consumption
of individual SCOAs appear to change. Concentrations of
formate, acetate, butyrate, and CH4 in the low micromolar
and submicromolar range in high-pH muds of Yinazao are
in the typical range of non-methanogenic marine sediments
(Glombitza et al., 2014, 2015), and provide no indications of
significant additional, non-diagenetic inputs. Only propionate
and H2 stand out, albeit only slightly. Propionate concentrations
(∼5–10 µM) exceed those in typical marine sediments (≤2 µM;
Glombitza et al., 2019), and are consistent with a low-
temperature thermogenic propionate input (80◦C; Carothers and July 2019 | Volume 10 | Article 1729 15 Organic Acids in Serpentinite Mud Eickenbusch et al. (and formate), as δ13C-values of both vary from ∼0 to ∼5‰,
which is in the range of DIC, but considerably higher than the
δ13C values of TOC and DOC (Figure 7). for >95% of this acetate to then be decarboxylated to CH4 +
CO2−
3 . Potential Sources and Production
Mechanisms of Deep SCOAs If recent field studies on multiple submarine locations and
laboratory experiments with olivine are a good indication, then it
is more likely that most of the CH4 in serpentinitic fluids of Asùt
Tesoru and Conical Seamount is released from fluid inclusions
within serpentinized mantle rock (McDermott et al., 2015;
McCollom, 2016; Wang et al., 2018). In addition, CH4 could form
in thermodynamically distinct chemical microenvironments with
H2-rich vapors, e.g., serpentinization fronts (McCollom et al.,
2016), rock fractures, or rock pores (Etiope and Whiticar, 2019). Akin to CH4 from fluid inclusions, abiotically produced CH4
from these microenvironments could then be liberated into mud
fluids as a result of serpentinitic rock-alteration. Geochemical Trends in Sulfate, Methane,
and Bulk Organic Carbon Pools g
Besides the increase in H2 and formate concentrations, a striking
change between Yinazao and Fantangisña high-pH mud fluids
is the 80% decrease in sulfate concentrations, from close to
seawater values (25 mM) at Yinazao to <5 mM at Fantangisña. This decrease is consistent with anhydrite (CaSO4) precipitation,
which has been proposed to remove most or all seawater sulfate
from fluids during subduction in the Mariana forearc, and is
only partially reversed by re-dissolution during fluid ascent
(Kawagucci et al., 2018). By comparison, sulfate removal by
thermochemical sulfate reduction, which is thermodynamically
favorable in all mud volcanoes (Table 2), is an unlikely driver,
both based on previous studies, which suggest that this process is
inhibited in high-pH, serpentinitic systems (Seyfried et al., 2007),
and given that sulfate concentrations increase again at higher
temperatures. This strong increase in sulfate concentrations
to values that exceed those in seawater in high-pH fluids of
Asùt Tesoru (∼31 mM; this study) and Conical Seamount (up
to ∼47 mM; Shipboard Scientific Party, 1990) is enigmatic. A
possible source is redissolution of anhydrite during fluid ascent. Furthermore, antigorite, which forms at >200◦C above the slab
at Asùt Tesoru (Debret et al., 2019), and breaks down during
serpentinization to generate oxidizing conditions that lead to
sulfate production (Debret and Sverjensky, 2017), might explain
the high sulfate concentrations in high-pH fluids of Asùt Tesoru
and Conical Seamount. This same mechanism of antigorite
breakdown could also explain the increase in DIC concentrations
at Asùt Tesoru (Figure 2D) and the high alkalinities of 33–62
meq kg−1 in high-pH subsurface pore fluids of Conical Seamount
(Shipboard Scientific Party, 1990). p
The TOC, DOC, and DIC isotopic compositions provide
general insights into the sources of organic and inorganic
carbon at Yinazao, Fantangisña, and Asùt Tesoru. Similar δ13C-
values (range: −21 to −30‰, with most values between −26
to −30 ‰) and similar, for the most part very low (0.01 wt. %) TOC contents indicate similar origins of TOC across all
three mud volcanoes, including high-pH and moderate-pH muds
(Figure 7, Supplementary Figure S5). These values are mostly
lower than TOC of marine phytoplankton and suggest that—if
they are of sedimentary origin—there is a significant terrestrial
TOC contribution. Geochemical Trends in Sulfate, Methane,
and Bulk Organic Carbon Pools By contrast, production of CH4 by thermal decarboxylation of
acetate, a reaction that has the same stoichiometry as biological
methanogenesis from acetate (Kharaka et al., 1993) and is
catalyzed by magnetite (McCollom and Seewald, 2003b), is
thermodynamically favorable (Table 2). Yet, this reaction would
require the production of millimolar acetate concentrations, and Frontiers in Microbiology | www.frontiersin.org DATA AVAILABILITY The datasets generated for this study are available on request to
the corresponding author. Supplementary Figure S1 | (A) Chloride concentration depth profiles at all three
mud volcanoes. (B) Depth profiles of sulfate-to-chloride concentration ratios (both
mM). High-pH fluids (summit sites) are indicated by red marker symbols,
moderate-pH fluids (flank sites) by black marker symbols. The dashed gray
vertical lines indicate typical seawater chloride concentrations (559 mM) and
seawater sulfate-to-chloride ratios [0.0517; assumes [sulfate] = 28.9 mM]. The
dashed red vertical lines indicate chloride concentration asymptotes for high-pH
fluids [Yinazao: 460 mM; Fantangisña: 480 mM; Asùt Tesoru (U1496A): 540 mM]. Chloride concentrations in high-pH fluids that are significantly below those in
seawater indicate a potential freshwater contribution from dewatering of deeply
buried clay minerals. CONCLUSIONS Our study produces novel insights into the controls on the
production of microbial energy substrates, in particular SCOAs,
in deeply buried subducting slab environments. Despite the July 2019 | Volume 10 | Article 1729 Frontiers in Microbiology | www.frontiersin.org 16 Organic Acids in Serpentinite Mud Eickenbusch et al. (SNSF; www.snf.ch) project No. 182091 and by the Deep
Carbon
Observatory
(www.deepcarbon.net). Funding
for this research was provided by ETH Zurich. CG and
BJ
were
supported
by
the
Danish
National
Research
Foundation
[DNRF104],
the
European
Research
Council
[ERC Advanced Grant #294200], and the Danish Council
for
Independent
Research
[DFF−7014-00196]. CM
was
supported by a NERC UK IODP Phase 2 Moratorium
Award [NE/P020909/1]. presence of high microbial energy substrate concentrations and
significant Gibbs energy yields of a wide range of catabolic
reactions, microorganisms are rare or absent, and no unequivocal
evidence for microbial activity could be detected. This has
implications for our understanding of deeply buried serpentinitic
environments as potential habitats or even deep hotspots of
microbial life, and suggests that the combination of temperature,
highly alkaline pH, and dispersal limitation may strongly
limit microbial population size in these environments. Instead,
due to the absence of strong diagenetic alteration during
fluid ascent over tens of millennia, high-pH fluids from mud
volcanoes offer a unique window into the abiotic production
mechanisms of microbial energy substrates by serpentinization
and by serpentinization-related processes within subduction
zones. Future studies will reveal the mechanisms by which some
of these energy compounds, e.g., acetate, CH4, are produced,
which unknown sources contribute to the strongly elevated DOC
concentrations in high-temperature, high-pH fluids, and what
the origins of the still enigmatic moderate-pH fluids are. ACKNOWLEDGMENTS We thank Madalina Jaggi for laboratory support. PS thanks the
Laboratoire d’Excellence LabexMER (ANR-10-LABX-19) and the
GPI Laboratories, Inc. (Grand Rapids, MI, USA) for funding and
analyses. OS thanks IODP-France for funding. FUNDING Supplementary Figure S4 | Concentration ratios of formate-to-acetate,
formate-to-propionate, and acetate-to-propionate vs. temperature in the
subducting slab (upper panel) and distance to the Mariana Trench (lower panel). Supplementary Figure S4 | Concentration ratios of formate-to-acetate,
formate-to-propionate, and acetate-to-propionate vs. temperature in the
subducting slab (upper panel) and distance to the Mariana Trench (lower panel). This
research
used
samples
and/or
data
provided
by
the
International
Ocean
Discovery
Program
(IODP). Additional support for the participation in IODP Expedition
came
from
SwissDrilling
(www.swissdrilling.ch),
which
is
funded
by
the
Swiss
National
Science
Foundation Supplementary Figure S5 | δ13C-isotopic compositions of TOC, DOC, and DIC
from moderate-pH (left column) and high-pH (right column) mud fluids of three
serpentinite mud volcanoes drilled during IODP Expedition 366. Supplementary Figure S5 | δ13C-isotopic compositions of TOC, DOC, and DIC
from moderate-pH (left column) and high-pH (right column) mud fluids of three
serpentinite mud volcanoes drilled during IODP Expedition 366. erpentinite mud volcanoes drilled during IODP Expedition 366. AUTHOR CONTRIBUTIONS PE and ML designed the study. PE, KT, OS, SSu, CM, SSa,
PS, ET, and ML took samples, performed measurements,
and/or contributed data analyses. SB, CG, BJ, and YM
provided technical advice and support with the analyses. PE
and
ML
wrote
the
manuscript
with
input
from
all co-authors. PE and ML designed the study. PE, KT, OS, SSu, CM, SSa,
PS, ET, and ML took samples, performed measurements,
and/or contributed data analyses. SB, CG, BJ, and YM
provided technical advice and support with the analyses. PE
and
ML
wrote
the
manuscript
with
input
from
all co-authors. Supplementary Figure S2 | Concentration profiles of pyruvate, lactate, and
valerate across boreholes of the three mud volcanoes. Supplementary Figure S2 | Concentration profiles of pyruvate, lactate, and
valerate across boreholes of the three mud volcanoes. Supplementary Figure S3 | Concentration profiles of CO across boreholes of
the three mud volcanoes. Supplementary Figure S3 | Concentration profiles of CO across boreholes of
the three mud volcanoes. Supplementary Figure S3 | Concentration profiles of CO across boreholes of
the three mud volcanoes. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fmicb. 2019.01729/full#supplementary-material The Supplementary Material for this article can be found
online
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conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Vanlint, D., Mitchell, R., Bailey, E., Meersman, F., Mcmillan, P. F., Michiels, C. W., et al. (2011). Rapid acquisition of gigapascal-high-pressure resistance by
Escherichia coli. mBio 2:10. doi: 10.1128/mBio.00130-10 Copyright © 2019 Eickenbusch, Takai, Sissman, Suzuki, Menzies, Sakai, Sansjofre,
Tasumi, Bernasconi, Glombitza, Jørgensen, Morono and Lever. 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. Copyright © 2019 Eickenbusch, Takai, Sissman, Suzuki, Menzies, Sakai, Sansjofre,
Tasumi, Bernasconi, Glombitza, Jørgensen, Morono and Lever. Frontiers in Microbiology | www.frontiersin.org July 2019 | Volume 10 | Article 1729 REFERENCES 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. Wang, D. T., Reeves, E. P., Mcdermott, J. M., Seewald, J. S., and Ono, S. (2018). Clumped isotopologue constraints on the origin of methane
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Evaluation of Carbon Dioxide-Based Urethane Acrylate Composites for Sealers of Root Canal Obturation
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Polymers
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Received: 24 January 2020; Accepted: 18 February 2020; Published: 21 February 2020 Received: 24 January 2020; Accepted: 18 February 2020; Published: 21 February 2020 Abstract: A new root canal sealer was developed based on urethane acrylates using polycarbonate
polyol (PCPO), a macrodiol prepared in the consumption of carbon dioxide as feedstock. The superior
mechanical properties and biostability nature of PCPO-based urethane acrylates were then
co-crosslinked with a difunctional monomer of tripropylene glycol diarylate (TPGDA) as sealers for
resin matrix. Moreover, nanoscale silicate platelets (NSPs) immobilized with silver nanoparticles
(AgNPs) and/or zinc oxide nanoparticles (ZnONPs) were introduced to enhance the antibacterial effect
for the sealers. The biocompatibility and the antibacterial effect were investigated by Alamar blue assay
and LDH assay. In addition, the antibacterial efficiency was performed by using Enterococcus faecalis
(E. faecalis) as microbial response evaluation. These results demonstrate that the PCPO-based urethane
acrylates with 50 ppm of both AgNP and ZnONP immobilized on silicate platelets, i.e., Ag/ZnO@NSP,
exhibited great potential as an antibacterial composite for the sealer of root canal obturation. Keywords: root canal obturation; composites; urethane acrylates; nanoscale silicate platelets; carbon
dioxide-based resins polymers
Article
Evaluation of Carbon Dioxide-Based Urethane
Acrylate Composites for Sealers of Root
Canal Obturation polymers Evaluation of Carbon Dioxide-Based Urethane
Acrylate Composites for Sealers of Root
Canal Obturation Hao-Hueng Chang 1,2, Yi-Ting Tseng 3, Sheng-Wun Huang 1, Yi-Fang Kuo 1, Chun-Liang Yeh 1
,
Chien-Hsin Wu 3
, Ying-Chi Huang 3, Ru-Jong Jeng 3,*
, Jiang-Jen Lin 4,* and Chun-Pin Lin 1,2,* 1
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei 100, Taiwan;
changhh@ntu.edu.tw (H.-H.C.); yelsaint@gmail.com (S.-W.H.); franca_kay@hotmail.com (Y.-F.K.);
staryeh0524@gmail.com (C.-L.Y.) 1
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei 100, Taiwan;
changhh@ntu.edu.tw (H.-H.C.); yelsaint@gmail.com (S.-W.H.); franca_kay@hotmail.com (Y.-F.K.);
staryeh0524@gmail.com (C.-L.Y.) 2
Department of Dentistry, National Taiwan University Hospital, Taipei 100, Taiwan
3
Institute of Polymer Science and Engineering and Advanced Research Center for Green Materials Science
and Technology, National Taiwan University, Taipei 100, Taiwan; kop11239@gmail.com (Y.-T.T.); 2
Department of Dentistry, National Taiwan University Hospital, Taipei 100, Taiwan
3
Institute of Polymer Science and Engineering and Advanced Research Center for Green Materials Science
and Technology, National Taiwan University, Taipei 100, Taiwan; kop11239@gmail.com (Y.-T.T.);
chwuoliver@gmail.com (C.-H.W.); inzmehuang@gmail.com (Y.-C.H.)
4
Department of Materials Science and Engineering, National Chung Hsing University, Taichung 400, Taiwan
*
Correspondence: rujong@ntu.edu.tw (R.-J.J.); jianglin@ntu.edu.tw (J.-J.L.); pinlin@ntu.edu.tw (C.-P.L.);
T l +886 2 3366 5884 (R J J ) +886 4228 57261 (J J L ) +886 2 2312 3456 (
t 67400) (C PL ) 3
Institute of Polymer Science and Engineering and Advanced Research Center for Green Materials Science
and Technology, National Taiwan University, Taipei 100, Taiwan; kop11239@gmail.com (Y.-T.T.);
chwuoliver@gmail.com (C.-H.W.); inzmehuang@gmail.com (Y.-C.H.) 3
Institute of Polymer Science and Engineering and Advanced Research Center for Green Materials Science
and Technology, National Taiwan University, Taipei 100, Taiwan; kop11239@gmail.com (Y.-T.T.);
chwuoliver@gmail.com (C.-H.W.); inzmehuang@gmail.com (Y.-C.H.)
4
Department of Materials Science and Engineering, National Chung Hsing University, Taichung 400, Taiwan
*
Correspondence: rujong@ntu.edu.tw (R.-J.J.); jianglin@ntu.edu.tw (J.-J.L.); pinlin@ntu.edu.tw (C.-P.L.);
Tel.: +886-2-3366-5884 (R.-J.J.); +886-4228-57261 (J.-J.L.); +886-2-2312-3456 (ext. 67400) (C.-P.L.) and Technology, National Taiwan University, Taipei 100, Taiwan; kop11239@gmail.com (Y.-T.T.);
chwuoliver@gmail.com (C.-H.W.); inzmehuang@gmail.com (Y.-C.H.)
4
Department of Materials Science and Engineering, National Chung Hsing University, Taichung 400, Taiwan
*
Correspondence: rujong@ntu.edu.tw (R.-J.J.); jianglin@ntu.edu.tw (J.-J.L.); pinlin@ntu.edu.tw (C.-P.L.);
Tel.: +886-2-3366-5884 (R.-J.J.); +886-4228-57261 (J.-J.L.); +886-2-2312-3456 (ext. 67400) (C.-P.L.) 4
Department of Materials Science and Engineering, National Chung Hsing University, Taichung 400, Taiwan
*
Correspondence: rujong@ntu.edu.tw (R.-J.J.); jianglin@ntu.edu.tw (J.-J.L.); pinlin@ntu.edu.tw (C.-P.L.);
Tel.: +886-2-3366-5884 (R.-J.J.); +886-4228-57261 (J.-J.L.); +886-2-2312-3456 (ext. 67400) (C.-P.L.)
Received: 24 January 2020; Accepted: 18 February 2020; Published: 21 February 2020
Polymers 2020, 12, 482; doi:10.3390/polym12020482 1. Introduction A tooth with damaged or injured dental pulp requires root canal therapy to cure or prevent apical
periodontitis. However, the root canal system has complex internal anatomy with high prevalence [1]. The successful root canal therapy includes correct diagnosis, adequate debridement, and dense filling
of three-dimensional space. The root canal obturation is to fill up the root canal and to prevent
microorganisms from re-entering into the canal system [2]. Obturation is the method used to fill and
seal a cleaned and shaped root canal using core filling materials and root canal sealer. The root canal
filling materials are usually composed of cone and sealer (Figure 1). Gutta-percha (GP) cone is the
most commonly used material for the obturation of the root canal therapy. Due to the insufficient
dentinal adhesion to GP cone, the uses of endodontic sealers are required to provide cohesive strength
between core material interface and root canal dentin wall to hold the obturation material together [3]. The root canal sealers are the filling materials that cover all areas of the canal, while acting as lubricants Polymers 2020, 12, 482; doi:10.3390/polym12020482 www.mdpi.com/journal/polymers www.mdpi.com/journal/polymers 2 of 19
o Ag c
e upta Polymers 2020, 12, 482
s AgNPs could
hibi i
i to reduce the friction resistance between the cone material and the canal wall [4,5]. As a result, the key
factor to the success of root canal therapy relies on the choice of the appropriate filling material that
can completely cover the root canal to prevent the infection. ing adenosine triphosphate (ATP) synthesis for microbial growth [21]. As a result, t
ealers were then prepared by urethane acrylate (UA) composites comprising the immo
articles on nanoscale platelets for the root canal obturation evaluations. gure 1. The illustration of filling materials for root canal ther
Figure 1. The illustration of filling materials for root canal therapy. ure 1. The illustration of filling materials for root canal the
Figure 1. The illustration of filling materials for root canal therapy. erials and Methods
nthesis of Urethane-acrylate (UA)
s shown in Figure 2, UAs were prepared from the reaction between the isocyanate gr
rone diisocyanate (IPDI) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (D
t, and hydroxyl groups of polyols to achieve urethane prepolymer end-cappe
nates, followed by the addition of hydroxyethylmethacrylate (HEMA). 1. Introduction In this stu
ed from polyols by using polytetramethylene ether glycol with a Mw of 650 (PTMEG
d as UAT65, while UA prepared from polycarbonate polyol with a Mw of 500 (PCPO
d as UAC50. The introduction of acrylate group was prepared from using HEMA as
molar ratio (2/1/1) of IPDI/polyol/HEMA. The synthesis of UAs was monitored by u
um (Jasco 4600 FT-IR Spectrophotometer with a Jasco ATR Pro 450-S accessory, Jasco,
The molecular weights were measured by using gel permeation chromatography (GP
Typical root canal sealers are composed of zinc-oxide eugenol, calcium hydroxide, or glass ionomer. Many studies have suggested that the conventional root canal sealers are easily dissolved [6] and have
no dentinal adhesion [7]. Recently, bioceramics such as tricalcium silicate-, dicalcium silicate-, calcium
phosphates-, colloidal silica-, or calcium hydroxide-based root canal sealers were developed [8,9]. The
moisture within the dentinal tubules was utilized to solidify the calcium silicate hydrate phase [10]. Subsequently, hydroxyapatite was precipitated within the hydrate phase to produce a composite-like
structure with reinforcing effects and good sealing ability. Moreover, the pH of the bioceramic sealer
during the setting process was usually higher than 12, which increased its bactericidal properties under
such circumstances. As compared to the typical root canal sealers, the presence of water is usually a
requirement in the canal space for the hydration reaction in order to enable the materials’ solidification
in the bioceramic sealers such as calcium silicate-based endodontic sealers [11]. However, the difficulty
on controlling the precise water content in the bioceramic sealers would result in uncertain setting
times along with microhardness. Furthermore, Pawar et al. [12] also suggested that the use of the
new bioceramic sealer usually exhibits a lower bond strength than the commonly used root filling
materials such as AH-Plus® during the push-out bond strength test. The development of composites
for dental-clinical performance still a challenging topic nowadays. g
y
g g
p
g
p y (
es dissolved in 1 wt % THF. (Waters chromatography system, two Waters Styrage
ns, and polystyrene as the standard with Mw 104, 2560, 7600, 18,000, and 37,900 by usi
nt at the flow rate of 1 mL/min.)
Apart from that, a series of novel endodontic sealers has been derived from synthetic polymers, such
as epoxy-, methacrylate-, and silicon-based resins. [13–16] Moreover, urethane-dimethacrylate-based
resins were also developed in order to achieve adhesion with dentin and resin properties [13,14]. 1. Introduction As
one of the key components in the success of advanced technologies, the urethane-based resins can be
easily obtained from a rapid formation between the isocyanate chemistry and raw materials with active
hydrogens. A large variety of properties can be tailored in the biomedical applications such as wound
dressings, artificial organs, and tissue scaffolds [17]. Although the improved bonding strength between
the sealers and dentin has been realized, the adhesive bonding with cone materials is still a challenging
issue. The gaps along with the sealer-dentin interfaces would attribute to the polymerization shrinkage
of the sealer, leading to the re-infection result after the root canal therapy. The incorporation of nanoscale antibacterial materials is one of the solutions to produce an
antibacterial root canal sealer material. The nanoscale silicate platelets (NSP) previously developed by
our research group were used as supports for nanoparticle dispersion, such as silver nanoparticles 3 of 19 Polymers 2020, 12, 482 (AgNPs) [18,19]. The nanometer-thin silicate platelets were intended for surface interactions in order
to achieve NSPs immobilized with AgNPs (Ag@NSP) [20]. In fact, Ag@NSP demonstrated excellent
antimicrobial activities against common bacteria. Their antibacterial activities depends heavily on the
particle size of AgNPs along with the weight ratios of the mobilized AgNPs on NSPs [21]. Moreover,
Ag@NSP with low cytotoxicity would further promote wound healing [22]. Apart from that, ZnONPs
are employed extensively in a variety of areas such as health products, cosmetics, and catalyst industry
owing to their optoelectronic properties, high catalytic efficiency, and antibacterial activities [23,24]. A considerable enhancement on the antibacterial bioactivity toward microorganisms along with
biocompatibility to human cells can be achieved by ZnONPs [25]. Therefore, ZnONPs were also
immobilized on the nanometer-thin silicate platelets for further enhancing antibacterial properties. Based on the above, nanohybrids such as Ag@NSP, ZnO@NSP or Ag/ZnO@NSP are have great potential
as antibacterial agents for the root canal applications. The purpose of this study was to develop a novel root canal sealer, using a biocompatible
resin matrix with NSPs as antibacterial components. The urethane resins were first prepared from
the aliphatic polycarbonate polyol (PCPO), which is a macrodiol prepared in the consumption of
carbon dioxide [26,27]. The urethane acrylates prepared from PCPO exhibited not only excellent
resistance to hydrolysis and good mechanical properties but significant improvement in biostability and
nontoxicity in many cases [28,29]. 1. Introduction It was also reported that the NSPs immobilized with nanoparticles
such as AgNPs could effectively prevent the nanoparticles from agglomeration into Ag clusters,
while exhibiting antimicrobial activity through dramatically decreasing glucose uptake and hindering
adenosine triphosphate (ATP) synthesis for microbial growth [21]. As a result, the root canal sealers
were then prepared by urethane acrylate (UA) composites comprising the immobilized nanoparticles
on nanoscale platelets for the root canal obturation evaluations. 2.2. Preparation of Antibacterial Sealer Based on UA
2.2. Preparation of Antibacterial Sealer Based on UA The UA samples (UAT65 and UAC50) were dissolved in acetone at 80 °C and mixed with
nanoscale silicate materials (Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP) to prepare UA-based
composites. The preparation and characterization of Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP were
performed according to the literature reported by our groups [21]. The sealers were then prepared
by the introduction of a diluent, tripropylene glycol diacrylate (TPGDA), and an initiator containing
camphorquinone (CQ) as a photosensitizer with ethyl-4-dimethylaminobenzoate (EDMA) for binary
initiation processes with a ratio of CQ/EDMAB = 1/2 (w/w), together with 0.5 wt %
azobisisobutyronitrile (AIBN) for ternary initiation processes. The characterizations of composites
were carried out by using the thermogravimetric analysis (TGA; TGA Q50 TA instrument, TA
Instrument, New Castle, DE, USA) to investigate the inorganic contents. For the curing process, the
sealer samples were first cast into Teflon molds in the dimension of 20-mm long, 2-mm wide, and
subsequently cured with a light emitting diode curing unit at an intensity of 800 mW/cm2 for 40 s
from the coronal aspect (SmartLite, Dentsply, PA). The degrees of curing conversions were
investigated by using FT-IR to calculate the peak area transition at 1638 cm−1 of aliphatic C=C double
bond of acrylate group according to the following equation (1) [30]:
The UA samples (UAT65 and UAC50) were dissolved in acetone at 80 ◦C and mixed with nanoscale
silicate materials (Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP) to prepare UA-based composites. The
preparation and characterization of Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP were performed according
to the literature reported by our groups [21]. The sealers were then prepared by the introduction
of a diluent, tripropylene glycol diacrylate (TPGDA), and an initiator containing camphorquinone
(CQ) as a photosensitizer with ethyl-4-dimethylaminobenzoate (EDMA) for binary initiation processes
with a ratio of CQ/EDMAB = 1/2 (w/w), together with 0.5 wt % azobisisobutyronitrile (AIBN) for
ternary initiation processes. The characterizations of composites were carried out by using the
thermogravimetric analysis (TGA; TGA Q50 TA instrument, TA Instrument, New Castle, DE, USA) to
investigate the inorganic contents. For the curing process, the sealer samples were first cast into Teflon
molds in the dimension of 20-mm long, 2-mm wide, and subsequently cured with a light emitting
diode curing unit at an intensity of 800 mW/cm2 for 40 s from the coronal aspect (SmartLite, Dentsply,
PA). 2.2. Preparation of Antibacterial Sealer Based on UA
2.2. Preparation of Antibacterial Sealer Based on UA The degrees of curing conversions were investigated by using FT-IR to calculate the peak area
transition at 1638 cm−1 of aliphatic C=C double bond of acrylate group according to the following
equation (1) [30]: Degree of conversion % (DC%) ൌቈ1 െሾ𝐴ଵଷ଼ሿafter curing
ሾ𝐴ଵଷ଼ሿbefore curing
ൈ100%
(1)
Degree of conversion % (DC%) =
1 −
[A1638]after curing
[A1638]before curing
× 100%
(1) Degree of conversion % (DC%) ൌቈ1 െሾ𝐴ଵଷ଼ሿafter curing
ሾ𝐴ଵଷ଼ሿbefore curing
ൈ100%
(1)
Degree of conversion % (DC%) =
1 −
[A1638]after curing
[A1638]before curing
× 100%
(1) (1)
(1) 2.1. Synthesis of Urethane-acrylate (UA) As shown in Figure 2, UAs were prepared from the reaction between the isocyanate groups of
isophorone diisocyanate (IPDI) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as catalyst,
and hydroxyl groups of polyols to achieve urethane prepolymer end-capped with isocyanates, followed
by the addition of hydroxyethylmethacrylate (HEMA). In this study, UA prepared from polyols by
using polytetramethylene ether glycol with a Mw of 650 (PTMEG 650) is denoted as UAT65, while
UA prepared from polycarbonate polyol with a Mw of 500 (PCPO 500) is denoted as UAC50. The
introduction of acrylate group was prepared from using HEMA as reagent in the molar ratio (2/1/1)
of IPDI/polyol/HEMA. The synthesis of UAs was monitored by using IR spectrum (Jasco 4600 FT-IR
Spectrophotometer with a Jasco ATR Pro 450-S accessory, Jasco, Tokyo, Japan). The molecular weights
were measured by using gel permeation chromatography (GPC) with samples dissolved in 1 wt % THF. (Waters chromatography system, two Waters Styragel linear columns, and polystyrene as the standard
with Mw 104, 2560, 7600, 18,000, and 37,900 by using THF as eluent at the flow rate of 1 mL/min.) 4 of 19 Polymers 2020, 12, 482 y
,
,
Figure 2. The preparation of urethane acrylate (UA). Figure 2. The preparation of urethane acrylate (UA). Figure 2. The preparation of urethane acrylate (UA). Figure 2. The preparation of urethane acrylate (UA). 2 4 Biocompatibility Test
2.4. Biocompatibility Test p
y
Biocompatibility tests were performed by using the ISO 10993-5 regulation through Alamar Blue
assay and lactate dehydrogenase (LDH) assay [34]. Alamar Blue assay was carried out according to
the following steps: first, 3T3 cells were cultured under 10% fetal bovine serum (FBS) Dulbecco’s
modified eagle’s medium (DMEM) at 37 °C in the presence of 5% CO2. All Cells were subcultured
twice before the following experiment. In the next step, the specimens were polymerized under
photo-polymerization machine on top of a round Teflon mold, which is 8 mm in diameter and 2 mm
in height, in a distance of 0.5 cm for 40 s. After the polymerization, the UA composites were then
removed from the mold, sterilized by UV-light for 24 h, and soaked in fresh medium at 37 °C for 24
h. In the final step, the medium containing 3T3 cells were added to 96-well plates with a cell number
of 104/well. After the cells fully adhered on the plate, the original cell culture medium was replaced
by the extract material. The cell incubation for Alamar blue assay cell activity analysis was conducted
at 37 °C for 1, 3, and 7 days in the presence of 5% CO2. Moreover, the evaluation of LDH assay for
cytotoxicity were also conducted for 1, 3, and 7 days by using 3T3 cells under similar culture condition
in 10% FBS DMEM at 37 °C in the presence of 5% CO2. Biocompatibility tests were performed by using the ISO 10993-5 regulation through Alamar Blue
assay and lactate dehydrogenase (LDH) assay [34]. Alamar Blue assay was carried out according to
the following steps: first, 3T3 cells were cultured under 10% fetal bovine serum (FBS) Dulbecco’s
modified eagle’s medium (DMEM) at 37 ◦C in the presence of 5% CO2. All Cells were subcultured
twice before the following experiment. In the next step, the specimens were polymerized under
photo-polymerization machine on top of a round Teflon mold, which is 8 mm in diameter and 2 mm
in height, in a distance of 0.5 cm for 40 s. After the polymerization, the UA composites were then
removed from the mold, sterilized by UV-light for 24 h, and soaked in fresh medium at 37 ◦C for 24 h. In the final step, the medium containing 3T3 cells were added to 96-well plates with a cell number of
104/well. 2.3. Curing Condition and Depth Test
2.3. Curing Condition and Depth Test To evaluate the feasibility of the sealers for root canal, the flow and viscosity tests were carried
out for UA resins according to ISO 6876:2001. Based on the specifications, the mixture (0.05 mL) of
UA resin and TPGDA (UAT65/TPGDA or UAC50/TPGDA) was placed on the center of glass plate
using a graduated syringe. After the initial mixing for 180 s (±5 s), another glass plate was placed on
top of the sealer, following a weight providing a total mass of 120 g (±2 g). After 10 min, the weight
was removed in order to measure the maximum and minimum diameter of the compressed disks of
To evaluate the feasibility of the sealers for root canal, the flow and viscosity tests were carried
out for UA resins according to ISO 6876:2001. Based on the specifications, the mixture (0.05 mL) of UA
resin and TPGDA (UAT65/TPGDA or UAC50/TPGDA) was placed on the center of glass plate using a
graduated syringe. After the initial mixing for 180 s (±5 s), another glass plate was placed on top of
the sealer, following a weight providing a total mass of 120 g (±2 g). After 10 min, the weight was
removed in order to measure the maximum and minimum diameter of the compressed disks of the 5 of 19 Polymers 2020, 12, 482 sealers [31]. Apart from that, the viscosities of the sealers were measured by using a syringe-based
viscometer based on Instron 3360 series universal testing system [32]. In Figure 3, the measurement of
maximal curing increment thickness for resin composites was conducted by using a ISO 4049:2009
method [33]. The curing process was performed with a 2 × 2 × 20 mm re-usable stainless-steel mold
with a top cover to prevent materials’ leakage. After curing process under a light source of halogen
lamp for 40 s, all samples were kept in saturated humidity at 37 ◦C for 24 h to measure the curing
depths. The mechanical properties were also measured by using the Universal Testing Instrumentals
according to ASTM D412-98a with a stretching rate at 100 mm/min. The tensile tests were conducted
for five times on an average. Polymers 2019, 11, x FOR PEER REVIEW
5 of 21
of maximal curing increment thickness for resin composites was conducted by using a ISO 4049:2009
method [33]. 2 4 Biocompatibility Test
2.4. Biocompatibility Test After the cells fully adhered on the plate, the original cell culture medium was replaced by
the extract material. The cell incubation for Alamar blue assay cell activity analysis was conducted
at 37 ◦C for 1, 3, and 7 days in the presence of 5% CO2. Moreover, the evaluation of LDH assay for
cytotoxicity were also conducted for 1, 3, and 7 days by using 3T3 cells under similar culture condition
in 10% FBS DMEM at 37 ◦C in the presence of 5% CO2. 2.3. Curing Condition and Depth Test
2.3. Curing Condition and Depth Test The curing process was performed with a 2 × 2 × 20 mm re-usable stainless-steel mold
with a top cover to prevent materials’ leakage. After curing process under a light source of halogen
lamp for 40 s, all samples were kept in saturated humidity at 37 °C for 24 h to measure the curing
depths. The mechanical properties were also measured by using the Universal Testing Instrumentals
according to ASTM D412-98a with a stretching rate at 100 mm/min. The tensile tests were conducted
for five times on an average. Figure 3. The test method for curing depths according to ISO 4049:2009 [33]. Figure 3. The test method for curing depths according to ISO 4049:2009 [33]. Figure 3. The test method for curing depths according to ISO 4049:2009 [33]. Figure 3. The test method for curing depths according to ISO 4049:2009 [33]. 2 5 Antibacterial Test
2.5. Antibacterial Test A
i a e ia
e
The antibacterial activity of UAT65/TPGDA or UAC50/TPGDA with various Ag@NSP,
ZnO@NSP, or Ag/ZnO@NSP contents was performed according to the Japanese Industrial Standard
as shown in Figure 4 (JIS Z 2801-2000) [32]. Gram-positive E. faecalis (ATCC 29212, Super Laboratory
Co., Taiwan) was the bacterial strain cultivated in brain heart infusion (BHI) broth. The number of
bacterial suspensions was adjusted to 104 colony-forming units per milliliter (CFU/mL). The
antibacterial test was carried out by the following steps. First, colonies were added into 5 mL HBI
broth, cultivated at 37 °C for 16–18 h. The medium of bacteria fluid of lysogeny broth (LB) was
replaced by phosphate-buffered saline (PBS) by using the centrifugation for three times (8,000 xg, 3
min), and diluted to the concentration of 10−5–10−7 (CFU/mL)
Subsequently, the as-prepared
The antibacterial activity of UAT65/TPGDA or UAC50/TPGDA with various Ag@NSP, ZnO@NSP,
or Ag/ZnO@NSP contents was performed according to the Japanese Industrial Standard as shown
in Figure 4 (JIS Z 2801-2000) [32]. Gram-positive E. faecalis (ATCC 29212, Super Laboratory Co.,
Taiwan) was the bacterial strain cultivated in brain heart infusion (BHI) broth. The number of bacterial
suspensions was adjusted to 104 colony-forming units per milliliter (CFU/mL). The antibacterial test
was carried out by the following steps. First, colonies were added into 5 mL HBI broth, cultivated at 37
◦C for 16–18 h. The medium of bacteria fluid of lysogeny broth (LB) was replaced by phosphate-buffered
saline (PBS) by using the centrifugation for three times (8,000 xg, 3 min), and diluted to the concentration
of 10−5–10−7 (CFU/mL). Subsequently, the as-prepared suspension was spread onto the BHI agar plates 6 of 19 Polymers 2020, 12, 482 (10 µL) and incubated cells at 37 ◦C for 16–18 h. The 0.5 McFarland (108 CFU/mL) bacterial fluid
was produced by using E. faecalis (ATCC 29212). Specimens were shaped to square (5 cm × 5 cm),
wiped with alcohol, and sterilized by the exposure of UV-light for 24 h. Then, 400 µL bacterial fluid
(104 CFU/mL) was added to the specimens, covered with a sterile square PE film (4 cm × 4 cm) and
incubated at 37 ◦C under relative humidity 90% for 24 h. After bacterial adhesion or proliferation, the
specimens were rinsed for three times by using PBS and then transformed to a new 24-well plate. 2 5 Antibacterial Test
2.5. Antibacterial Test Each
specimen was soaked in 2.5% glutaraldehyde and reacted at 4 ◦C for 1 h, followed by the removal
of glutaraldehyde by rinsing with PBS for three times. Before the investigation of morphology for
the evaluation of antibacterial activity, the samples were prepared by using ethanol-wet bonding
technique [35]. The freeze-drying processes were conducted for 24 h by the increased concentration of
(w/w) ethanol from 30%, 50%, 70%, 90%, 95%, 99% to 100%. In addition, the statistical analysis was
performed by Statistical Analysis Software (SAS). One-way analysis of variance (ANOVA) was used to
analyze the difference between bacterial groups, and Duncan’s multiple tests were used to distinguish
various bacterial groups. The p-value <0.05 is regarded as statistically significant. Polymers 2019, 11, x FOR PEER REVIEW
6 of 21
with a sterile square PE film (4 cm × 4 cm) and incubated at 37 °C under relative humidity 90% for 24
h. After bacterial adhesion or proliferation, the specimens were rinsed for three times by using PBS
and then transformed to a new 24-well plate. Each specimen was soaked in 2.5% glutaraldehyde and
reacted at 4 °C for 1 h, followed by the removal of glutaraldehyde by rinsing with PBS for three times. Before the investigation of morphology for the evaluation of antibacterial activity, the samples were
prepared by using ethanol-wet bonding technique [35]. The freeze-drying processes were conducted
for 24 h by the increased concentration of (w/w) ethanol from 30%, 50%, 70%, 90%, 95%, 99% to 100%. In addition, the statistical analysis was performed by Statistical Analysis Software (SAS). One-way
analysis of variance (ANOVA) was used to analyze the difference between bacterial groups, and
Duncan’s multiple tests were used to distinguish various bacterial groups. The p-value <0.05 is
regarded as statistically significant. Figure 4. The process of antibacterial evaluation. Figure 4. The process of antibacterial evaluation. Figure 4. The process of antibacterial evaluation. Figure 4. The process of antibacterial evaluation. 3.1. Synthesis of Urethan-acrylate (UA)
3.1. Synthesis of Urethan-acrylate (UA) The preparation of urethane acrylate was monitored by using the IR spectra as shown in Figure
5. For the spectrum of the initial mixture of IPDI and PCPO 500, two distinct peaks at 2260 and 1742
cm-1 were present for isocyanate, and carbonyl group of carbonate ester, respectively. After 2 h into
the reaction, a newly emerged shoulder at 1718 cm−1 was observed, indicating the formation of
urethane carbonyl group [36,37]. Moreover, the urethane prepolymer end-capped with isocyanates
was further reacted with HEMA to provide the product of UAC50 as evident by the near
disappearance of isocyanate group and the formation of a new adsorption peak at 1638 cm−1 of vinyl
group. The additional TPGDA in the mixture of UAC50/TPGDA = 70/30 (w/w) resulted in a stronger
absorption intensity of vinyl group. The weight average molecular weights were measured by using
GPC, leading to the results of 3320 g/mol for UAC50 and 3620 g/mol for UAT65. The preparation of urethane acrylate was monitored by using the IR spectra as shown in Figure 5. For the spectrum of the initial mixture of IPDI and PCPO 500, two distinct peaks at 2260 and 1742
cm−1 were present for isocyanate, and carbonyl group of carbonate ester, respectively. After 2 h
into the reaction, a newly emerged shoulder at 1718 cm−1 was observed, indicating the formation of
urethane carbonyl group [36,37]. Moreover, the urethane prepolymer end-capped with isocyanates
was further reacted with HEMA to provide the product of UAC50 as evident by the near disappearance
of isocyanate group and the formation of a new adsorption peak at 1638 cm−1 of vinyl group. The
additional TPGDA in the mixture of UAC50/TPGDA = 70/30 (w/w) resulted in a stronger absorption
intensity of vinyl group. The weight average molecular weights were measured by using GPC, leading
to the results of 3320 g/mol for UAC50 and 3620 g/mol for UAT65. 7 of 19
7 of 21 Polymers 2020, 12, 482
Polymers 2019, 11, x FOR Figure 5. FTIR spectra of UAC50 and UAC50/TPGDA. 3.2. Preparation and Thermal Properties of UA Composites
Figure 5. FTIR spectra of UAC50 and UAC50/TPGDA. 3.2. Preparation and Thermal Properties of UA Composites
Figure 5. FTIR spectra of UAC50 and UAC50/TPGDA. 3.2. Preparation and Thermal Properties of UA Composites 3.2. Preparation and Thermal Properties of UA Composites
3.2. Preparation and Thermal Properties of UA Composites
3.2. 3.1. Synthesis of Urethan-acrylate (UA)
3.1. Synthesis of Urethan-acrylate (UA) Preparation and Thermal Properties of UA Composites The determination of the processing effectiveness and the quantitative fillers within the matrix
are usually investigated by using thermogravimetric analysis (TGA) for the polymer composites [38]. The UA composites were prepared by mixing 5 wt % or 10 wt % in total composites by using the
nanometer-thin silicate platelets immobilized with nanoparticles such as AgNPs (Ag@NSP), ZnONPs
(ZnO@NSP), or AgNPs together with ZnONPs (Ag/ZnO@NSP) [20,22,25,39,40]. In TGA thermograms
(Figure 6), the UA composites exhibited 5% weight loss at about 300 °C, indicating that good thermal
stability was available for the following tests. Moreover, the addition of nanoparticles immobilized
on the nanometer-thin silicate platelets provided higher char yield (%) dependent on the addition of
inorganic contents. The determination of the processing effectiveness and the quantitative fillers within the matrix
are usually investigated by using thermogravimetric analysis (TGA) for the polymer composites [38]. The UA composites were prepared by mixing 5 wt % or 10 wt % in total composites by using the
nanometer-thin silicate platelets immobilized with nanoparticles such as AgNPs (Ag@NSP), ZnONPs
(ZnO@NSP), or AgNPs together with ZnONPs (Ag/ZnO@NSP) [20,22,25,39,40]. In TGA thermograms
(Figure 6), the UA composites exhibited 5% weight loss at about 300 ◦C, indicating that good thermal
stability was available for the following tests. Moreover, the addition of nanoparticles immobilized
on the nanometer-thin silicate platelets provided higher char yield (%) dependent on the addition of
inorganic contents. The determination of the processing effectiveness and the quantitative fillers within the matrix
are usually investigated by using thermogravimetric analysis (TGA) for the polymer composites [38]. The UA composites were prepared by mixing 5 wt % or 10 wt % in total composites by using the
nanometer-thin silicate platelets immobilized with nanoparticles such as AgNPs (Ag@NSP), ZnONPs
(ZnO@NSP), or AgNPs together with ZnONPs (Ag/ZnO@NSP) [20,22,25,39,40]. In TGA thermograms
(Figure 6), the UA composites exhibited 5% weight loss at about 300 °C, indicating that good thermal
stability was available for the following tests. Moreover, the addition of nanoparticles immobilized
on the nanometer-thin silicate platelets provided higher char yield (%) dependent on the addition of
inorganic contents. Figure 6. Thermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. Figure 6. Thermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. Figure 6. Thermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. Figure 6. Thermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. Figure 6. 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t The endodontic sealers are required to exhibit several properties to meet the desired performance. The flow behavior is one of the most important factors for sealers to penetrate into small irregularities
of the root canal system and dentinal tubules as shown in Figure 7. According to the regulation of ISO
4049:2009, the flow diameter should be over 20 mm after compression under a weight disc for 10 min. The tests were carried out by the introduction of difunctional TPGDA in order to increase flow diameter
and crosslinking density. As a result, the samples with higher ratio of TPGDA exhibited larger flow
diameter, such as UAT65/TPGDA = 80/20, 70/30, or 60/40; or UAC50/TPGDA = 70/30 or 60/40, since the
difunctional TPGDA monomer is a diluent with a lower molecular weight when compared with the
UA resins. between the root canal, periodontal ligament, and the apical foramen. According to ISO 4049:2009,
the viscosity should range from 1000 to 2000 cp to meet the desired operation [32]. In Figure 8,
UAT65/TPGDA (70/30) and UAC50/TPGDA (70/30) were the samples of choice for further
investigations since the addition of the optimized TPGDA content would achieve the viscosity range
mentioned above. It is important to note that flow and viscosity properties depends on not only the
ratios between UA resins and TPGDA but the use of polyols such as PTMEG or PCPO. The PCPO-
based UA resins exhibited somewhat lower flow diameter and higher viscosity under the same ratio
of UA/TPGDA composition. This is because the incorporation of the PCPO-based polyol with
carbonate groups brought about more hydrogen bonding interactions with the urethane linkages. This would restrain the molecular mobility of the UA segments [41,42]. Figure 7. Flow analysis of UA resins with various weight ratios of UAT65/tripropylene glycol
diacrylate (TPGDA) or UAC50/TPGDA according to ISO 4049:2009. Figure 7. Flow analysis of UA resins with various weight ratios of UAT65/tripropylene glycol diacrylate
(TPGDA) or UAC50/TPGDA according to ISO 4049:2009. Figure 7. Flow analysis of UA resins with various weight ratios of UAT65/tripropylene glycol
diacrylate (TPGDA) or UAC50/TPGDA according to ISO 4049:2009. Figure 7. Flow analysis of UA resins with various weight ratios of UAT65/tripropylene glycol diacrylate
(TPGDA) or UAC50/TPGDA according to ISO 4049:2009. Moreover, viscosity is another important factor for the root canal sealer to establish connection
between the root canal, periodontal ligament, and the apical foramen. 3.1. Synthesis of Urethan-acrylate (UA)
3.1. Synthesis of Urethan-acrylate (UA) Thermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. Figure 6. Thermogravimetric analysis (TGA) thermograms of UAC50- and UAC50-based composites. Polymers 2020, 12, 482
TPGDA exhibited
UAC50/TPGDA = 8 of 19
/40; or
lower 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t This is because the low conversion degree of acrylate functional groups with a low
concentration initiator resulted in insufficient crosslinking density for obtaining good mechanical
performance, whereas the excessive photoinitiators would provide the localized absorption and
crosslinking density over the percolation threshold leading to the adverse effect on the mechanical
It is important to note that the DC% values were close to 70% for the above-mentioned samples. This is
because the low conversion degree of acrylate functional groups with a low concentration initiator
resulted in insufficient crosslinking density for obtaining good mechanical performance, whereas the
excessive photoinitiators would provide the localized absorption and crosslinking density over the
percolation threshold, leading to the adverse effect on the mechanical properties [43]. crosslinking density over the percolation threshold, leading to the adverse effect on the mechanical
properties [43]. Table 1. Mechanical properties and DC% for the UAT65/TPGDA (w/w = 70/30) and
TPGDAC50/TPGDA (w/w = 70/30) samples after curing with different dosages of photoinitiator. Entry
UA Formulation
Photoinitiator
(phr 1)
Tensile Strength
(MPa)
Young’s Modulus
(MPa)
DC 2 (%)
UA
/
A
Since the acrylate-based photopolymerization depends deeply on the sealer transparency, the
curing depths of UAT65/TPGDA resin (w/w = 70/30) and UAC50/TPGDA resin (w/w = 70/30) were
assessed by using various contents of antibacterial NSPs immobilized with nanoparticles including
Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP (Figures 9–11). It is reported that the photo crosslinking
activity relies on the use of different types of metal nanoparticles and the rate of photo crosslinking,
and the DC% also varies with the additives under similar processing condition [44]. In this study, the
curing depths decreased with increasing content of antibacterial NSP agents. 1
UAT65/TPGDA
(w/w = 70/30)
1
51.71 ± 3.03
362.81 ± 37.10
46.27 ± 3.07
2
UAT65/TPGDA
(w/w = 70/30)
3
56.15 ± 3.26
361.88 ± 32.38
64.91 ± 1.06
3
UAT65/TPGDA
(w/w = 70/30)
6
45.96 ± 2.22
33.49 ± 1.65
67.58 ± 2.27
4
UAT65/TPGDA
(w/w = 70/30)
9
36.78 ± 1.65
1.92 ± 0.42
70.35 ± 1.76
These UA composite composed of various antibacterial nanomaterials are denoted as Ag@NSP-n,
ZnO@NSP-n, or Ag/ZnO@NSP-n, where the “n” is denoted as the parts per million (ppm) to the total
weight of UAT65/TPGDA or UAC50/TPGDA. According to the regulation of ISO 4049:2009, the curing
depth should be larger than 10 mm. 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t According to ISO 4049:2009,
the viscosity should range from 1000 to 2000 cp to meet the desired operation [32]. In Figure 8,
UAT65/TPGDA (70/30) and UAC50/TPGDA (70/30) were the samples of choice for further investigations
since the addition of the optimized TPGDA content would achieve the viscosity range mentioned
above. It is important to note that flow and viscosity properties depends on not only the ratios between
UA resins and TPGDA but the use of polyols such as PTMEG or PCPO. The PCPO-based UA resins
exhibited somewhat lower flow diameter and higher viscosity under the same ratio of UA/TPGDA
composition. This is because the incorporation of the PCPO-based polyol with carbonate groups
brought about more hydrogen bonding interactions with the urethane linkages. This would restrain
the molecular mobility of the UA segments [41,42]. 9 of 19 Polymers 2020, 12, 482 Figure 8. Viscosity analysis of UA resins with various weight ratios of UAT65/TPGDA or
UAC50/TPGDA according to ISO 4049:2009. Figure 8. Viscosity analysis of UA resins with various weight ratios of UAT65/TPGDA or UAC50/TPGDA
according to ISO 4049:2009. Figure 8. Viscosity analysis of UA resins with various weight ratios of UAT65/TPGDA or
UAC50/TPGDA according to ISO 4049:2009. Figure 8. Viscosity analysis of UA resins with various weight ratios of UAT65/TPGDA or UAC50/TPGDA
according to ISO 4049:2009. The curing conditions of UAT65/TPGDA (70/30) or UAC50/TPGDA (70/30) were carried out by
using ternary initiation processes for photo crosslinking. The degree of acrylate conversion (DC%)
would be based on the peak area at 1638 cm−1 of aliphatic C=C double bond of acrylate content as a
function of various initiator concentrations (1, 3, 6, and 9 phr) before and after curing (Table 1). Optimum mechanical properties with a tensile strength of 56.15 ± 3.26 MPa and a Young’s modulus
of 361.88 ± 32.38 MPa were achieved for the sample UAT65/TPGDA (70/30) cured with 3 phr initiator
concentration. It is important to note that the DC% values were close to 70% for the above-mentioned
samples. This is because the low conversion degree of acrylate functional groups with a low
concentration initiator resulted in insufficient crosslinking density for obtaining good mechanical
performance, whereas the excessive photoinitiators would provide the localized absorption and
crosslinking density over the percolation threshold, leading to the adverse effect on the mechanical
The curing conditions of UAT65/TPGDA (70/30) or UAC50/TPGDA (70/30) were carried out by
using ternary initiation processes for photo crosslinking. 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t The degree of acrylate conversion (DC%)
would be based on the peak area at 1638 cm−1 of aliphatic C=C double bond of acrylate content as a
function of various initiator concentrations (1, 3, 6, and 9 phr) before and after curing (Table 1). Optimum
mechanical properties with a tensile strength of 56.15 ± 3.26 MPa and a Young’s modulus of 361.88 ±
32.38 MPa were achieved for the sample UAT65/TPGDA (70/30) cured with 3 phr initiator concentration. It is important to note that the DC% values were close to 70% for the above-mentioned samples. This is
because the low conversion degree of acrylate functional groups with a low concentration initiator
resulted in insufficient crosslinking density for obtaining good mechanical performance, whereas the
excessive photoinitiators would provide the localized absorption and crosslinking density over the
percolation threshold, leading to the adverse effect on the mechanical properties [43]. The curing conditions of UAT65/TPGDA (70/30) or UAC50/TPGDA (70/30) were carried out by
using ternary initiation processes for photo crosslinking. The degree of acrylate conversion (DC%)
would be based on the peak area at 1638 cm−1 of aliphatic C=C double bond of acrylate content as a
function of various initiator concentrations (1, 3, 6, and 9 phr) before and after curing (Table 1). Optimum mechanical properties with a tensile strength of 56.15 ± 3.26 MPa and a Young’s modulus
of 361 88 ± 32 38 MPa were achieved for the sample UAT65/TPGDA (70/30) cured with 3 phr initiator
The curing conditions of UAT65/TPGDA (70/30) or UAC50/TPGDA (70/30) were carried out by
using ternary initiation processes for photo crosslinking. The degree of acrylate conversion (DC%)
would be based on the peak area at 1638 cm−1 of aliphatic C=C double bond of acrylate content as a
function of various initiator concentrations (1, 3, 6, and 9 phr) before and after curing (Table 1). Optimum
mechanical properties with a tensile strength of 56.15 ± 3.26 MPa and a Young’s modulus of 361.88 ±
32.38 MPa were achieved for the sample UAT65/TPGDA (70/30) cured with 3 phr initiator concentration. of 361.88 ± 32.38 MPa were achieved for the sample UAT65/TPGDA (70/30) cured with 3 phr initiator
concentration. It is important to note that the DC% values were close to 70% for the above-mentioned
samples. 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t These UA composite composed of various antibacterial nanomaterials are denoted as Ag@NSP-
n, ZnO@NSP-n, or Ag/ZnO@NSP-n, where the “n” is denoted as the parts per million (ppm) to the
total weight of UAT65/TPGDA or UAC50/TPGDA. According to the regulation of ISO 4049:2009, the
curing depth should be larger than 10 mm. As a result, the limitation for the addition of a maximum
amount of Ag@NSP is 500 ppm (Ag@NSP-500) as shown in Figure 9. Similar results were also
obtained for the composites incorporated with ZnO@NSP or Ag/ZnO@NSP (Figures 10 and 11). This
indicates that the presence of different nanomaterials such as Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP
in the UA composites did not influence curing depths much. Figure 9. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag@NSP contents (ppm). Figure 9. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag@NSP contents (ppm). Table 1. Mechanical properties and DC% for the UAT65/TPGDA (w/w = 70/30) and TPGDAC50/TPGDA
(w/w = 70/30) samples after curing with different dosages of photoinitiator. Entry
UA
Formulation
Photoinitiator
(phr 1)
Tensile Strength
(MPa)
Young’s Modulus
(MPa)
DC 2 (%)
1
UAT65/TPGDA
(w/w = 70/30)
1
51.71 ± 3.03
362.81 ± 37.10
46.27 ± 3.07
2
UAT65/TPGDA
(w/w = 70/30)
3
56.15 ± 3.26
361.88 ± 32.38
64.91 ± 1.06
3
UAT65/TPGDA
(w/w = 70/30)
6
45.96 ± 2.22
33.49 ± 1.65
67.58 ± 2.27
4
UAT65/TPGDA
(w/w = 70/30)
9
36.78 ± 1.65
1.92 ± 0.42
70.35 ± 1.76
5
UAC50/TPGDA
(w/w = 70/30)
1
46.30 ± 3.18
1072.25 ± 46.54
57.89 ± 0.41
6
UAC50/TPGDA
(w/w = 70/30)
3
60.54 ± 4.72
1042.02 ± 39.62
72.44 ± 1.57
7
UAC50/TPGDA
(w/w = 70/30)
6
61.09 ± 1.15
14.01 ± 2.65
79.03 ± 2.57
8
UAC50/TPGDA
(w/w = 70/30)
9
59.04 ± 2.75
8.61 ± 0.41
80.08 ± 2.79
1: parts per hundred; 2: after the exposure under UV light for 40 s. Polymers 2019, 11, x FOR PEER REVIEW
10 of 2
7
UAC50/TPGDA
(w/w = 70/30)
6
61.09 ± 1.15
14.01 ± 2.65
79.03 ± 2.57
8
UAC50/TPGDA
(w/w = 70/30)
9
59.04 ± 2.75
8.61 ± 0.41
80.08 ± 2.79
1: parts per hundred; 2: after the exposure under UV light for 40 s. 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t As a result, the limitation for the addition of a maximum amount
of Ag@NSP is 500 ppm (Ag@NSP-500) as shown in Figure 9. Similar results were also obtained for
the composites incorporated with ZnO@NSP or Ag/ZnO@NSP (Figures 10 and 11). This indicates
that the presence of different nanomaterials such as Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP in the UA
composites did not influence curing depths much. 10 of 19 Polymers 2020, 12, 482 y
Table 1. Mechanical properties and DC% for the UAT65/TPGDA (w/w = 70/30) and TPGDAC50/TPGDA
(w/w = 70/30) samples after curing with different dosages of photoinitiator. Entry
UA
Formulation
Photoinitiator
(phr 1)
Tensile Strength
(MPa)
Young’s Modulus
(MPa)
DC 2 (%)
1
UAT65/TPGDA
(w/w = 70/30)
1
51.71 ± 3.03
362.81 ± 37.10
46.27 ± 3.07
2
UAT65/TPGDA
(w/w = 70/30)
3
56.15 ± 3.26
361.88 ± 32.38
64.91 ± 1.06
3
UAT65/TPGDA
(w/w = 70/30)
6
45.96 ± 2.22
33.49 ± 1.65
67.58 ± 2.27
4
UAT65/TPGDA
(w/w = 70/30)
9
36.78 ± 1.65
1.92 ± 0.42
70.35 ± 1.76
5
UAC50/TPGDA
(w/w = 70/30)
1
46.30 ± 3.18
1072.25 ± 46.54
57.89 ± 0.41
6
UAC50/TPGDA
(w/w = 70/30)
3
60.54 ± 4.72
1042.02 ± 39.62
72.44 ± 1.57
7
UAC50/TPGDA
(w/w = 70/30)
6
61.09 ± 1.15
14.01 ± 2.65
79.03 ± 2.57
8
UAC50/TPGDA
(w/w = 70/30)
9
59.04 ± 2.75
8.61 ± 0.41
80.08 ± 2.79
1: parts per hundred; 2: after the exposure under UV light for 40 s. Polymers 2019, 11, x FOR PEER REVIEW
10 of 21
7
UAC50/TPGDA
(w/w = 70/30)
6
61.09 ± 1.15
14.01 ± 2.65
79.03 ± 2.57
8
UAC50/TPGDA
(w/w = 70/30)
9
59.04 ± 2.75
8.61 ± 0.41
80.08 ± 2.79
1: parts per hundred; 2: after the exposure under UV light for 40 s. Since the acrylate-based photopolymerization depends deeply on the sealer transparency, the
curing depths of UAT65/TPGDA resin (w/w = 70/30) and UAC50/TPGDA resin (w/w = 70/30) were
assessed by using various contents of antibacterial NSPs immobilized with nanoparticles including
Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP (Figures 9–11). It is reported that the photo crosslinking
activity relies on the use of different types of metal nanoparticles and the rate of photo crosslinking,
and the DC% also varies with the additives under similar processing condition [44]. In this study, the
curing depths decreased with increasing content of antibacterial NSP agents. 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t Since the acrylate-based photopolymerization depends deeply on the sealer transparency, the
curing depths of UAT65/TPGDA resin (w/w = 70/30) and UAC50/TPGDA resin (w/w = 70/30) wer
assessed by using various contents of antibacterial NSPs immobilized with nanoparticles including
Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP (Figures 9–11). It is reported that the photo crosslinking
activity relies on the use of different types of metal nanoparticles and the rate of photo crosslinking
and the DC% also varies with the additives under similar processing condition [44]. In this study, th
curing depths decreased with increasing content of antibacterial NSP agents. These UA composite composed of various antibacterial nanomaterials are denoted as Ag@NSP
n, ZnO@NSP-n, or Ag/ZnO@NSP-n, where the “n” is denoted as the parts per million (ppm) to the
total weight of UAT65/TPGDA or UAC50/TPGDA. According to the regulation of ISO 4049:2009, the
curing depth should be larger than 10 mm. As a result, the limitation for the addition of a maximum
amount of Ag@NSP is 500 ppm (Ag@NSP-500) as shown in Figure 9. Similar results were also
obtained for the composites incorporated with ZnO@NSP or Ag/ZnO@NSP (Figures 10 and 11). Thi
indicates that the presence of different nanomaterials such as Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP
i
the UA o
o ite did
ot i flue
e u i
de th
u h the UA composites did not influence curing depths much. Figure 9. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag@NSP contents (ppm). Figure 9. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag@NSP contents (ppm). Figure 9. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag@NSP contents (ppm). Figure 9. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag@NSP contents (ppm). 11 of 19
11 of 21
11 of 21 Polymers 2020, 12, 482
P l
2019 11
FO
Polymers 2019, 11, x FOR Figure 10. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various ZnO@NSP contents (ppm). Figure 10. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various ZnO@NSP contents (ppm). Figure 10. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various ZnO@NSP contents (ppm). Figure 10. 3.3. Curing Conditions and Depths for UA Composites
Moreover, viscosity is another important factor
between the root canal periodontal ligament and t Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various ZnO@NSP contents (ppm). Figure 10. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various ZnO@NSP contents (ppm). Figure 10. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various ZnO@NSP contents (ppm). Figure 11. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
Figure 11. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag/ZnO@NSP contents (ppm). Figure 11. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag/ZnO@NSP contents (ppm). Figure 11. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
Figure 11. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag/ZnO@NSP contents (ppm). Figure 11. Curing depths of UAT65/TPGDA resins (w/w = 70/30) and UAC50/TPGDA resins (w/w =
70/30) with various Ag/ZnO@NSP contents (ppm). 70/30) with various Ag/ZnO
/
)
g/
3 4 Bi
tibilit A
l
i
3.4. Biocompatibility Analysis 70/30) with various Ag/ZnO
)
g
3 4 Bi
tibilit A
l
i
3.4. Biocompatibility Analysis 3.4. Biocompatibility Analysis
Grossman [45] advocated that an ideal root canal filling material should not irritate periradicular
tissues. In addition, Faccioni et al. found that root canal materials with metal ions might influence
cell
metabolism
and
differentiation
[46]. Other
studies
found
that
the
incomplete
photopolymerization reaction resulted in the release of uncured monomers and initiators, which
3.4. Biocompatibility Analysis
Grossman [45] advocated that an ideal root canal filling material should not irritate periradicular
tissues. In addition, Faccioni et al. found that root canal materials with metal ions might influence
cell
metabolism
and
differentiation
[46]. Other
studies
found
that
the
incomplete
photopolymerization reaction resulted in the release of uncured monomers and initiators, which
would affect the mitochondrial enzyme activity [47 48] Therefore the concentration of antibacterial
Grossman [45] advocated that an ideal root canal filling material should not irritate periradicular
tissues. In addition, Faccioni et al. found that root canal materials with metal ions might influence cell
metabolism and differentiation [46]. Other studies found that the incomplete photopolymerization
reaction resulted in the release of uncured monomers and initiators, which would affect the
mitochondrial enzyme activity [47,48]. Therefore, the concentration of antibacterial agent in sealers 12 of 19 Polymers 2020, 12, 482 depends deeply on the biocompatibility of composites. In this study, the tests of Alamar Blue assay
and LDH assay were conducted for the biocompatibility tests for UAT65/TPGDA (w/w = 70/30) and
UAC50/TPGDA resins (w/w = 70/30) with various concentrations of antimicrobial agents such as
Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP as shown in Figures 12 and 13, respectively. Polymers 2019, 11, x FOR PEER REVIEW
13 of 21 Metabolic activity (% of control)
(a)
Day 1
Day 3
Day 7
Polymers 2019, 11, x FOR PEER REVIEW
14 of 21
Figure 12. Alamar blue assay of (a) UAT65/TPGDA (w/w = 70/30) and (b) UAC50/TPGDA (w/w = 70/30)
resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (p < 0.05). Metabolic activity (% of control)
(b)
Day 1
Day 3
Day 7
re 12. Alamar blue assay of (a) UAT65/TPGDA (w/w = 70/30) and (b) UAC50/TPGDA (w/w = 70
ns with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (p < 0.05). Metabolic activity (% of control)
(a)
Day 1
Day 3
Day 7
Polymers 2019, 11, x FOR PEER REVIEW
14 of 21 Figure 12. 70/30) with various Ag/ZnO
)
g
3 4 Bi
tibilit A
l
i
3.4. Biocompatibility Analysis Alamar blue assay of (a) UAT65/TPGDA (w/w = 70/30) and (b) UAC50/TPGDA (w/w = 70/30)
resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (p < 0.05). Metabolic activity (% of control)
(b)
Day 1
Day 3
Day 7
Figure 12. Alamar blue assay of (a) UAT65/TPGDA (w/w = 70/30) and (b) UAC50/TPGDA (w/w =
resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (p < 0.05). (b) Figure 12. Alamar blue assay of (a) UAT65/TPGDA (w/w = 70/30) and (b) UAC50/TPGDA (w/w = 70/30)
resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (p < 0.05). Figure 12. Alamar blue assay of (a) UAT65/TPGDA (w/w = 70/30) and (b) UAC50/TPGDA (w/w = 70/30)
resins with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP (p < 0.05). 13 of 19 Polymers 2020, 12, 482 0
20
40
60
80
100
120
Cytotoxicity (% of control)
(a)
Day 1
Day 3
Day 7
Polymers 2019, 11, x FOR PEER REVIEW
16 of 21
Figure 13. Lactate dehydrogenase (LDH) assay of (a) UAT65/TPGDA (w/w = 70/30) and (b)
UAC50/TPGDA (w/w = 70/30) with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP
(p < 0.05). 3 5 Antibacterial Analysis
0
20
40
60
80
100
120
Cytotoxicity (% of control)
(b)
Day 1
Day 3
Day 7
Figure 13. Lactate dehydrogenase (LDH) assay of (a) UAT65/TPGDA (w/w = 70/30) and (b)
UAC50/TPGDA (w/w = 70/30) with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP
(p < 0.05). 0
20
40
60
80
100
120
Cytotoxicity (% of control)
(a)
Day 1
Day 3
Day 7
Polymers 2019, 11, x FOR PEER REVIEW
16 of 21 (a) 0
20
40
60
80
100
120
Cytotoxicity (% of control)
(b)
Day 1
Day 3
Day 7 (b) Figure 13. Lactate dehydrogenase (LDH) assay of (a) UAT65/TPGDA (w/w = 70/30) and (b)
UAC50/TPGDA (w/w = 70/30) with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP
(p < 0.05). 3.5. Antibacterial Analysis
Figure 13. Lactate dehydrogenase (LDH) assay of (a) UAT65/TPGDA (w/w = 70/30) and (b)
UAC50/TPGDA (w/w = 70/30) with various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP
(p < 0.05). The antibacterial activities of the composites based on UAC50/TPGDA (w/w = 70/30) with
nanoparticles-on-platelet nanohybrids (Ag@NSP, ZnO@NSP, Ag/ZnO@NSP) were analyzed by
treating the composites with Gram-positive E. faecalis (ATCC 29212) according to Japanese Industrial
Standard (JIS Z 2801-2000) as shown in Table 2. 70/30) with various Ag/ZnO
)
g
3 4 Bi
tibilit A
l
i
3.4. Biocompatibility Analysis According to the biocompatibility tests in previous
section, the use of antibacterial NSP agents should not be higher than 100 ppm for UAC50/TPGDA
(w/w = 70/30) resins. Indeed, the AgNPs-based composites with 75 ppm (Ag@NSP-75) or 100 ppm
(Ag@NSP-100) inorganic additives exhibited antibacterial activity. However, ZnONPs-based
composites even with 1000 ppm (ZnO@NSP-1000) were not able to exhibit antibacterial activity. In
fact, the antibacterial activity was feasible only when the use of high concentration of ZnONPs over
2000 ppm (ZnO@NSP-2000 and ZnO@NSP-3000), indicating that the Ag@NSP-based composites
would be better candidates for the root canal sealer applications. In addition, the simultaneous
In the Alamar blue assay test, both pristine resins of UAT65/TPGDA (w/w = 70/30) (Figure 12a) and
UAC50/TPGDA (w/w = 70/30) (Figure 12b) were substantially free of cytotoxicity. As the antimicrobial
agents were incorporated into the resins to form UA composites, reduced metabolic activities were
observed, as shown in Figure 12. Furthermore, the composite with Ag@NSP exhibited the poorest
biocompatibility when compared to other samples, especially in the example for both UAT65/TPGDA
and UAC50/TPGDA incorporated with 500 ppm additives. The metabolic activities were higher than
70% for all the UA composites with 100 ppm or less than 100 ppm antibacterial NSP agents. This Polymers 2020, 12, 482 14 of 19 14 of 19 indicates that good biocompatibility could be achieved with the addition of a certain content of the
antimicrobial agents such as Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP to the composites. In the LDH assay test, the cytotoxicity is also dependent on the addition of various concentrations of
Ag@NSP, ZnO@NSP, and Ag/ZnO@NSP to the composites (Figure 13). Given the fact that UA composites
with 100 ppm would exhibit good biocompatibility, the cytotoxicity of the UA composites with 100 ppm
antimicrobial agents on 3T3 cells was investigated and observed in the following order: Ag@NSP-100
> Ag/ZnO@NSP-100 > ZnO@NSP-100. This implies that the composites comprising AgNPs would
exhibit poor cytotoxicity performance. In addition, the cytotoxicity of the composites based on the
UAC50/TPGDA (w/w = 70/30) are lower than that of the composites based on UAT65/TPGDA (w/w
= 70/30), especially in the example for both UAT65/TPGDA (~90% of control) and UAC50/TPGDA
(~80% of control) incorporated with 500 ppm Ag@NSP. As a matter of fact, polycarbonate-based
polyurethanes (PUs) with better biocompatibility correspond to the weaker immune response when
compared with polyether-based PUs according to the literature [49]. 3.5. Antibacterial Analysis The antibacterial activities of the composites based on UAC50/TPGDA (w/w = 70/30) with
nanoparticles-on-platelet nanohybrids (Ag@NSP, ZnO@NSP, Ag/ZnO@NSP) were analyzed by treating
the composites with Gram-positive E. faecalis (ATCC 29212) according to Japanese Industrial Standard
(JIS Z 2801-2000) as shown in Table 2. According to the biocompatibility tests in previous section, the use
of antibacterial NSP agents should not be higher than 100 ppm for UAC50/TPGDA (w/w = 70/30) resins. Indeed, the AgNPs-based composites with 75 ppm (Ag@NSP-75) or 100 ppm (Ag@NSP-100) inorganic
additives exhibited antibacterial activity. However, ZnONPs-based composites even with 1000 ppm
(ZnO@NSP-1000) were not able to exhibit antibacterial activity. In fact, the antibacterial activity was
feasible only when the use of high concentration of ZnONPs over 2000 ppm (ZnO@NSP-2000 and
ZnO@NSP-3000), indicating that the Ag@NSP-based composites would be better candidates for the
root canal sealer applications. In addition, the simultaneous immobilization of AgNPs and ZnONPs
on silicate platelets, i.e., Ag/ZnO@NSP, led to much better antimicrobial results even for the composite
with an antibacterial agent concentration as low as 50 ppm (Ag/ZnO@NSP-50). The direct contact of UAC50/TPGDA composites with the bacterial population could be visualized
by using scanning electron microscopy (SEM) as shown in Figure 14. According to the investigation
above, this study was conducted by using E. faecalis on the surfaces of UA/TPGDA composites with 50
ppm Ag@NSP, 75 ppm Ag@NSP, and 50 ppm Ag/ZnO@NSP after 6h and 24h. For the UAC50/TPGDA
resin without the use of antibacterial agents, a rapid growth of bacterial in number was observed as
shown in Figure 14a,e. For the composite (Ag@NSP-50) with 50 ppm of Ag@NSP, a certain amount of
bacteria appeared first after 6h (Figure 14b). Subsequently, these bacteria increased in number and
aggregated after 24 h (Figure 14f). For the composite (Ag@NSP-75) with 75 ppm of Ag@NSP, a certain
amount of bacteria appeared to be aggregated and deformed after 6h, and subsequently these bacteria
remained aggregated without the sign of number increase as shown in Figure 14c,g, respectively. It is
likely that the nanoparticles can re-charge from NSPs to inactivate and rupture bacterial aggregates [51]. It was reported that the combined use of different types of nanoparticles could adsorb onto the
cytoderm of the bacteria and even penetrate the cytomembrane to disturb the normal function of
cells, leading to cell apoptosis [50]. 70/30) with various Ag/ZnO
)
g
3 4 Bi
tibilit A
l
i
3.4. Biocompatibility Analysis This is because the α-carbon
atoms of the polyether-based PUs (such as UAT65) are highly susceptible to oxidation by oxygen
radicals to form esters, which result in unstable chemical structures for polymers [50]. As a result, the
carbonate containing UAC50/TPGDA system would be the material of choice for dental root canal
sealers instead of the ether-containing UAT65/TPGDA resins. 3.5. Antibacterial Analysis This is evidenced by the presence of only 50 ppm Ag/ZnO@NSP
in the UAC50/TPGDA composite capable of exhibiting a satisfactory antibacterial effect against E. faecalis (Table 2). For the composites (Ag/ZnO@NSP-50) with 50 ppm of Ag/ZnO@NSP, once again a
certain amount of bacteria appeared to be aggregated and deformed after 6h, and subsequently these
bacteria remained aggregated without the sign of number increase as shown in Figure 14d,f. The 15 of 19 15 of 19 Polymers 2020, 12, 482 simultaneous immobilization of AgNPs and ZnONPs on silicate platelets could not only enhance the
antibacterial activities and reduce the dose of AgNPs, but act as a promoter in the antibacterial effect
for the Ag/ZnO@NSP-based composites as well. Polymers 2019, 11, x FOR PEER REVIEW
18 of 21 Figure 14. SEM images (2500x) of E. faecalis on the surfaces of UAC50/TPGDA = (w/w = 70/30) w
various antibacterial agents: control (a,e); 50 ppm Ag@NSP (b,f); 75 ppm Ag@NSP (c,g); 50 p
Ag/ZnO@NSP (d,h) for 6 h and 24 h, respectively. onclusions
Figure 14. SEM images (2500x) of E. faecalis on the surfaces of UAC50/TPGDA = (w/w = 70/30) w
various antibacterial agents: control (a,e); 50 ppm Ag@NSP (b,f); 75 ppm Ag@NSP (c,g); 50 pp
Ag/ZnO@NSP (d,h) for 6 h and 24 h, respectively. Figure 14. SEM images (2500x) of E. faecalis on the surfaces of UAC50/TPGDA = (w/w = 70/30) with
various antibacterial agents: control (a,e); 50 ppm Ag@NSP (b,f); 75 ppm Ag@NSP (c,g); 50 ppm
Ag/ZnO@NSP (d,h) for 6 h and 24 h, respectively. Figure 14. SEM images (2500x) of E. faecalis on the surfaces of UAC50/TPGDA = (w/w = 70/30) with
various antibacterial agents: control (a,e); 50 ppm Ag@NSP (b,f); 75 ppm Ag@NSP (c,g); 50 ppm
Ag/ZnO@NSP (d,h) for 6 h and 24 h, respectively. 16 of 19 Polymers 2020, 12, 482 Table 2. Antibacterial activity of the composites based on UAC50/TPGDA (w/w = 70/30) resins with
various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP. ble 2. Antibacterial activity of the composites based on UAC50/TPGDA (w/w = 70/30) resins with
rious concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP. Table 2. Antibacterial activity of the composites based on UAC50/TPGDA (w/w = 70/30) resins with
various concentrations of Ag@NSP, ZnO@NSP, or Ag/ZnO@NSP. 4. Conclusions The purpose of this study is to develop a new root canal sealer with a biocompatible resin based on
a macrodiol of polycarbonate (PCPO) that could be prepared through the utilization of carbon dioxide
as feedstock. With good biocompatibility and biostability, these carbonate-containing urethane acrylate
resins would be able to exhibit superior performance to the reference, polyether (PTMEG)-based resins
in the preparation of root canal obturation sealers. The successful incorporation of nanoparticles
immobilized on nanoscale platelets in the resin matrix resulted in the root canal obturation sealers
with satisfactory biocompatibility and antibacterial effect. As a result, the PCPO-based urethane
acrylate was selected to be the resin sealer matrix. Moreover, the incorporation of ZnONPs and AgNPs
simultaneously immobilized on silicate platelets into the PCPO-based urethane acrylates would not
only enhance the antibacterial activities, but also serve as a promoter in the antibacterial effect. Based
on the above, the UAC50/TPGDA (70/30 = w/w) resin with 50 ppm Ag/ZnO@NSP has a great potential
as an antibacterial root canal sealer. Author Contributions: Conceptualization, H.-H.C., R.-J.J., and C.-P.L.; methodology, Y.-T.T.; validation, H.-H.C. and C.-P.L.; data curation, S.-W.H. and Y.-F.K.; formal analysis, Y.-C.H., H.-H.C. and Y.-T.T.; writing—original
draft preparation, Y.-T.T.; writing—review and editing, C.-L.Y. and C.-H.W.; supervision, J.-J.L. and C.-P.L. All
authors have read and agreed to the published version of the manuscript. Funding: This work was financially supported by the “Advanced Research Center for Green Materials Science and
Technology” from The Featured Area Research Center Program within the framework of the Higher Education
Sprout Project by the Ministry of Education (108L9006) and the Ministry of Science and Technology in Taiwan
(MOST 108-3017-E-002-002, MOST 106-2221-E-002-189-MY3). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results. 3.5. Antibacterial Analysis Entry
Type of Composites (1)
Antibacterial Activity (2)
1
Ag@NSP-50
×
2
Ag@NSP-75
#
3
Ag@NSP-100
#
4
ZnO@NSP-100
×
5
ZnO@NSP-200
×
6
ZnO@NSP-300
×
7
ZnO@NSP-500
×
8
ZnO@NSP-1000
×
9
ZnO@NSP-2000
#
10
ZnO@NSP-3000
#
11
Ag/ZnO@NSP-10
×
12
Ag/ZnO@NSP-25
×
13
Ag/ZnO@NSP-50
#
14
Ag/ZnO@NSP-75
#
15
Ag/ZnO@NSP-100
#
(1): composites using UAC50 as polymer matrix; (2): “#“ indicating the inhibition of bacteria; “ד indicating the
growth of bacteria. Entry
Type of Composites (1)
Antibacterial Activity (2) 1.
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<i>In Situ</i> Identification of Paleoarchean Biosignatures Using Colocated Perseverance Rover Analyses: Perspectives for <i>In Situ</i> Mars Science and Sample Return
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Astrobiology
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In situ Identification of Paleoarchean Biosignatures
Using Colocated Perseverance Rover Analyses:
Perspectives for in situ Mars Science and Sample Return Keyron Hickman-Lewis,1,2 Kelsey R. Moore,3,4 Joseph J. Razzell Hollis,3 Michael L. Tuite,3
Luther W. Beegle,3 Rohit Bhartia,5 John P. Grotzinger,4 Adrian J. Brown,6
Svetlana Shkolyar,7,8 Barbara Cavalazzi,2,9 and Caroline L. Smith1,10 Keyron Hickman-Lewis et al., 2022; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of
the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited. y,
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Geology, Geophysics and Geochemistry Lab, NASA Goddard Space Flight Center, Greenbelt, Maryland, USA. NASA Jet Propulsion Laboratory, Pasadena, California, USA.
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California, USA
Photon Systems Inc., Covina, California, USA. p
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Dipartimento di Scienze Biologiche, Geologiche e Ambientali, Universita` di Bologna, Bologna, Italy.
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USA 1Department of Earth Sciences, The Natural History Museum, London, United Kingdom.
2 1Department of Earth Sciences, The Natural History Museum, London, United Kingdom.
2Dipartimento di Scienze Biologiche, Geologiche e Ambientali, Universita` di Bologna, Bologna, Italy.
3NASA Jet Propulsion Laboratory, Pasadena, California, USA.
4Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California, USA.
5Photon Systems Inc., Covina, California, USA.
6Plancius Research, Severna Park, Maryland, USA.
7Department of Astronomy, University of Maryland, College Park, Maryland, USA.
8Planetary Geology, Geophysics and Geochemistry Lab, NASA Goddard Space Flight Center, Greenbelt, Maryland, USA
9Department of Geology, University of Johannesburg, Johannesburg, South Africa.
10School of Geographical and Earth Sciences, University of Glasgow, Glasgow, United Kingdom. Research Article Open camera or QR reader and
scan code to access this article
and other resources online. ASTROBIOLOGY
Volume 22, Number 9, 2022
Mary Ann Liebert, Inc.
DOI: 10.1089/ast.2022.0018 ASTROBIOLOGY
Volume 22, Number 9, 2022
Mary Ann Liebert, Inc. DOI: 10.1089/ast.2022.0018 y
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partment of Geology, University of Johannesburg, Johannesburg, South Africa. y
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9Department of Geology, University of Johannesburg, Johannesburg, South Africa.
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Plancius Research, Severna Park, Maryland, USA. Plancius Research, Severna Park, Maryland, USA.
7Department of Astronomy, University of Maryland, College Park, Maryland, USA.
8Planetary Geology, Geophysics and Geochemistry Lab, NASA Goddard Space Flight Center, Greenbelt, Maryland, USA
9 p
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10School of Geographical and Earth Sciences, University of Glasgow, Glasgow, United Kingdom. Plancius Research, Severna Park, Maryland, USA.
7Department of Astronomy, University of Maryland, College Park, Maryland, USA.
8 1. Introduction Such discoveries would address
wider questions in astrobiology concerning the nature and
distribution of life in the Solar System and would help con-
strain the early planetary conditions, evolution, and the ha-
bitability of rocky bodies (see reviews in Lammer et al.,
2009; Cockell et al., 2016). I For astrobiological research, the analytical process con-
ducted by Perseverance seeks to identify rocks with high
biosignature preservation potential, both for morphological
and chemical relics of ancient life. Biosignature preserva-
tion potential varies markedly as a function of the forma-
tional and diagenetic histories of the host rock (Westall
et al., 2015, 2021; Hays et al., 2017); however, it is widely
accepted that fine-grained or finely crystalline rocks un-
dergoing early and rapid mineralization or cementation
create taphonomic windows of preservation for organo-
sedimentary structures, organic materials, (extra)cellular
remains, and in exceptional circumstances cellular body
fossils (e.g., Anderson et al., 2011; Wacey et al., 2014;
Westall et al., 2015; Hickman-Lewis et al., 2018a, 2020;
McMahon et al., 2018). Indeed, such lithofacies in Gale
crater feature the preservation of organic materials (Freis-
sinet et al., 2015; Eigenbrode et al., 2018). For this reason,
an array of likely fine-grained sediments in Jezero crater,
mostly associated with the western delta and its remnant
buttes, are considered essential MSR targets and will form a
crucial part of the sample cache gathered during the Per-
severance rover mission (Williford et al., 2018; Herd et al.,
2021; Mangold et al., 2021). ;
)
The Perseverance rover is equipped with a synergistic
suite of highly complementary instruments capable of
characterizing the geology of Jezero crater at scales from
kilometer to micron. Geomorphology and outcrop-scale
features are observed collaboratively by the cameras of
MastCam-Z and the SuperCam Remote Micro-Imager
(RMI) (Bell et al., 2021; Wiens et al., 2021), and subsurface
geology is defined by the RIMFAX ground-penetrating ra-
dar (Hamran et al., 2020). Mineralogy is determined re-
motely by MastCam-Z multispectral imaging, SuperCam
LIBS, VISIR, and Raman spot analyses (Bell et al., 2021;
Wiens et al., 2021). Proximal science is then conducted
using the rover arm, on which are mounted SHERLOC
(Scanning Habitable Environments with Raman and Lumi-
nescence for Organics and Chemicals), a deep-UV Raman
and fluorescence spectrometer, and PIXL (Planetary In-
strument for X-ray Lithochemistry), an X-ray fluorescence
spectrometer (Allwood et al., 2020; Bhartia et al., 2021). 1. Introduction enabling micron-scale correlations between rock fabric and
texture; organic content; elemental, chemical, and mineral
phase distributions. Once a high-priority sample is identified
through use of Mastcam-Z and SuperCam analysis, the
science team makes the decision whether to perform prox-
imity science. The rover is placed into position to analyze a
rock target with the proximity instruments and potentially
collect a core for eventual sample return. Prior to SHER-
LOC and PIXL analyses, the abrasion tool and gas Dust
Removal Tool (gDRT) create a fresh surface of 45 mm in
diameter and up to 13 mm deep. The abrading bit produces a
smooth surface with a relief of <250 microns to enable in-
focus maps and co-registration of SHERLOC and PIXL. Proximal science operations are also governed by limita-
tions in the rover slip potential, tip-tilt, and the geomor-
phology of the target. I
dentifying and characterizing traces of life through-
out the Solar System are among the fundamental chal-
lenges facing astrobiology. The search for life beyond Earth
is focused on Mars, which is uniquely compelling in that it
appears to have been habitable during its early history (the
Noachian–Hesperian period; 4.1–3.2 Ga), when it was war-
mer and stable bodies of water existed at its surface (Carr and
Head, 2010; Westall et al., 2013; Cockell, 2014; Grotzinger
et al., 2014, 2015). In this regard, the NASA Mars 2020
Perseverance rover mission has as its principal objectives the
reconstruction of Noachian–Hesperian paleoenvironments
at an ancient martian locality, Jezero crater (Isidis Planitia),
and the potential identification of fossil traces of life (bio-
signatures) in sedimentary strata therein (Williford et al.,
2018; Farley et al., 2020). Through sampling and caching a
diverse suite of geological samples within hermetically
sealed tubes, Mars 2020 also represents the first step toward
near-future international collaborations on Mars Sample
Return (MSR) (Farley et al., 2020; Grady, 2020; Meyer et al.,
2022). Potential organic- and biosignature-bearing sedi-
mentary rocks are high-priority targets for MSR since they
may answer major outstanding questions in astrobiology,
including (i) the nature of habitable niches early in Mars’
history; (ii) whether biosignatures and/or (pre)biotic organic
materials are preserved on Mars; and (iii) if life emerged on
Mars, for how long the biosphere persisted (Grotzinger et al.,
2014; Herd et al., 2021). HICKMAN-LEWIS ET AL. 2 Abstract The NASA Mars 2020 Perseverance rover is currently exploring Jezero crater, a Noachian–Hesperian locality
that once hosted a delta–lake system with high habitability and biosignature preservation potential. Persever-
ance conducts detailed appraisals of rock targets using a synergistic payload capable of geological character-
ization from kilometer to micron scales. The highest-resolution textural and chemical information will be
provided by correlated WATSON (imaging), SHERLOC (deep-UV Raman and fluorescence spectroscopy), and
PIXL (X-ray lithochemistry) analyses, enabling the distributions of organic and mineral phases within rock
targets to be comprehensively established. Herein, we analyze Paleoarchean microbial mats from the *3.42 Ga
Buck Reef Chert (Barberton greenstone belt, South Africa)—considered astrobiological analogues for a
putative ancient martian biosphere—following a WATSON–SHERLOC–PIXL protocol identical to that
conducted by Perseverance on Mars during all sampling activities. Correlating deep-UV Raman and fluo-
rescence spectroscopic mapping with X-ray elemental mapping, we show that the Perseverance payload has
the capability to detect thermally and texturally mature organic materials of biogenic origin and can highlight
organic–mineral interrelationships and elemental colocation at fine spatial scales. We also show that the
Perseverance protocol obtains very similar results to high-performance laboratory imaging, Raman spec-
troscopy, and mXRF instruments. This is encouraging for the prospect of detecting microscale organic-
bearing textural biosignatures on Mars using the correlative micro-analytical approach enabled by WATSON,
SHERLOC, and PIXL; indeed, laminated, organic-bearing samples such as those studied herein are con-
sidered plausible analogues of biosignatures from a potential Noachian–Hesperian biosphere. Were similar
materials discovered at Jezero crater, they would offer opportunities to reconstruct aspects of the early
martian carbon cycle and search for potential fossilized traces of life in ancient paleoenvironments. Such
samples should be prioritized for caching and eventual return to Earth. Key Words: Mars—Astrobiology—
Mars 2020—Biosignatures—Buck Reef Chert—Microbial mats. Astrobiology 22, xxx–xxx. 1 1. Introduction SHERLOC has two high-resolution imagers: the Wide-
Angle Topographic Sensor for Operations and eNgineering
(WATSON), which is similar to the MAHLI multi-scale
color camera on the Mars Science Laboratory rover, and the
Autofocus and Contextual Imager (ACI), a grayscale fixed
focus high-resolution imager for the colocation of laser-
induced spectra to rock textures. Through imaging, SHER-
LOC and PIXL are capable of conducting colocated
deep-ultraviolet (DUV) Raman and fluorescence mapping
and
X-ray
elemental
lithochemistry
analyses,
thereby g
The biogeochemistry of a potential fossil martian bio-
sphere remains a topic of scientific hypothesis; however,
some general expectations include that it would inhabit
aqueous environments, bear similarity to relatively ‘‘primi-
tive’’ bacteria and archaea (photoautotrophs, chemoautotrophs,
and/or chemolithotrophs), and exhibit polyextremophily and/or
polextremotolerance (Schulze-Makuch et al., 2008; Westall
et al., 2015; Cavalazzi et al., 2019). In this regard, the
earliest traces of life on Earth, dating to almost 3.5 Ga (e.g.,
Javaux, 2019 and references therein) are considered excel-
lent potential analogues for the types of ecosystems that may
once have colonized habitable environments on Noachian–
Hesperian Mars. Controversies surrounding the most ancient
traces of life (Brasier et al., 2006; Brasier and Wacey, 2012)
mean that such fossils also serve as a benchmark for the
stringency and rigor of analysis required to establish the
origins of putative martian biosignatures. Correlative microanalysis, that is, the systematic ac-
quisition of spatially colocated, mutually corroborative
data, has emerged as a compelling approach in the eval-
uation of the biogenicity, metabolic networks, and ta-
phonomy of microbial biosignatures in deep time (Brasier
et al., 2015; Hickman-Lewis et al., 2019; Wacey et al., CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 3 3 goals of both the mission and sample return: to detect
organic, chemical, and mineralogical biosignatures (dis-
tribution, composition, and texture) in their local pa-
leoenvironmental context. 2019; Cavalazzi et al., 2021) and is expected to be an
essential strategy in the search for life on Mars. WAT-
SON, SHERLOC, and PIXL can correlatively study min-
eralogical, molecular, and elemental distributions and
concentrations in rock targets on Mars; these detections
can be ascribed to specific phases and textures, and this
can be considered a planetary science equivalent of ter-
restrial correlative microanalysis. Herein, we analyze
three samples of microbial mat–rich chert from the 3.42
Ga Buck Reef Chert (Barberton greenstone belt, South
Africa; Fig. 1) using WATSON, SHERLOC, and PIXL
emulator instruments and a Perseverance-representative
analytical strategy. 1. Introduction Since the Buck Reef Chert contains
uncontroversial evidence of a Paleoarchean microbial
ecosystem and is characterized by exceptional preserva-
tion of fine-scale organic fabrics (Fig. 2) interpreted as
relics of productive phototrophic microbialites (Walsh,
1992; Tice and Lowe, 2004, 2006; Tice, 2009; Greco
et al., 2018), the microstructures within are excellent as-
trobiological analogues for the primitive microbial con-
sortia
possible
within
the
constraints
of
Noachian
habitability and biogeochemistry. This study aims (i) to
demonstrate the utility of correlated SHERLOC and PIXL
analyses on astrobiologically relevant materials for Mars
by obtaining a dataset similar to that acquired on Mars in
advance of sampling; (ii) to address the challenge of
biosignature detection and interpretation using in situ
approaches on Mars; and (iii) to briefly highlight some of
the similarities and differences between in situ and labo-
ratory datasets with reference to the Perseverance rover
mission and present, non-exhaustively, how complemen-
tary analyses might be conducted following sample return. Our findings thus have direct bearing on the major science cant biomass (Hofmann and Bolhar, 2007; Ledevin et al.,
2019). Bulk carbon isotope values from mat-rich chert range
from -36.9& to -20.1& (mean = -29.9&), implying bio-
logical processing with major contributions from photo-
synthetic
microbes
employing
hydrogen-based
carbon
fixation (Tice and Lowe, 2006b), though likely with 2.1. Geological context and microbial paleontology The studied samples (F10, F8, and F6) were obtained
from the BARB-3 core collected during the International
Continental Scientific Drilling Program (ICDP) Barberton
Drilling Project ‘‘Peering into the Cradle of Life’’ (Fig. 1),
the stratigraphy and microbial mat fabrics of which were
presented in Hofmann et al. (2013) and Greco et al. (2018). The Buck Reef Chert is the lowermost unit of the Kromberg
Formation (Onverwacht Group, Barberton greenstone belt)
and comprises four lithofacies: (i) a basal evaporite facies;
(ii) the lower platformal facies; (iii) the basinal facies; and
(iv) the upper platform facies, overlain by volcanic strata
of the Kromberg Formation (Tice and Lowe, 2004, 2006;
Hofmann et al., 2013). )
Detailed descriptions of the morphologies and preserva-
tion of Buck Reef Chert microbial mats show that these
mats comprise carbonaceous laminae that, at thin section-
scale, form thick sequences of crinkly–wavy carbonaceous
laminations intercalated with layers of fine volcanogenic
sediment and microcrystalline chemical chert (Tice and
Lowe 2006a; Greco et al., 2018). Individual laminations
comprise stacks of finely layered carbonaceous materials
consistent with biofilm growth (Fig. 2). Siderite and pyrite
are intimately associated with the microbial mats, most
likely due to heightened ambient concentrations of ferrous
iron at the time of deposition (Beukes, 2004; Tice and FIG. 1. Geological and stratigraphic context of the studied Buck Reef Chert samples. Simplified geological map of the
Barberton greenstone belt showing the location of the BARB-3 core, from which the studied samples were sourced; inset
shows location within southern Africa. Simplified stratigraphic log of the BARB-3 core showing the position of the studied
samples within the basinal (F6) and lower platformal (F8, F10) facies. Modified from Greco et al. (2018). FIG. 1. Geological and stratigraphic context of the studied Buck Reef Chert samples. Simplified geological map of the
Barberton greenstone belt showing the location of the BARB-3 core, from which the studied samples were sourced; inset
shows location within southern Africa. Simplified stratigraphic log of the BARB-3 core showing the position of the studied
samples within the basinal (F6) and lower platformal (F8, F10) facies. Modified from Greco et al. (2018). HICKMAN-LEWIS ET AL. 4 Buck Reef Chert microbial mat fabrics. (A) Overview image of thin section F10. (B) Overview ima
8. (C) Overview image of thin section F6. (D) Carbon-rich microbial mats in sample F10. (E) Siderite
l mats in sample F6. FIG. 2. Buck Reef Chert microbial mat fabrics. 2.2. SHERLOC-like deep-UV Raman
and fluorescence spectroscopy 2.2. SHERLOC-like deep-UV Raman
and fluorescence spectroscopy SHERLOC-like measurements were conducted using the
SHERLOC Brassboard instrument at the NASA Jet Pro-
pulsion Laboratory. The Brassboard is an optical analogue
of the SHERLOC flight instrument, adapted to work
under terrestrial conditions. Like the flight instrument, the
Brassboard generates spatially resolved spectral maps of
laser-induced fluorescence and Raman scattering, along with
co-boresighted high-resolution (10.1 mm/pixel) microscopy
images. The Brassboard uses a pulsed NeCu 248.6 nm laser as
the excitation source, firing 40 ms pulses at 80 Hz, focused to a
nominal working distance of 48mm using a SHERLOC-
equivalent f/7 objective lens. The focused laser spot is annular
in shape, with an outer diameter of 110 mm and an estimated
illuminated area of 7540 mm2, delivering an energy dose of 30–
45 J/m2 per pulse (Bhartia et al., 2021; Razzell Hollis et al. 2021). Fluorescence and Raman scattered photons from or-
ganics and minerals in the illuminated volume are collected by
the same objective lens, dispersed by a diffractive grating, and
detected by a multi-stage thermoelectrically cooled (-54C) ,
)
The thin sections shown in Fig. 2A–2C correspond to the
core samples shown in subsequent figures. Optical micros-
copy observations of these thin sections confirm that they
contain well-preserved microbial mats that are either
kerogen-rich (Fig. 2D) or siderite-replaced (Fig. 2E). Of the
studied samples, F10 and F8 are dominated by kerogen-rich
microbial mats, whereas F6 contains a greater amount of
siderite in association with mats. Samples for SHERLOC-
and PIXL-like analyses were prepared as roughly cut core
fragments (e.g., Fig. 3A) as a broad approximation of the
abrasion process conducted by the Perseverance rover. Samples were not polished after cutting, and residual dam-
age to the analyzed surface was left unmitigated; this is
analogous to the imperfect nature of abrasion patches made
by Perseverance, where chatter marks and other abrasion- FIG. 3. Overview of SHERLOC analyses conducted on sample F10. (A) Simulated WATSON image of the sample
showing the locations of SHERLOC survey scans (S1–2) and detail scans (D1–4). (B) Full SHERLOC spectra (Raman plus
DUV fluorescence) for each region of interest. (C) Raman spectra for each region of interest. analyses conducted on sample F10. (A) Simulated WATSON image of the sample
survey scans (S1–2) and detail scans (D1–4). (B) Full SHERLOC spectra (Raman plus
of interest. (C) Raman spectra for each region of interest. FIG. 3. CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 5 related surface phenomena mildly disrupt rock fabrics and
textures. Prior to analysis, samples were cleaned with iso-
propyl alcohol and rinsed with ultrapure water. contributions from secondary heterotrophic reworking of
biomass. The mats also exhibit plastic deformation indica-
tive of a primary EPS-bound composition, as well as ero-
sional phenomena, such as roll-ups and tear-ups, consistent
with the reworking of microbial mats under wave and cur-
rent activity (Simonson and Carney, 1999; Tice and Lowe,
2004). Combined, this evidence is consistent with domi-
nantly photosynthetic mat growth under variable energy
regimes that encouraged both growth and degradation of
mats (cf. Tice and Lowe, 2004; Tice, 2009; Hickman-Lewis
et al., 2016, 2018b). 2.1. Geological context and microbial paleontology (A) Overview image of thin section F10. (B) Overview image o
section F8. (C) Overview image of thin section F6. (D) Carbon-rich microbial mats in sample F10. (E) Siderite-re
microbial mats in sample F6. Buck Reef Chert microbial mat fabrics. (A) Overview image of thin section F10. (B) Overview ima FIG. 2. Buck Reef Chert microbial mat fabrics. (A) Overview image of thin section F10. (B) Overview image of thin
section F8. (C) Overview image of thin section F6. (D) Carbon-rich microbial mats in sample F10. (E) Siderite-replaced
microbial mats in sample F6. Lowe, 2004). Trace and rare earth element studies through
black and white banded cherts in the Buck Reef Chert imply
that the paleoenvironment was either a near-continental
platform (Tice and Lowe, 2004) or an epeiric sea (Grosch
et al., 2011) nourished by inputs from hydrothermal effluent
that presumably contributed to the development of signifi- cant biomass (Hofmann and Bolhar, 2007; Ledevin et al.,
2019). Bulk carbon isotope values from mat-rich chert range
from -36.9& to -20.1& (mean = -29.9&), implying bio-
logical processing with major contributions from photo-
synthetic
microbes
employing
hydrogen-based
carbon
fixation (Tice and Lowe, 2006b), though likely with 3.1. In situ Perseverance-like protocol In this study, we aimed to closely replicate the analysis of
abrasion patches by the Perseverance rover. It is important
to note that each abrasion patch on Mars is analyzed in a
slightly different manner due to the inherent limitations of
rover operations (time of day chosen for light conditions and
detector temperature, state of rover charge, availability of
high-priority targets, and data volume restrictions), and thus
the concept of routine analysis used herein should be taken
as notional. Nonetheless, the strategy of analysis applied
herein bears crucial similarities to Perseverance operations
as follows: (i) instrumental acquisition parameters were kept
identical or highly similar to those of SHERLOC and PIXL
operating on Mars; (ii) a small number of regions of interest
were studied, reflecting the limited time and power available
for such analytical procedures on Mars; (iii) SHERLOC
conducts nested analyses, starting with large-area survey scans
before performing detail analyses on points of interest; and
(iv) SHERLOC and PIXL can conduct colocated analyses
using the same analytical footprint. As such, the datasets re-
ported herein can be considered representative of those we
would expect from similar geological materials on Mars. Data processing was conducted by using a combination of
custom software and python scripts, utilizing the SciPy,
NumPy, and LMFIT packages (Jones et al., 2001; Van der
Walt et al., 2011; Newville et al., 2014). Cosmic rays were
removed automatically using a pre-established algorithm
(Uckert et al., 2019); then Raman and fluorescence spectra
were recombined after trimming regions containing only dark
noise. For Raman peak analysis, the background signal was
removed by subtraction of a polynomial baseline between 0
and 4000cm-1; then any Raman peaks more than >3 times the
background noise were fitted using Gaussian functions to
determine their precise positions and intensities. The 600–
800 cm-1 region was ignored due to overlap with the second-
ary laser emission line at 252.93nm (692cm-1). Spectral
intensity maps were generated by taking the summed intensities
of three spectral bands in each spectrum and assigning those
intensities to the R, G, B values of the corresponding pixel,
normalized to the 2% and 98% percentiles for all three bands
across the entire map. For fluorescence maps, R, G, B values
represent the 335–345 nm, 305–315 nm, and 275–285 nm
bands, respectively; for Raman maps, R, G, B values represent
the 1500–1700 cm-1, 900–1100cm-1, and 425–475 cm-1
bands. 2.2. SHERLOC-like deep-UV Raman
and fluorescence spectroscopy Overview of SHERLOC analyses conducted on sample F10. (A) Simulated WATSON image of the sample
showing the locations of SHERLOC survey scans (S1–2) and detail scans (D1–4). (B) Full SHERLOC spectra (Raman plus
DUV fluorescence) for each region of interest. (C) Raman spectra for each region of interest. HICKMAN-LEWIS ET AL. 6 commercial Synapse Symphony II (Horiba) housing a 42-10
BIUV 512 · 2048 pixel CCD from e2v (CCD identical to
SHERLOC). Similar to the SHERLOC flight spectrometer, the
Raman and fluorescence regions are separated on the detector
through curved projection of the spectrum onto the CCD for
overall instrument compactness and enabled better separation
of the weaker Raman scattered photons from the stronger
fluorescence photons (Razzell Hollis et al., 2021). The Raman
measurements span 246.8–317.8 nm (-290 to 8760 cm-1), and
fluorescence measurements span 296.2–357.1 nm (6470 to
12200cm-1). The spectral calibration has a mean absolute error
of 0.018– 0.010nm (2.7– 1.6 cm-1) in the Raman region, as-
sessed using spectral standards of acetonitrile, powdered cal-
cite, highly oriented pyrolytic graphite, and a secondary laser
emission line at 252.93 nm (Razzell Hollis et al., 2021). commercial Synapse Symphony II (Horiba) housing a 42-10
BIUV 512 · 2048 pixel CCD from e2v (CCD identical to
SHERLOC). Similar to the SHERLOC flight spectrometer, the
Raman and fluorescence regions are separated on the detector
through curved projection of the spectrum onto the CCD for
overall instrument compactness and enabled better separation
of the weaker Raman scattered photons from the stronger
fluorescence photons (Razzell Hollis et al., 2021). The Raman
measurements span 246.8–317.8 nm (-290 to 8760 cm-1), and
fluorescence measurements span 296.2–357.1 nm (6470 to
12200cm-1). The spectral calibration has a mean absolute error
of 0.018– 0.010nm (2.7– 1.6 cm-1) in the Raman region, as-
sessed using spectral standards of acetonitrile, powdered cal-
cite, highly oriented pyrolytic graphite, and a secondary laser
emission line at 252.93 nm (Razzell Hollis et al., 2021). partment. All analyses were conducted at 2 mbar pressure, both
because conducting X-ray fluorescence (XRF) under low
pressure to vacuum conditions allows for better detection of
lighter elements and because this closely approximates PIXL-
style analyses conducted on Mars where surface pressures are
*6 mbar. All analyses were conducted using two silicon drift
detectors with X-ray tube voltages of 50 kV and currents of
400 mA with no primary beam filters. 2.2. SHERLOC-like deep-UV Raman
and fluorescence spectroscopy Detectors were set to
measure 90 keV and 130 kcps. Large area maps were analyzed
using high-resolution mapping with a 20 mm spot size. Regions
of interest (colocated with SHERLOC-like microspectroscopy)
and line scans wereanalyzed usinga 120mm spot size to closely
replicate PIXL analyses conducted in situ on Mars. Data were
analyzed using Bruker M4 Tornado software. Each sample was characterized following a similar
methodology to SHERLOC operations in situ on Mars. First,
a survey scan (36 · 36 points, 7 · 7 mm, 100 pulses per
point, 200 mm spacing between points) was performed to
rapidly map strong Raman and fluorescence signals across a
large area. Then, one or more regions of interest were se-
lected based on visible morphology and fluorescence, and a
detail scan (10 · 10 points, 1 · 1 mm, 800 pulses per point,
100 mm spacing between points) was performed in each
region to maximize signal from lower-yield Raman scat-
tering and obtain a representative Raman spectrum at higher
spatial resolution. High-resolution laboratory maps of 4800
points across a larger area (16 · 3 mm) with 100 mm spacing,
at 100 pulses per point, were also acquired. 3.1.1. Sample F10 WATSON-like imaging. Images obtained of the core
sample and regions of interest (e.g., Figs. 3A, 4A, 4E) show
a regularly laminated structure comprising thin dark-toned
layers intercalated with thin and thick light-toned layers. The dark-toned layers are characterized by a relatively ho-
mogeneous appearance, whereas the light-toned layers fea-
ture
some
apparent
granularity. Some
folding
and
undulatory character is exhibited by laminations in the lower
portion of the sample (Fig. 3A). SHERLOC-like deep-UV Raman and fluorescence spec-
troscopy. Two SHERLOC survey scans (7 · 7 mm) were
acquired to characterize millimeter-scale organic variability,
within which four SHERLOC detail scans (1 · 1 mm) were
obtained to reveal fine-scale heterogeneity (Figs. 3 and 4). DUV fluorescence is generally dominated by a peak cen-
tered on *305 nm, except detail 3, which is dominated by a
peak at *270 nm (Fig. 3B). Fluorescence mapping high-
lights three signatures with maxima at *300, *340, and
*270 nm;
the
340 nm
signal
shows
correlation
with 3.1. In situ Perseverance-like protocol Quartz/organic ratio maps were generated by dividing
the quartz signal at 450 cm-1 by the summed signal of quartz at
450 cm-1 and organic material at 1600cm-1. A dip in fluo-
rescence signal was observed at 297 nm in all spectra acquired
on the Brassboard and attributed to an instrumental artifact. 2.3. PIXL-like elemental mapping PIXL-like analyses were conducted on a Bruker M4 Tornado
mX-Ray Fluorescence spectrometer instrument at the California
Institute of Technology Geology and Planetary Sciences De- CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 7 FIG. 4. Simulated SHERLOC analyses of sample F10. (A–D) WATSON-like image and fluorescence, Raman, and
Raman quartz/organic ratio maps for survey scan S1. (E–H) WATSON-like image and fluorescence, Raman, and Raman
quartz/organic ratio maps for survey scan S2. (I–J) Raman and Raman quartz/organic ratio maps for SHERLOC details 1
and 2; see (A) for localization. (K–L). Raman and Raman quartz/organic ratio maps for SHERLOC details 3 and 4; see (E)
for localization. See legend for color code used in this and all subsequent DUV Raman and fluorescence maps. Scale shown
in (H) is applicable to all quartz/organic ratio maps. FIG. 4. Simulated SHERLOC analyses of sample F10. (A–D) WATSON-like image and fluorescence, Raman, and
Raman quartz/organic ratio maps for survey scan S1. (E–H) WATSON-like image and fluorescence, Raman, and Raman
quartz/organic ratio maps for survey scan S2. (I–J) Raman and Raman quartz/organic ratio maps for SHERLOC details 1
and 2; see (A) for localization. (K–L). Raman and Raman quartz/organic ratio maps for SHERLOC details 3 and 4; see (E)
for localization. See legend for color code used in this and all subsequent DUV Raman and fluorescence maps. Scale shown
in (H) is applicable to all quartz/organic ratio maps. laminations (Fig. 4). Detail 2 may show evidence for sp2-
bonded carbon spectrum in the form of a UV edge of a
feature tail that peaks beyond 360 nm (Fig. 3B). Survey 2 and detail 3 show that the 270 nm DUV fluores-
cence and 1350cm-1 Raman signals are strongly colocated
with a cross-cutting vein, which is also enriched in quartz (at
450 cm-1) relative to the surrounding matrix (Figs. 3, 4E–4H). The corresponding Raman spectra show peaks for quartz
(450cm-1) and carbonaceous materials (the carbon G-band at
*1600cm-1) (Fig. 3C). A weak peak at *1080cm-1 is in-
terpreted as carbonate; this wavenumber is low relative to the
dominant Raman signal of calcite (*1089cm-1) and dolomite
(*1097–1099 cm-1) and most likely arises from siderite. We
acknowledge an additional complication in the interpretation
of Fe carbonates: the overlap between a carbonate peak at
1080cm-1 and minor peaks of quartz at 1084 and 1165cm-1. 2.3. PIXL-like elemental mapping Without detection of other carbonate modes, this peak might
be considered most consistent with quartz, but when combined
with detection of Fe by the PIXL analogue instrument (and its
insensitivity to C, see below), Fe carbonate is the most plau-
sible explanation. The spectra within detail 3 also exhibit the
carbon D-band at *1350cm-1. Raman spectroscopic map-
ping highlights the dominance of the G-band signature
throughout the sample (Fig. 4), which shows a broad correla-
tion with the trend and spatial distribution of dark-toned layers,
that is, their intercalation with thin quartz laminae. PIXL-like elemental mapping. Two PIXL scans were
acquired,
one
7 · 7 mm
full
scan
overlapping
with
SHERLOC survey scan 2 and a line scan through multiple
layers of the sample (Fig. 5). Si concentrations are in-
versely correlated with Fe and Mn, while Fe and Mn are
strongly correlated throughout the sample. Ca was de-
tected in trace amounts in the Fe-Mn layers, while S, Al,
and Ti were detected in trace amounts but with no clear
distributions. CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 9 9 with fluorescence; however, one dark lamination adjacent to
the underlying light quartz-rich layer is clearly defined in
both maps (Fig. 7A–7D). In survey 2, a clearer correlation
between the Raman and fluorescence signals is observed;
the Raman organic signal is particularly strongly correlated
with visible dark-toned laminations (Fig. 7E–7H). SHERLOC-like deep-UV Raman and fluorescence spec-
troscopy. Three SHERLOC survey scans and five SHER-
LOC detail scans were obtained (Figs. 6 and 7), within
which fluorescence is centered on a dominant peak at
*305 nm and a weaker peak at *340 nm (Fig. 6B). In
survey 1, distinct fluorescence signals are associated with
primary laminations and secondary damage, probably due to
cutting or abrasion of the surface; the former is better-
defined than the latter (Fig. 7A–7D). Survey 2 also shows a
moderate
correlation
between
fluorescence
signals
at
305 nm and two dark-toned laminations (Fig. 7E–7H). Two detail scans (3 and 4) within survey scan 2 confirm
the correlation of the orange-red fluorescence hotspot
(which has a maximum at 305 nm but relatively strong
signal >320 nm) with a quartz-dominated Raman signal
and the yellow-green fluorescence hotspot (which is
dominated by the peak at 305 nm) with an organic-rich
Raman signal. The corresponding Raman measurements show the car-
bonaceous material G-band, which is enriched in the dark-
toned layers, and quartz, which is enriched in the lighter-
toned intercalated layers (Figs. 6 and 7). A weak peak at
*1080 cm-1 is again interpreted as carbonate, likely sider-
ite. In survey 1, the Raman signal shows limited correlation PIXL-like elemental mapping. Three PIXL scans were
acquired, two 7 · 7 mm full scans overlapping with SHER-
LOC survey scans 1 and 2 (Fig. 8A–8E, 8J–8N) and a line . 7. Simulated SHERLOC analyses of sample F8. (A–D) WATSON-like image and fluorescence, Raman, and Raman
tz/organic ratio maps for survey scan S1. (E–H) WATSON-like image and fluorescence, Raman, and Raman quartz/
nic ratio maps for survey scan S2. (I–L) WATSON-like image and fluorescence, Raman, and Raman quartz/organic
maps for survey scan S3. (M) Raman and Raman quartz/organic ratio maps for SHERLOC detail 1; see (A) for
ization. (N–O). Raman and Raman quartz/organic ratio maps for SHERLOC details 3 and 4; see (E) for localization. Q). Raman and Raman quartz/organic ratio maps for SHERLOC details 5 and 6; see (I) for localization. Scale shown in
is applicable to all quartz/organic ratio maps. 3.1.2. Sample F8 WATSON-like imaging. Similar to F10, sample F8 ex-
hibits regions of intercalated undulatory dark-toned and
light-toned laminations; however, thick light-toned lamina-
tions between the darker regions of this sample exhibit a
more homogeneous texture consistent with a finer degree of
granularity. FIG. 5. Simulated PIXL analyses of sample F10. (A) WATSON-like image showing the location of PIXL scans. (B–E)
PIXL-like scan showing the distributions of Si, Fe, and Mn. (F–I) PIXL-like line scan showing the distributions of Si, Fe,
and Mn. FIG. 5. Simulated PIXL analyses of sample F10. (A) WATSON-like image showing the location of PIXL scans. (B–E)
PIXL-like scan showing the distributions of Si, Fe, and Mn. (F–I) PIXL-like line scan showing the distributions of Si, Fe,
and Mn. FIG. 6. Overview of SHERLOC analyses for sample F8. (A–B) Simulated WATSON images of two faces of the
sample showing the locations of SHERLOC survey scans (S1–3) and detail scans (D1–6). (C) Full SHERLOC spectra of
each region of interest. (D) Raman spectra of each region of interest. Note the absence of the D-band in all Raman
spectra. FIG. 6. Overview of SHERLOC analyses for sample F8. (A–B) Simulated WATSON images of two faces of the
sample showing the locations of SHERLOC survey scans (S1–3) and detail scans (D1–6). (C) Full SHERLOC spectra of
each region of interest. (D) Raman spectra of each region of interest. Note the absence of the D-band in all Raman
spectra. 8 CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES FIG. 7. Simulated SHERLOC analyses of sample F8. (A–D) WATSON-like image and fluorescence, Raman, and Raman
quartz/organic ratio maps for survey scan S1. (E–H) WATSON-like image and fluorescence, Raman, and Raman quartz/
organic ratio maps for survey scan S2. (I–L) WATSON-like image and fluorescence, Raman, and Raman quartz/organic
ratio maps for survey scan S3. (M) Raman and Raman quartz/organic ratio maps for SHERLOC detail 1; see (A) for
localization. (N–O). Raman and Raman quartz/organic ratio maps for SHERLOC details 3 and 4; see (E) for localization. (P–Q). Raman and Raman quartz/organic ratio maps for SHERLOC details 5 and 6; see (I) for localization. Scale shown in
(D) is applicable to all quartz/organic ratio maps. 10 HICKMAN-LEWIS ET AL. . Simulated PIXL analyses of sample F8. (A) WATSON-like image showing the location of PIXL scans on f
PIXL-like scan showing the distributions of Si, Fe, and Mn. (F–I) PIXL-like line scan showing the distributions
d Mn. (J) WATSON-like image showing the location of PIXL scans on face 2. (K–N) PIXL-like scan showin
utions of Si, Fe, and Mn. f
l F8 (A) WATSON lik i
h
i
th l
ti
f PIXL FIG. 8. Simulated PIXL analyses of sample F8. (A) WATSON-like image showing the location of PIXL scans on fac
(B–E) PIXL-like scan showing the distributions of Si, Fe, and Mn. (F–I) PIXL-like line scan showing the distributions o
Fe, and Mn. (J) WATSON-like image showing the location of PIXL scans on face 2. (K–N) PIXL-like scan showing
distributions of Si, Fe, and Mn. FIG. 8. Simulated PIXL analyses of sample F8. (A) WATSON-like image showing the location of PIXL scans on face 1. (B–E) PIXL-like scan showing the distributions of Si, Fe, and Mn. (F–I) PIXL-like line scan showing the distributions of Si,
Fe, and Mn. (J) WATSON-like image showing the location of PIXL scans on face 2. (K–N) PIXL-like scan showing the
distributions of Si, Fe, and Mn. FIG. 8. Simulated PIXL analyses of sample F8. (A) WATSON-like image showing the location of PIXL scans on face 1. (B–E) PIXL-like scan showing the distributions of Si, Fe, and Mn. (F–I) PIXL-like line scan showing the distributions of Si,
Fe, and Mn. (J) WATSON-like image showing the location of PIXL scans on face 2. (K–N) PIXL-like scan showing the
distributions of Si, Fe, and Mn. is dominated by dark-toned material. CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES One light-toned
layer and one gray-toned layer are conformably in-
tercalated
with
the
bulk
rock
fabric. Several
pale
cross-cutting veins also obliquely disrupt the primary
fabric; however, the sample is otherwise relatively
featureless. scan passing through a thick light-toned layer and thick
dark-toned layer (Fig. 8F–8I). Si is anti-correlated with Fe
and Mn, while Fe and Mn are strongly correlated. Fe-Mn
laminae are also found to have small amounts of Ca, while
Al and Ti occur in trace quantities as discrete spots
throughout the analyzed regions. SHERLOC-like deep-UV Raman and fluorescence spec-
troscopy. Three survey scans and six detail scans were ob-
tained, all of which exhibit weak fluorescence at *310 nm. In
all regions, fluorescence is strongest along the fractures, for 3.1.3. Sample F6 The corresponding Raman scans show a weak organic
signal, quartz, and a more prominent peak at *1080 cm-1
in regions with weak quartz signals that is again interpreted
as carbonate, most likely siderite (Figs. 9 and 10). Unlike
samples F10 and F8, the weak organic and quartz signals
exhibit no systematic variation within the relatively ho-
mogeneous matrix, correlating instead with layering or
fractures. The corresponding Raman scans show a weak organic
signal, quartz, and a more prominent peak at *1080 cm-1
in regions with weak quartz signals that is again interpreted
as carbonate, most likely siderite (Figs. 9 and 10). Unlike
samples F10 and F8, the weak organic and quartz signals
exhibit no systematic variation within the relatively ho-
mogeneous matrix, correlating instead with layering or
fractures. 3.2.1. Sample F10. High-resolution XRF mapping of the
whole sample surface at a spatial resolution of 30 mm
(Fig. 12) confirmed the spatial correlation of Fe and Mn and
the inverse relationship of Si to Fe and Mn. In addition,
numerous S-rich laminae and S-rich particles were identi-
fied, as well as trace Al, Ca, Ti, and Zn. PIXL-like elemental mapping. One 7 · 7 mm PIXL scan
was acquired, overlapping with SHERLOC survey scan 4
(Fig. 11). As in samples F10 and F8, Si is anti-correlated
with Fe and Mn, while Fe and Mn are strongly correlated,
Mn being enriched in certain layers (Fig. 11E). 3.2.2. Sample
F8. High-resolution,
large-area
Raman
mapping through a dark-toned region (Fig. 13A–13C)
showed a clear arrangement of layers with different inten-
sities of organic signal, the highest intensities localized
within the darkest layers, as well as several small quartz-rich
domained distributed throughout the region. These maps
also show that the light-toned blocks that sandwich the dark
region consist mainly of quartz. Mapping the Raman in-
tensity ratio between quartz and organics demonstrates that
mid-toned layers have similar relative concentrations of
organic material and quartz; this suggests that the Raman
signal is being modulated by variation in laser attenuation 3.1.3. Sample F6 WATSON-like imaging. In contrast to samples F10
and F8, sample F6 has a faintly laminated texture and CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 11 FIG. 9. Overview of SHERLOC analyses for sample F6. (A) Simulated WATSON image of the sample showing the
locations of SHERLOC survey scans (S1–4) and detail scans (D1–6). One pound coin for scale. (B) Full SHERLOC spectra
of each region of interest. (C) Raman spectra of each region of interest. Note the absence of the D-band in all Raman
spectra. s for sample F6. (A) Simulated WATSON image of the sample showing the
) and detail scans (D1–6). One pound coin for scale. (B) Full SHERLOC spectra
ctra of each region of interest. Note the absence of the D-band in all Raman FIG. 9. Overview of SHERLOC analyses for sample F6. (A) Simulated WATSON image of the sample showing the
locations of SHERLOC survey scans (S1–4) and detail scans (D1–6). One pound coin for scale. (B) Full SHERLOC spectra
of each region of interest. (C) Raman spectra of each region of interest. Note the absence of the D-band in all Raman
spectra. Raman spectroscopy and mXRF were undertaken to confirm
the results of emulated SHERLOC and PIXL analyses and
provide higher-quality data. The higher resolution and im-
proved detection capabilities of laboratory instruments rel-
ative
to
miniaturized
rover
instruments
are
more
representative of the level of analysis with which core
samples may be studied following MSR. Raman spectroscopy and mXRF were undertaken to confirm
the results of emulated SHERLOC and PIXL analyses and
provide higher-quality data. The higher resolution and im-
proved detection capabilities of laboratory instruments rel-
ative
to
miniaturized
rover
instruments
are
more
representative of the level of analysis with which core
samples may be studied following MSR. example, along the edges of the large fracture in survey 1 and
throughoutthecross-cuttingfracturesinotherregionsofinterest
(Figs. 9 and 10). Dark regions in this sample exhibit relatively
limited fluorescence. Detail scans confirm the relative hetero-
geneity of regions; that is, quartz-rich regions have minimal
organic signal, and organic regions have minimal quartz signal. example, along the edges of the large fracture in survey 1 and
throughoutthecross-cuttingfracturesinotherregionsofinterest
(Figs. 9 and 10). Dark regions in this sample exhibit relatively
limited fluorescence. Detail scans confirm the relative hetero-
geneity of regions; that is, quartz-rich regions have minimal
organic signal, and organic regions have minimal quartz signal. 3.2. High-resolution and large-area laboratory
characterization The design and operating conditions of the SHERLOC
and PIXL instruments on board the rover introduce limita-
tions in terms of scan area, duration, and spatial resolution. Complementary laboratory analyses using high-resolution HICKMAN-LEWIS ET AL. 12 FIG. 10. Simulated SHERLOC analyses of sample F6. (A–D) WATSON-like image and fluorescence, Raman, and
Raman quartz/organic ratio maps for survey scan S1. (E–H) WATSON-like image and fluorescence, Raman, and Raman
quartz/organic ratio maps for survey scan S3. (I–L) WATSON-like image and fluorescence, Raman, and Raman quartz/
organic ratio maps for survey scan S4. (M–N) Raman and Raman quartz/organic ratio maps for SHERLOC details 1 and 2;
see (A) for localization. (O–P). Raman and Raman quartz/organic ratio maps for SHERLOC details 3 and 4; see (E) for
localization. (Q–R). Raman and Raman quartz/organic ratio maps for SHERLOC details 5 and 6; see (I) for localization. Scale shown in (H) is applicable to all quartz/organic ratio maps. FIG. 10. Simulated SHERLOC analyses of sample F6. (A–D) WATSON-like image and fluorescence, Raman, and
Raman quartz/organic ratio maps for survey scan S1. (E–H) WATSON-like image and fluorescence, Raman, and Raman
quartz/organic ratio maps for survey scan S3. (I–L) WATSON-like image and fluorescence, Raman, and Raman quartz/
organic ratio maps for survey scan S4. (M–N) Raman and Raman quartz/organic ratio maps for SHERLOC details 1 and 2;
see (A) for localization. (O–P). Raman and Raman quartz/organic ratio maps for SHERLOC details 3 and 4; see (E) for
localization. (Q–R). Raman and Raman quartz/organic ratio maps for SHERLOC details 5 and 6; see (I) for localization. Scale shown in (H) is applicable to all quartz/organic ratio maps. by the rock rather than concentration. This is consistent with
the XRF observation of Fe carbonates in the lower-signal
layers, since Fe-rich minerals are known to strongly absorb
DUV light (Cloutis et al., 2008; Shkolyar et al., 2018;
Carrier et al., 2019). intensity is lower in the dark-toned region—possibly due to
the presence of Fe carbonate (cf. XRF maps in Fig. 14D). The more granular mid-toned layer (upper right of Fig. 14A)
appears identical to the dark-toned region. A fracture cutting
through mid-toned layer is slightly enriched in quartz and
was possibly introduced at the same time as the quartz-rich
layer. High-resolution XRF mapping of the whole sample sur-
face (Fig. 13D, 13E) again showed correlation between Fe
and Mn, and inverse correlation between Si and Fe/Mn. 3.2. High-resolution and large-area laboratory
characterization Some Al-rich laminae were identified, and trace Ca and Ti
was noted throughout the sample. High-resolution XRF mapping of the whole sample sur-
face showed overlap between very strong signals of Fe and
Mn, which anticorrelate with Si. Although Si is present
throughout the sample, its relatively high concentrations
with the light-toned region (vein) are most visually appar-
ent. Some Al and trace Ca and Ti were also observed
throughout the sample. 3.2.3. Sample
F6. High-resolution,
large-area
Raman
mapping of a contact between light- and dark-toned layers
(Fig. 14A–14C) showed that the light-toned layer is gener-
ally rich in the quartz signal, while the organic signal is
more apparent in the dark-toned region; overall, the signal In summary, throughout the three samples analyzed,
strikingly similar signals and identical distributions in FIG. 11. Simulated PIXL analyses of sample F6. (A) WATSON-like image showing the location of the PIXL scan. One
pound coin for scale. (B–E) PIXL-like scan showing the distributions of Si, Fe, and Mn. FIG. 11. Simulated PIXL analyses of sample F6. (A) WATSON-like image showing the location of the PIXL scan. One
pound coin for scale. (B–E) PIXL-like scan showing the distributions of Si, Fe, and Mn. FIG. 12. Optical images and laboratory XRF scans for two faces of sample F10. The face analyzed in (A) corresponds to
Figs. 3–5, while the face in (B) was not analyzed using the Perseverance-like protocol. FIG. 12. Optical images and laboratory XRF scans for two faces of sample F10. The face analyzed in (A) corresponds to
Figs. 3–5, while the face in (B) was not analyzed using the Perseverance-like protocol. 13 HICKMAN-LEWIS ET AL. 14 FIG. 13. (A–C) Optical image, laboratory Raman scan, and quartz-organic ratio map for a whole face of sample F8. (D–
E) Optical images and laboratory XRF scans for two faces of sample F8. The face analyzed corresponds to Figs. 6–8. FIG. 13. (A–C) Optical image, laboratory Raman scan, and quartz-organic ratio map for a whole face of sample F8. (D–
E) Optical images and laboratory XRF scans for two faces of sample F8. The face analyzed corresponds to Figs. 6–8. organic and elemental lithochemistry were observed, vali-
dating the results obtained by the SHERLOC and PIXL
emulators. alogies that might be anticipated to occur at the mildly acidic
water–rock interfaces of early martian lakes (Agangi et al.,
2021). 3.2. High-resolution and large-area laboratory
characterization It is further of note that the studied samples were
sourced from core samples rather than outcrops and have
therefore been spared the vicissitudes of alteration and deg-
radation at Earth’s surface and should be expected to retain
primary lithochemical characteristics. 4.1. Mars analogy of the Buck Reef Chert There is no perfect terrestrial analogue for planetary explo-
ration. Due to variably significant and/or subtle differences in
chemistry, mechanical properties, and paleoenvironmental set-
ting, there are always differences between a proposed analogue
sample and the extraterrestrial material to which it refers (Fou-
cher et al., 2021). Rather than assuming ideal analogy, samples
chosen as analogues should be defined as such on the basis of the
characteristics in which they are analogous to the target object
and recognized for inevitable shortcomings; such materials are
termed ‘‘functional analogues’’ (Foucher et al., 2021). p
y
There are nonetheless shortcomings to the analogy of the
Buck Reef Chert. Firstly, the thermal maturity of the sample
(lower greenschist grade metamorphism, as is the case for the
entirety of the Barberton greenstone belt) is greater than that
expected at the surface of Mars. As such, the biogeochemical
signals obtained from our analyses may be considered a
‘‘worst-case scenario’’ for biosignature detection on Mars. Secondly, while silica-rich compositions have been identified
in martian rocks (Squyres et al., 2008; Ruff and Farmer, 2016;
Tarnas et al., 2019), it is unclear whether these materials are
cherts sensu stricto. Despite these caveats, for the reasons
outlined we consider the BARB-3 core Buck Reef Chert
samples to be appropriate astrobiological analogues for Mars. We consider the Buck Reef Chert to be an appropriate
martian astrobiological analogue for the following reasons: (i)
Its Paleoarchean age is approximately contemporaneous with
the Noachian–Hesperian period during which Mars is con-
sidered to have been habitable; thus the biosignatures within
can be expected to have attained a similar, but not greater, level
of evolutionary sophistication than anticipated biosignatures
on Mars (cf. Westall et al., 2015). (ii) Its mechanism of pres-
ervation by silicification bears similarity to outcrops of pro-
posed astrobiological significance on Mars, among which
silica-rich deposits around Jezero crater detected in orbital data
(Tarnas et al., 2019) and along the Mars Exploration Rover
traverses (Squyres et al., 2008; Ruff and Farmer, 2016) are key
examples. (iii) Its carbonate-rich mineralogy and the preva-
lence of siderite parallel both key outcrops of interest at the
Jezero crater margins to be investigated by Perseverance in the
near future (Horgan et al., 2020) and the carbonate miner- 4.2. Biosignature detection Our emulated WATSON–SHERLOC–PIXL analyses dem-
onstrate that correlation among imaging, organic character-
ization, and the mapped distribution of organic, mineral, and
elemental signatures enables the detection of organic-rich
laminated fabrics within a geologically (thermally and diage-
netically) mature silica–carbonate matrix. Whether this con-
stitutes a definitive biosignature detection is challenging to
evaluate at this stage since the unambiguous identification of
a biosignature should rely upon numerous high-resolution
datasets, some using techniques incompatible with the power
and implementation constraints of rover engineering and CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 15 FIG. 14. (A–C) Optical image,
laboratory Raman scan, and quartz-
organic ratio map for a larger region
of interest within sample F6. (D)
Optical images and laboratory XRF
scans for one face of sample F8. ingle analytical
he detection of
et al., 2010 and
acquired under
at the analyzed
he hallmarks of
r environments:
aminated archi-
nations between
icrostratigraphy
rrestrial micro-
e et al., 2013;
Noffke, 2021). Although this microstratigraphy is also
consistent with many other finely laminated sediments
that show alternating light and dark layers as a result of
grain size or mineralogical variations (Grotzinger and
Knoll, 1999), the dark-toned laminations exhibit undu-
latory, non-isopachous morphologies that are largely
inconsistent with the isopachous characteristic of abiotic
laminations (Grotzinger and Knoll, 1999; Pope et al.,
2000; Sugitani et al., 2007; Perri et al., 2013). None-
theless, thin section preparation following MSR would
be required to achieve resolutions enabling the identifi-
cation of unambiguously biogenic micromorphologies
(Fig. 2). The observation of a dominantly fine-grained FIG. 14. (A–C) Optical image,
laboratory Raman scan, and quartz-
organic ratio map for a larger region
of interest within sample F6. (D)
Optical images and laboratory XRF
scans for one face of sample F8. Noffke, 2021). Although this microstratigraphy is also
consistent with many other finely laminated sediments
that show alternating light and dark layers as a result of
grain size or mineralogical variations (Grotzinger and
Knoll, 1999), the dark-toned laminations exhibit undu-
latory, non-isopachous morphologies that are largely
inconsistent with the isopachous characteristic of abiotic
laminations (Grotzinger and Knoll, 1999; Pope et al.,
2000; Sugitani et al., 2007; Perri et al., 2013). None-
theless, thin section preparation following MSR would
be required to achieve resolutions enabling the identifi-
cation of unambiguously biogenic micromorphologies
(Fig. 2). The observation of a dominantly fine-grained technology. 4.2. Biosignature detection If encountered on Mars, similar
materials would be paramount candidates for caching and
subsequent sample return to Earth. probable
microbial
biosignatures
using
correlated
WATSON–SHERLOC–PIXL data is achievable during in
situ mission operations. In the event that an unambiguous
biosignature identification cannot be made following this
protocol, we nonetheless show that materials with high
biosignature preservation potential—key targets in the
search for astrobiologically relevant samples (Westall et al.,
2015; Hays et al., 2017)—can be identified during standard
mission operations. Additionally, we highlight that fine-
grained, finely laminated, siliceous sedimentary rocks, such
as the Buck Reef Chert, may be excellent paleoenviron-
mental archives and can assist in the reconstruction of the
depositional energy, chemistry, and redox of the ambient
sub-aqueous environment. If encountered on Mars, similar
materials would be paramount candidates for caching and
subsequent sample return to Earth. sedimentary texture also suggests higher biosignature
preservation potential (e.g., McMahon et al., 2018; Bo-
sak et al., 2021), although this is again dependent upon
mineralogy and rapidity and timing of mineralization. gy
p
y
g
SHERLOC-like fluorescence and Raman mapping con-
firms that the dark-toned layers are carbonaceous in origin
and indicates that the organic material is thermally mature
(i.e., moderately graphitic carbonaceous materials rich in
polyaromatic sp2-bonded carbon). As such, the dark-
toned layers can be considered syngenetic with the host
rock and are of a composition comparable to the Raman-
determined kerogenous nature of ancient microbial mats
from numerous Precambrian localities (e.g., Schopf et al.,
2005; Marshall et al., 2010; Delarue et al., 2016). Cross-
cutting fabric relationships between microbial lamina-
tions and later veining further enable the sequence of
events within the samples to be established; the veins are
demonstrably secondary and associated with a distinct
silica-enriched lithochemistry and post-depositional, less
mature, organic content (e.g., Figs. 3 and 4). SHERLOC-like fluorescence and Raman mapping con-
firms that the dark-toned layers are carbonaceous in origin
and indicates that the organic material is thermally mature
(i.e., moderately graphitic carbonaceous materials rich in
polyaromatic sp2-bonded carbon). As such, the dark-
toned layers can be considered syngenetic with the host
rock and are of a composition comparable to the Raman-
determined kerogenous nature of ancient microbial mats
from numerous Precambrian localities (e.g., Schopf et al.,
2005; Marshall et al., 2010; Delarue et al., 2016). 4.2. Biosignature detection Cross-
cutting fabric relationships between microbial lamina-
tions and later veining further enable the sequence of
events within the samples to be established; the veins are
demonstrably secondary and associated with a distinct
silica-enriched lithochemistry and post-depositional, less
mature, organic content (e.g., Figs. 3 and 4). Laboratory studies carried out to supplement SHERLOC–
WATSON–PIXL analyses demonstrate the importance of
MSR since these more thorough and higher-resolution an-
alyses will provide pivotal supporting evidence for the
characterization and detection of biosignatures upon return
to Earth. In the present case, previous laboratory analyses,
simulating post-MSR approaches, have already shown that
the studied laminated fabrics are of biogenic origin, their
exceptional preservation by virtue of early and rapid silici-
fication (Walsh and Lowe, 1999; Tice and Lowe, 2004,
2006a; Greco et al., 2018). PIXL-like elemental mapping demonstrates that organic
laminations (identified by SHERLOC-like analyses) are
predominantly preserved within silica and are intercalated
with siderite-rich layers that also contain organic material. These iron carbonate layers are likely a result of growth on
a carbonate platform under mildly acidic aqueous chem-
istry (cf. Tice and Lowe, 2004, 2006a; Westall et al.,
2018). The ubiquitous presence of laminated microcrys-
talline silica throughout the samples suggests a rapidly
lithifying chemical agent of preservation, again suggesting
that silica may have precipitated in the primary environ-
ment and acted as a means of early organic preservation. 4.2. Biosignature detection Although it is often true that no single analytical
dataset can provide a ‘‘smoking gun’’ for the detection of
ancient life (e.g., Brasier et al., 2006; Schopf et al., 2010 and
references therein), these emulator data acquired under
mission-representative conditions show that the analyzed
Buck Reef Chert samples bear many of the hallmarks of
fossilized microbial mats from shallow-water environments: WATSON-like images show that the laminated archi-
tecture of the rock comprises regular alternations between
dark- and light-toned layers, that is, a microstratigraphy
consistent with modern and fossil terrestrial micro-
bialites and biolaminites (e.g., Noffke et al., 2013; HICKMAN-LEWIS ET AL. HICKMAN-LEWIS ET AL. 16 FIG. 15. Example simulation of correlated WATSON imaging, SHERLOC DUV-Raman spectroscopy, and PIXL element
mapping conducted during a Mars 2020 Perseverance sampling cycle. Analyses were conducted on the same region of interest
(sample F8) following an identical protocol to that conducted during each sampling event on Mars. (A) WATSON-like image of
an abraded sample surface. (B–D) WATSON-like close-up image with fluorescence, and Raman maps for survey scan 2. (E–H)
Detail scans 3 and 4 within survey region S2. (I–L) PIXL elemental mapping showing the distribution of Si, Fe, and Mn. FIG. 15. Example simulation of correlated WATSON imaging, SHERLOC DUV-Raman spectroscopy, and PIXL element
mapping conducted during a Mars 2020 Perseverance sampling cycle. Analyses were conducted on the same region of interest
(sample F8) following an identical protocol to that conducted during each sampling event on Mars. (A) WATSON-like image of
an abraded sample surface. (B–D) WATSON-like close-up image with fluorescence, and Raman maps for survey scan 2. (E–H)
Detail scans 3 and 4 within survey region S2. (I–L) PIXL elemental mapping showing the distribution of Si, Fe, and Mn. probable
microbial
biosignatures
using
correlated
WATSON–SHERLOC–PIXL data is achievable during in
situ mission operations. In the event that an unambiguous
biosignature identification cannot be made following this
protocol, we nonetheless show that materials with high
biosignature preservation potential—key targets in the
search for astrobiologically relevant samples (Westall et al.,
2015; Hays et al., 2017)—can be identified during standard
mission operations. Additionally, we highlight that fine-
grained, finely laminated, siliceous sedimentary rocks, such
as the Buck Reef Chert, may be excellent paleoenviron-
mental archives and can assist in the reconstruction of the
depositional energy, chemistry, and redox of the ambient
sub-aqueous environment. 4.3. Implementation of the Perseverance protocol The Perseverance-like characterization presented in this
study emulates the final stage of high-resolution analysis
during the rover’s targeted operation on Mars and is typi-
cally conducted on abraded patches. Complementary prior
orbital and outcrop studies provide the context to understand
the broader paleoenvironment of the sample. Analyses on Our results, following a sequence identical to that of the
standard Perseverance characterization protocol prior to
sampling events, imply that the detection of possible and CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 17 abraded patches will be supported by this plethora of data
from other payload instruments and considerations that fa-
cilitate detailed assessments of the origin of a putative
biosignature. At this advanced stage of in situ character-
ization, identification of the key attributes noted above
would provide a logical motivation for caching in light of
the potential presence of a biosignature. Following obser-
vations of the Kodiak delta remnant and delta front early in
the Mars 2020 prime mission, outcrop-scale observations by
Perseverance are already ground-truthing the presence of
habitable conditions (a fluvio-deltaic setting) potentially
containing
fine-grained
sediments
with
probable
bio-
signature preservation potential (Mangold et al., 2021;
Williford et al., 2021). Ongoing outcrop characterization
throughout the mission will allow the selection of localities
with astrobiological potential for analysis and sampling to
be refined. Based on the above emulated in situ analysis
scenario, combined WATSON–SHERLOC–PIXL datasets
can be considered a minimum combination of analyses re-
quired to make a confident decision for sample caching. Following return to Earth and laboratory analysis, further
high-resolution and high-sensitivity analyses on these sam-
ples would be conducted to confirm whether or not they
represent preserved microbial mats or microbialites/stro-
matolites, or whether the observed laminated features are
abiotic in origin (Cady et al., 2003; Allwood et al., 2015;
Hays et al., 2017; Williford et al., 2018; Steele et al., 2022). clusion that a sample should be cached because it contains
potential biosignatures, while the analysis of those samples
on Earth would show that fabrics within these samples are
biogenic. The only major differences between the mission-
like and laboratory analyses presented are as follows: (i) the
impossibility to observe individual microscopic biofilms, for
which invasive sample preparation (thin sections) and mi-
croscopy are necessary; and (ii) limits of scan size imposed
by rover arm instrumental configuration and power con-
straints. 4.3. Implementation of the Perseverance protocol In this regard,
before each sampling activity, Perseverance will execute the
Sample Threshold Observation Protocol (STOP) suite of
analyses, which is designed to ensure complete fundamental
characterization of the sample, thereby ensuring that it
meets the optimal criteria prior to a sampling and caching
commitment. The presented WATSON, SHERLOC, and
PIXL analyses are contained within the STOP list, and
Fig. 15 presents an example correlated dataset that could be
obtained prior to sampling. Although mission time con-
straints mean that there will probably not be more than one
set of SHERLOC and one set of PIXL analyses in a single
STOP list (e.g., the datasets shown in Fig. 15), we strongly
advocate for further opportunistic analyses where particu-
larly compelling features are observed, for example, lami-
nated carbonaceous fabrics or sedimentary textures that may
have arisen from biological activity It is probable that samples hosting putative biosignatures
will be known long prior to their return to Earth, during
which time an appropriate analytical protocol can be de-
veloped (cf. Rummel and Kminek, 2018; Kminek et al.,
2022; Meyer et al., 2022), likely on a sample-by-sample
basis. A wide range of returned sample science could be
proposed further to in situ analyses conducted by Perse-
verance in order to confirm or disprove their potential bio-
genic nature. Since the amount of returned sample will be
limited, this analytical protocol should focus on obtaining
information still missing after Perseverance analyses, that is,
to conduct analyses that are impossible when using in situ
instrumentation. Such data will probably include micro-
morphological analyses requiring thin section preparation
(as shown in Fig. 2) or the extraction of ultrathin rock
sections using a focused ion beam (e.g., Cavalazzi, 2007;
Schiffbauer and Xiao, 2009; Cavalazzi et al., 2021), isotope
geochemistry requiring the identification and isolation of
individual mineral phases or fabric elements (e.g., Valley
et al., 1994; van den Boorn et al., 2010; Lowe et al., 2020),
high-resolution tomography requiring minimally invasive
sample preparation (e.g., Hickman-Lewis et al., 2016b,
2019; Maldanis et al., 2020), and organic geochemistry re-
quiring the solvent extraction of organic aliquots (e.g., Duda
et al., 2018). Under time-pressured mission conditions and competing
science priorities, developing an effective analytical strategy
is essential to ensure rapid, yet accurate, assessments of rock
fabric and composition; to define specific sampling locali-
ties; and to identify potential biosignatures. 4.3. Implementation of the Perseverance protocol In this regard,
before each sampling activity, Perseverance will execute the
Sample Threshold Observation Protocol (STOP) suite of
analyses, which is designed to ensure complete fundamental
characterization of the sample, thereby ensuring that it
meets the optimal criteria prior to a sampling and caching
commitment. The presented WATSON, SHERLOC, and
PIXL analyses are contained within the STOP list, and
Fig. 15 presents an example correlated dataset that could be
obtained prior to sampling. Although mission time con-
straints mean that there will probably not be more than one
set of SHERLOC and one set of PIXL analyses in a single
STOP list (e.g., the datasets shown in Fig. 15), we strongly
advocate for further opportunistic analyses where particu-
larly compelling features are observed, for example, lami-
nated carbonaceous fabrics or sedimentary textures that may
have arisen from biological activity. 4.3. Implementation of the Perseverance protocol Additionally, the studied samples have undergone a
more severe thermal history (lower greenschist grade meta-
morphism) than is expected at the surface of Mars, and we
can therefore anticipate that organic materials on Mars would
generally be less degraded than those preserved within the
Buck Reef Chert microbialites. Regardless, our study dem-
onstrates that micron- to millimeter-scale correlated analyses
on Mars using Perseverance instrumentation can reach a high
level of detail in lithological characterization, facilitating the
optimal selection of potential biosignature-bearing samples
for return to Earth. Such samples would fulfil the major as-
trobiology goals of the Mars 2020 mission (Williford et al.,
2018; Farley et al., 2020) and are certain to provide detailed
insights into the geochemistry and elemental cycles, partic-
ularly the carbon cycle, of early Mars. abraded patches will be supported by this plethora of data
from other payload instruments and considerations that fa-
cilitate detailed assessments of the origin of a putative
biosignature. At this advanced stage of in situ character-
ization, identification of the key attributes noted above
would provide a logical motivation for caching in light of
the potential presence of a biosignature. Following obser-
vations of the Kodiak delta remnant and delta front early in
the Mars 2020 prime mission, outcrop-scale observations by
Perseverance are already ground-truthing the presence of
habitable conditions (a fluvio-deltaic setting) potentially
containing
fine-grained
sediments
with
probable
bio-
signature preservation potential (Mangold et al., 2021;
Williford et al., 2021). Ongoing outcrop characterization
throughout the mission will allow the selection of localities
with astrobiological potential for analysis and sampling to
be refined. Based on the above emulated in situ analysis
scenario, combined WATSON–SHERLOC–PIXL datasets
can be considered a minimum combination of analyses re-
quired to make a confident decision for sample caching. Following return to Earth and laboratory analysis, further
high-resolution and high-sensitivity analyses on these sam-
ples would be conducted to confirm whether or not they
represent preserved microbial mats or microbialites/stro-
matolites, or whether the observed laminated features are
abiotic in origin (Cady et al., 2003; Allwood et al., 2015;
Hays et al., 2017; Williford et al., 2018; Steele et al., 2022). Under time-pressured mission conditions and competing
science priorities, developing an effective analytical strategy
is essential to ensure rapid, yet accurate, assessments of rock
fabric and composition; to define specific sampling locali-
ties; and to identify potential biosignatures. References As the Perseverance rover continues its operations in
Jezero crater, numerous studies use datasets based on sim-
ulated payload analyses as a means to investigate geological
materials encountered and anticipated along its traverse
(e.g., Mandon et al., 2021; Razzell Hollis et al., 2021;
Anderson et al., 2022) as well as to optimize the treatment
and interpretation of datasets under mission conditions and
constraints (e.g., Allwood et al., 2020; Flannery et al.,
2021). For astrobiology, the obtainment of datasets such as
those reported on the Strelley Pool stromatolites (Allwood
et al., 2015) and the Buck Reef Chert (this study) will
provide guidance for the interpretation of similar bio-
signatures if encountered on Mars. Such studies also provide
initial guidance for sample return. At present, preparations
for MSR are being formulated and refined with emphasis on
analytical strategies, curation and sample storage, and
planetary protection (Rummel and Kminek, 2018; Smith
et al., 2021; Kminek et al., 2022; Meyer et al., 2022; Tait
et al., 2022). Defining the datasets to which we will have
access from only in situ rover analyses will facilitate the
prioritization of analyses conducted on returned materials
and could conceivably incentivize the development of in-
strumentation or analytical approaches enabling missing
data to be acquired. In this regard, the analysis of limited
and ‘‘precious’’ early Earth samples is particularly prudent
since these materials face many of the same challenges that
will be faced by materials returned from Mars: in both cases,
it is necessary to extract a maximum amount and quality of
data from a minimal volume of sample material, using non-
invasive and nondestructive approaches insofar as possible. Further studies using Perseverance-like protocols on similar
terrestrial samples should further help constrain the nature
and interpretation of data gathered in situ on Mars and best
prepare astrobiologists for the challenge of assessing po-
tential traces of life in returned martian samples. Agangi A, Hofmann A, Ossa Ossa F, et al. (2021) Mesoarch-
aean acidic volcanic lakes: a critical ecological niche in early
land colonization. Earth Planet Sci Lett 556, doi:10.1016/
j.epsl.2020.116725. Allwood AC, Clark B, Flannery DT, et al. (2015) Texture-
specific elemental analysis of rocks and soils with PIXL: the
Planetary Instrument for X-ray Lithochemistry on Mars 2020. In 2015 IEEE Aerospace Conference Proceedings. IEEE,
Piscataway, NJ, doi:10.1109/AERO.2015.7119099. Allwood AC, Wade LA, Foote MC, et al. (2020) PIXL: Pla-
netary Instrument for X-Ray Lithochemistry. HICKMAN-LEWIS ET AL. 18 studied samples’ laminated architecture, composition, and
paleoenvironmental signatures are consistent with habitable
settings containing mature organic materials of biological
origin. This suggests that a similar level of interpretation will
be possible at the surface of Mars. Previous, more conven-
tional studies of samples from the Buck Reef Chert confirm
that these horizons contain well-preserved organic-rich bio-
signatures (microbial mats) of Paleoarchean age, which are
considered to serve as analogues of possible primitive bio-
signatures that might be found on Mars. Our protocol cap-
tures a high degree of this biogenic interpretation. lowship (ST/V00560X/1 – Correlative microscopy analyti-
cal strategies for Mars Sample Return). The samples used in
this study are part of the sample collection of the INACMa
(Inorganic
Nanoparticles
in
Archaean
Carbonaceous
Matter—a key to early life) project to BC (EU-FP7 Grant
no. 618657). JRH carried out research at the Jet Propulsion
Laboratory, California Institute of Technology, under a
contract with the National Aeronautics and Space Admin-
istration (80NM0018D0004). KRM and JPG acknowledge
the Simons Foundation and the Simons Collaboration on the
Origins of Life. SS acknowledges support from NASA un-
der award number 80GSFC21M0002. We thank Axel Hof-
mann (University of Johannesburg) for the provision of
samples from the BARB-3 core and Callum Hatch (NHM)
for technical support in sample preparation. g
g
g
p
Organic compounds formed via microbial metabolism are
among the prime targets for sample return, and our study of
the Buck Reef Chert suggests that, if such materials exist in
the rocks at Jezero crater, they could be identified by using the
Perseverance analytical protocol. If similar targets are identi-
fied on Mars, they would offer opportunities both to recon-
struct aspects of the early martian carbon cycle and to search
for potential fossilized traces of life in ancient paleoenviron-
ments. Under such circumstances, the protocol of correlated
analyses presented herein could be deployed to prioritize
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Mars. Science 375:172–177. McMahon S, Bosak T, Grotzinger JP, et al. CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES Horgan BNH, Anderson RA, Dromart G, et al. (2020) The
mineral diversity of Jezero crater: evidence for possible la-
custrine carbonates on Mars. Icarus 339:113526. Greco F, Cavalazzi B, Hofmann A, et al. (2018) Raman spectros-
copy in palaeontology: a case study from Archaean biostructures. Bollettino della Societa` Paleontologica Italiana 57:59–74. Javaux EJ (2019) Challenges in evidencing the earliest traces of
life. Nature 572:451–460. Grosch EG, Kosler J, McLoughlin N, et al. (2011) Paleoarchean
detrital zircon ages from the earliest tectonic basin in the
Barberton Greenstone Belt, Kaapvaal craton, South Africa. Precambrian Res 191:85–99. Jones E, Oliphant T, and Peterson P (2001) SciPy: Open Source
Scientific Tools for Python. Available online at http://
www.scipy.org Grotzinger JP and Knoll AH (1999) Stromatolites in Precam-
brian carbonates: evolutionary mileposts or environmental
dipsticks? Annu Rev Earth Planet Sci 27:313–358. Kminek G, Meyer M, Beaty DW, et al. (2022) Mars Sample
Return (MSR): planning for returned sample science. Astro-
biology 22(Supplement), doi:10.1089/ast.2021.0198. CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES 21 Williford KH, Farley KA, Stack KM, et al. (2021) A tour of
ancient habitable environments in and around Jezero crater,
Mars [abstract 1599]. In 52nd Lunar and Planetary Science
Conference. Lunar and Planetary Institute, Houston. Wacey D, Saunders M, Roberts M, et al. (2014) Enhanced
cellular preservation by clay minerals in 1 billion-year-old
lakes. Sci Rep 4, doi:10.1038/srep05841. Wacey D, Eiloart K, and Saunders M (2019) Comparative multi-
scale analysis of filamentous microfossils from the c. 850 Ma
Bitter Springs Group and filaments from the c. 3460 Ma Apex
chert. Journal of the Geological Society 176:1247–1260. Address correspondence to:
Keyron Hickman-Lewis
Department of Earth Sciences
The Natural History Museum
Cromwell Road
South Kensington
London
SW7 5BD
United Kingdom Address correspondence to:
Keyron Hickman-Lewis
Department of Earth Sciences
The Natural History Museum
Cromwell Road
South Kensington
London
SW7 5BD
United Kingdom Walsh MM (1992) Microfossils and possible microfossils from
the early Archean Onverwacht group, Barberton mountain
land, South Africa. Precambrian Res 54:271–293. Walsh MM and Lowe DR (1999) Modes of accumulation of
carbonaceous matter in the early Archaean: a petrographic
and geochemical study of the carbonaceous cherts of the
Swaziland Supergroup. In Geologic Evolution of the Bar-
berton Greenstone Belt, South Africa, Geological Society of
America Special Paper 329, edited by DR Lowe and GR
Byerly. Geological Society of America, Boulder, CO, pp
115–132. E-mail: keyron.hickman-lewis@nhm.ac.uk E-mail: keyron.hickman-lewis@nhm.ac.uk Westall F, Loizeau D, Foucher F, et al. (2013) Habitability on
Mars from a microbial point of view. Astrobiology 13:887–897. Westall F, Loizeau D, Foucher F, et al. (2013) Habitability on
Mars from a microbial point of view. Astrobiology 13:887–897. Westall F, Foucher F, Bost N, et al. (2015) Biosignatures on
Mars: what, where, and how? Implications for the search for
martian life. Astrobiology 15:998–1029. Submitted 27 January 2022
Accepted 13 June 2022 Westall F, Foucher F, Bost N, et al. (2015) Biosignatures on
Mars: what, where, and how? Implications for the search for
martian life. Astrobiology 15:998–1029. Westall F, Hickman-Lewis K, Hinman N, et al. (2018) A
hydrothermal-sedimentary context for the origin for life. Astrobiology 18:259–293. HICKMAN-LEWIS ET AL. Valley JW, Chiarenzelli JR, and McLelland JM (1994) Oxygen
isotope geochemistry of zircon. Earth Planet Sci Lett 126:
187–206. Ruff SW and Farmer JD (2016) Silica deposits on Mars with
features resembling hot spring biosignatures at El Tatio in
Chile. Nat Commun 7, doi:10.1038/ncomms13554. van den Boorn SHJM, van Bergen MJ, Vroon PZ, et al. (2010) Silicon isotope and trace element constraints on the
origin of *3.5 Ga cherts: implications for early Archaean
marine environments. Geochim Cosmochim Acta 74:1077–
1103. Rummel JD and Kminek G (2018) It’s time to develop a new
‘‘draft test protocol’’ for a Mars Sample Return mission (or
two.). Astrobiology 18:377–380. Schiffbauer JF and Xiao S (2009) Novel application of fo-
cused ion beam electron microscopy (FIB-EM) in preparation
and analysis of microfossil ultrastructures: a new view of
complexity in early eukaryotic organisms. Palaios 24:616–
626. Van der Walt S, Colbert SC, and Varoquaux G (2011) The
NumPy array: a structure for efficient numerical computation. Computing in Science and Engineering 13:22–30. Address correspondence to:
Keyron Hickman-Lewis
Department of Earth Sciences
The Natural History Museum
Cromwell Road
South Kensington
London
SW7 5BD
United Kingdom CORRELATED SHERLOC-PIXL ANALYSES OF MICROBIAL BIOSIGNATURES Abbreviations Used
DUV ¼ deep-ultraviolet
MSR ¼ Mars Sample Return
PIXL ¼ Planetary Instrument for X-ray
Lithochemistry
SHERLOC ¼ Scanning Habitable Environments with
Raman and Luminescence for Organics
and Chemicals
STOP ¼ Sample Threshold Observation Protocol
WATSON ¼ Wide-Angle Topographic Sensor for
Operations and eNgineering
XRF ¼ X-ray fluorescence Abbreviations Used
DUV ¼ deep-ultraviolet
MSR ¼ Mars Sample Return
PIXL ¼ Planetary Instrument for X-ray
Lithochemistry
SHERLOC ¼ Scanning Habitable Environments with
Raman and Luminescence for Organics
and Chemicals
STOP ¼ Sample Threshold Observation Protocol
WATSON ¼ Wide-Angle Topographic Sensor for
Operations and eNgineering
XRF ¼ X-ray fluorescence Westall F, Hickman-Lewis K, Cavalazzi B, et al. (2021) On
biosignatures for Mars. Int J Astrobiol 20:377–393. Wiens RC, Maurice S, Robinson SH, et al. (2021) The Super-
Cam instrument suite on the NASA Mars 2020 rover: body
unit and combined system tests. Space Sci Rev 217, doi:
10.1007/s11214-020-00777-5. Williford KH, Farley KA, Stack KM, et al. (2018) The
NASA Mars 2020 rover mission and the search for extra-
terrestrial life. In From Habitability to Life on Mars, edited
by NA Cabrol and EA Grin. Elsevier, Amsterdam, pp 275–
308.
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Next Generation Device Grade Silicon-Germanium on Insulator
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Scientific reports
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cc-by
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OPEN SUBJECT AREAS:
MATERIALS FOR DEVICES
SILICON PHOTONICS
CHARACTERIZATION AND
ANALYTICAL
TECHNIQUES
MATERIALS FOR OPTICS Callum G. Littlejohns1, Milos Nedeljkovic1, Christopher F. Mallinson2, John F. Watts2,
Goran Z. Mashanovich1, Graham T. Reed1 & Frederic Y. Gardes1 1Optoelectronics Research Centre, University of Southampton, Southampton, SO17 1BJ, UK, 2The Surface Analysis Laboratory,
Department of Mechanical Engineering Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK. High quality single crystal silicon-germanium-on-insulator has the potential to facilitate the next generation
of photonic and electronic devices. Using a rapid melt growth technique we engineer tailored single crystal
silicon-germanium-on-insulator structures with near constant composition over large areas. The proposed
structures avoid the problem of laterally graded SiGe compositions, caused by preferential Si rich solid
formation, encountered in straight SiGe wires by providing radiating elements distributed along the
structures. This method enables the fabrication of multiple single crystal silicon-germanium-on-insulator
layers of different compositions, on the same Si wafer, using only a single deposition process and a single
anneal process, simply by modifying the structural design and/or the anneal temperature. This facilitates a
host of device designs, within a relatively simple growth environment, as compared to the complexities of
other methods, and also offers flexibility in device designs within that growth environment. Received
14 September 2014
Accepted
12 January 2015
Published
6 February 2015 Received
14 September 2014
Accepted
12 January 2015
Published
6 February 2015 S S
i1-xGex has many attractive characteristics which can be exploited for numerous applications including
wavelength sensitive photonic devices1, high mobility complementary metal oxide semiconductor (CMOS)
devices2 and lattice matching for epitaxial III-V growth3,4. It possesses full miscibility across its entire
composition range which allows for the tuning of properties such as the bandgap and lattice constant between
those of bulk Si and bulk Ge. This means that depending on the composition, SiGe alloys can be either optically
absorbing or transparent at telecommunication wavelengths (1550 nm or 1310 nm) which enables the fabrica-
tion of active devices for both modulation5,6 and detection6,7. SiGe has both a higher hole and electron mobility
than Si, meaning that it will ultimately lead to faster electronic devices, e.g. transistors8, and is also a CMOS
compatible material. p
A number of methods have been proposed to produce silicon-germanium-on-insulator (SGOI) films on Si
wafers, including layer transfer9,10 and Ge condensation10,11. A complementary technique for fabricating localized
SGOI regions on a Si wafer is rapid melt growth (RMG). Correspondence and
requests for materials
should be addressed to
C.G.L. (c.littlejohns@
soton.ac.uk) OPEN RMG, also referred to as liquid phase epitaxy (LPE), is a
technique that was invented in the 1960’s12 and further developed in the 1970s for the fabrication of detectors13,
LEDs14 and laser diodes15. The technique, originally used for III–V crystal growth, was pioneered by Liu et al.16 for
localized germanium-on-insulator (GOI) growth in 2004 and has since been studied by various groups17,18 and
adapted for SGOI growth19,20. In order to realise RMG a polycrystalline Ge layer is deposited onto a patterned
insulating layer (in this case SiO2), with areas of the Si substrate exposed to act as a seed for crystal regrowth. The
Ge is patterned and encapsulated in micro-crucibles so that when it is melted and subsequently cooled it mimics
the template of the Si crystal structure. This process is described in Figure 1. To date, RMG of Ge has been
demonstrated for gate all around P-MOSFETs21,22, P-channel finFETs23 and waveguide integrated heterojunction
photodiodes24. These devices demonstrate the possibility of using RMG to obtain high quality Ge single crystal
layers on localised insulator islands located on top of silicon substrates or silicon-on-insulator (SOI) substrates. This enables a bridge between electronic components and photonic components; the latter group IV based
components being predominantly confined to the SOI platform25–31. This vision is clearly demonstrated by
Going et al. in a gate photoMOSFET32 where a Ge gated NMOS phototransistor is integrated on a SOI photonics
platform. RMG is very attractive for the heterogeneous integration of SiGe based devices on insulator for electronics and
photonics applications because it is possible to grow defect-free single crystal material, by the mechanism shown
in Figure 2a, with the condition that the regrowth propagation speed is sufficient enough to avoid random
nucleation in the liquid ahead of the epitaxial growth front20. This high material quality can lead to significant
improvements in device characteristics such as leakage current and quantum efficiency. However, until now SCIENTIFIC REPORTS | 5 : 8288 | DOI: 10.1038/srep08288 1 Figure 1 | Summary of the SiGe rapid melt growth fabrication process. (b), A comparison of the Raman Iratio on the left hand
axis (solid) and AES Si:Ge peak ratio on the right hand axis (dashed) for the
central strip of a tailored tree-like structure (black squares) and a straight
strip, with the branches omitted (red circles), as a function of distance from
the Si seed. This shows the dramatic improvement in composition tree-like structure; as described in ref. 19. All structures were annealed at
955uC. (c), Average Raman Iratio along the centre strip of a tailored tree-like
structure as a function of anneal temperature showing increased Si
composition at higher temperatures due to the increased Si diffusion from
the seed. Inset is a plot of the Raman Iratio as a function of distance from the
seed for a range of anneal temperatures from which the average Iratio values
were calculated. tree-like structure; as described in ref. 19. All structures were annealed at
955uC. (c), Average Raman Iratio along the centre strip of a tailored tree-like
structure as a function of anneal temperature showing increased Si g
composition at higher temperatures due to the increased Si diffusion from
the seed. Inset is a plot of the Raman Iratio as a function of distance from the
seed for a range of anneal temperatures from which the average Iratio values
were calculated. accurate control of the SiGe composition in the RMG layers has
proved to be extremely challenging. This is due to the large separa-
tion between the SiGe composition in the solid and liquid phase at
any given temperature, shown by the SiGe phase diagram in
Figure 2b. This large composition separation between phases results
in preferential Si rich solid formation at the growth front with rejec-
tion of Ge into the liquid, leading to a gradation of the SiGe com-
position in the regrowth direction of a straight strip as the Si is
consumed19,20. By using the tailored tree-like structure shown in
Figure 2c we have demonstrated for the first time (Figure 3a) that
the SiGe composition (shown on the graph as I(SiGe)/I(GeGe)) can
be engineered to a near constant value along the central strip of the
tree-like structure, with the graded composition characteristics prev-
iously demonstrated in straight strips appearing only in the branches
of the tree. Figure 1 | Summary of the SiGe rapid melt growth fabrication process. Figure 2 | Summary of the SiGe recrystallization process. (a), A cross-section schematic of the recrystallization process showing the growth front
initiating at the Si seed, which is essential to ensure single crystal epitaxial regrowth, and propagating along the SiGe structure. Growth is initiated at the
seed for two reasons: firstly the Si substrate acts as a heat sink ensuring more rapid cooling in the seed area; secondly, Si diffusion into the Ge structure
increases the solidification temperature so that material with a slightly higher Si composition solidifies first (i.e. areas nearer the Si seed). Random
nucleation occurs when the growth front propagation speed is too slow. (b), Phase diagram of SiGe alloys showing separation of the solidus and liquidus
curves – adapted from ref. 38. (c), An SEM image of the tailored tree-like structure showing the radiating elements which result in a near constant SiGe
composition in the central strip. Figure 2 | Summary of the SiGe recrystallization process. (a), A cross-section schematic of the recrystallization process showing the growth front
initiating at the Si seed, which is essential to ensure single crystal epitaxial regrowth, and propagating along the SiGe structure. Growth is initiated at the
seed for two reasons: firstly the Si substrate acts as a heat sink ensuring more rapid cooling in the seed area; secondly, Si diffusion into the Ge structure
increases the solidification temperature so that material with a slightly higher Si composition solidifies first (i.e. areas nearer the Si seed). Random
nucleation occurs when the growth front propagation speed is too slow. (b), Phase diagram of SiGe alloys showing separation of the solidus and liquidus
curves – adapted from ref. 38. (c), An SEM image of the tailored tree-like structure showing the radiating elements which result in a near constant SiGe
composition in the central strip. SCIENTIFIC REPORTS | 5 : 8288 | DOI: 10.1038/srep08288 2 Figure 3 | Study of the SiGe composition in tailored tree-like structures. (a), Raman Iratio as a function of distance from the Si seed for a tailored
tree-like structure annealed at 955uC, showing near constant SiGe
composition in the central strip (solid), and graded composition
characteristics in the branches (dashed). Each dashed line represents an
individual branch. Results The SiGe composition in our structures was characterized using
532 nm Raman spectroscopy, as well Auger Electron Spectroscopy
(AES) in order to confirm the integrity of the data. The Ge concen-
tration can be calculated from the Raman spectra using the equation
proposed by Mooney et al.33 by taking the ratio of the Si-Ge mode
intensity and Ge-Ge mode intensity (the peaks located at approxi-
mately 380 cm21 and 300 cm21 respectively) as described by
Equation (1): Iratio~ I SiGe
ð
Þ
I GeGe
ð
Þ ~ 2 1{x
ð
Þ
kx
ð1Þ ð1Þ Here, I(SiGe) and I(GeGe) are the integrated intensities of the Si-Ge
and Ge-Ge modes respectively, x is the Ge concentration and k,
which is dependent on the excitation wavelength, is an experimental
setup specific constant that can be determined from samples of
known composition. For the purpose of this paper only the ratios
of the integrated intensities, Iratio, are considered. Nevertheless, for
high Ge compositions (x . 0.75) the correlation between Iratio and x
is almost linear for all values of k quoted in the literature rendering
this analysis an accurate representation of the actual SiGe composi-
tion17. This has been confirmed by comparing secondary ion mass
spectrometry (SIMS) composition data to Iratio for some blanket SiGe
on Si samples, grown using reduced pressure chemical vapour depos-
ition (RPCVD), with compositions in the range 0.75 , x , 1. A
similar characterization process was carried out using AES. For this
analysis the peak ratios between Si and Ge (found at approximately
1615 eV and 1145 eV respectively) were calculated after a linear
background subtraction was performed. Again it is expected that this
ratio closely relates to the SiGe composition34. Figure 3 | Study of the SiGe composition in tailored tree-like structures. (a), Raman Iratio as a function of distance from the Si seed for a tailored
tree-like structure annealed at 955uC, showing near constant SiGe
composition in the central strip (solid), and graded composition
characteristics in the branches (dashed). Each dashed line represents an
individual branch. (b), A comparison of the Raman Iratio on the left hand
axis (solid) and AES Si:Ge peak ratio on the right hand axis (dashed) for the
central strip of a tailored tree-like structure (black squares) and a straight
strip, with the branches omitted (red circles), as a function of distance from
the Si seed. This is to be expected since the branches of the tree-like
structure are effectively straight strips seeding from the central part
of the tree-like structure. In practice the branches would be removed
once the RMG process has been performed, leaving only the constant
composition SGOI material for device fabrication. Throughout this
paper the centre strip of the tree-like structure will be referred to as
‘central strip’ and a straight strip, with no branches, will be referred to
as ‘straight strip’. Discussion In order to discuss the cause of these results it is firstly important to
understand the recrystallization and Si diffusion mechanisms that
are exhibited in the conventional RMG process. Once the Ge melts
upon heating to become liquid, Si diffusion into the Ge from the seed
dramatically increases to form a liquid SiGe pool. It can be approxi-
mated that the SiGe composition is uniform in the liquid pool due to
the high diffusivities of both Si36 and Ge37 in liquid SiGe. At a given
alloy composition, determined by the anneal temperature, the SiGe
will begin to solidify in the seed area, due to the slightly higher Si
concentration found here, and mimic the crystal structure of the
underlying Si substrate. Since the diffusivity of both Si and Ge in
solid SiGe is many orders of magnitude lower than in liquid SiGe it
can be approximated that there is no diffusion in the solid phase. Therefore, once the SiGe in the seed area has solidified the Si supply
from the substrate is cut off leading to a finite Si ‘pool’ available in the
SiGe melt. As described above, the SiGe phase diagram shows that
there is a preferential growth of Si rich solid which results in complete
depletion of the Si ‘pool’ before the end of a straight strip. However if
radiating elements are added to the strip to form a tailored tree-like
structure (see Figure 2c) the cooling rate of the structure is increased
and therefore the regrowth front propagation velocity is increased. This means that complete diffusion of the rejected Ge into the bulk of
the liquid does not occur and therefore the SiGe composition
becomes more consistent. It is also important to consider the penetration depths associated
with the respective measurement techniques. AES has a sampling
depth of several nanometers and thus will show only surface
information. A 532 nm laser has a sampling depth of several tens
of nanometers in high Ge percentage SiGe but is dependent on the
composition35. Therefore it is possible that there is a vertical distri-
bution of composition but this is predicted to be negligible since the
epitaxial growth front propagates laterally and not vertically. Figure 3c shows that we have also demonstrated that the composi-
tion of the localized SGOI structures can be controlled by the peak
anneal temperature, with more Si rich alloys formed at higher anneal
temperatures. Results This shows the dramatic improvement in composition
consistency when using the tailored tree-like structure. The straight strip
data has been standardized using data from 10 mm, 20 mm, 50 mm and
100 mm long strips in order to match the length of the 60 mm long tailored In Figure 3b both the Raman Iratio and the AES Si/Ge Peak ratio are
shown as a function of distance from the seed for the centre strip of
the tailored structure and a straight strip, with the tree branches
omitted, of the same width. This clearly demonstrates a dramatic SCIENTIFIC REPORTS | 5 : 8288 | DOI: 10.1038/srep08288 3 www.nature.com/scientificreports improvement of the composition consistency when using the tai-
lored structure. This figure establishes that the AES data is in agree-
ment with the Raman data, within measurment tolerances, so we
have displayed only the Raman data for all other samples. For quan-
tification of Si the more intense Si LMM Auger transition is usually
used. This would provide a more precise value for concentration as
its peak height is more sensitive to concentration. However this
transition cannot be used in this system as a consequence of the large
energy overlap between the Ge MNN and Si LMM transitions. As a
result the lower sensitivity Si KLL transition must be used instead. However, at low Si concentrations this peak is very weak and the
region must be force fitted to provide a value of intensity. Therefore
the discrepancy between the AES results and the Raman results at
low Si concentrations is likely due to the poor sensitivity of the Si KLL
peak to concentration changes in this composition range. the seed area and do not propagate along the SiGe strip. The high
resolution cross-section confirms single crystal, defect free SGOI
along the centre strip of the tree structure. Ge diffusion into the Si
substrate can also be observed. Electron back-scatter diffraction
(EBSD) measurements, shown in Figure 4b, confirm the single crys-
tal orientation of the SiGe tailored structures. Methods The fact that the SiGe composition is consistent in the centre strip
of the tree-like structure but then follows the characteristics of a
straight strip in the branches suggests that the regrowth front pro-
pagates firstly along the centre stripand secondly along the individual
branches, with the centre strip acting as a seed for each branch. In
order to confirm this hypothesis an experiment was designed to
study the solidification of the tailored structures. In this experiment
we used a poly-silicon (poly-Si) seed on a thick insulating layer rather
than a bulk Si seed. This has the effect of slowing down the cooling
rate, and therefore the regrowth front propagation velocity, of the
SiGe structures, whilst still maintaining single crystal regrowth17, by
reducing the heat sinking properties of the bulk Si wafer. The result is
that random nucleation occurs in the liquid SiGe ahead of the
regrowth front. The nucleation points are observable on scanning
electron microscope (SEM) images, as shown in Figure 5. The low
nucleation count observed in the centre strip of the tree-like structure
compared with the high nucleation count observed in the branches is
consistent with our hypothesis that the centre strip solidifies prior to
the branches, because only in the branches is the regrowth rate slow
enough to allow random nucleation to occur. Material characterization. The SiGe composition has been characterized firstly using
532 nm Raman spectroscopy with a spot size of approximately 0.5 mm. The ratio of
the Si-Ge and Ge-Ge mode intensities, Iratio, for each point has been calculated by
fitting a Lorentzian curve to each peak to calculate the area under the peak. In order to
confirm the integrity of this data Auger electron spectroscopy (AES) was also
performed using a Thermo Scientific Microlab 350 Scanning Auger Microscope, with
a spot size of 20 nm. High resolution Si KLL and Ge LMM spectra were collected from
1300–1650 and 650–1205 eV, respectively, with a step size of 2 eV, a retard ratio of 4
and a dwell time of 100 ms per channel. The air formed surface oxide and surface
carbon contamination were removed with 20 seconds of argon ion sputtering using a
3 kV beam energy and 1 mA sample current prior to measurement. Methods concentration reaches the solidification point determined by the
SiGe phase diagram, a more Si rich composition is achieved (see
Figure 3c). This would therefore enable tuning of the band edge
and lattice parameters of the layer to suit the design requirements
simply by modifying the anneal temperature. It is to be noted here
that, whilst the quoted temperatures are accurate relative to one
another, they should be considered arbitrary temperatures in the
absolute sense, because they were recorded using a pyrometer mea-
suring the infra-red radiation from the back of the Si substrate during
annealing. This recorded temperature does not match the SiGe tem-
perature since the optical absorption coefficients of the two materials
are different and will vary depending on the alloy composition. Rapid melt growth fabrication process. The SiGe tailored structures were grown on
6 inch (100) Si wafers. The wafers were cleaned using a conventional RCA clean prior
to processing to remove any contaminants from the substrate surface. A 50 nm SiO2
layer was then deposited using plasma enhanced chemical vapour deposition (PE-
CVD). The SiO2 layer was then patterned using standard UV photolithography and a
dilute (2051) HF wet etch in order to expose the underlying Si to act as a seed for the
SiGe recrystallization process. A 400 nm Ge layer was deposited using a non-selective
PE-CVD process and patterned using standard UV photolithography and an
inductively coupled plasma (ICP) etch, leaving Ge structures overlapping the Si seeds. A 1 mm SiO2 layer was then deposited by PE-CVD in order to encapsulate the Ge
structures, forming micro-crucibles. The wafers were subsequently heated in a rapid
thermal annealer (RTA) in order to melt the encapsulated Ge and initiate
recrystallization. The temperature of the RTA was stabilized at 500uC before ramping
to the maximum temperature (in the range 955uC to 1133uC) at a rate of
approximately 100uC/s. The wafers were held at the maximum temperature for 1
second before cooling naturally. Finally, the top SiO2 layer was removed using a 2051
HF wet etch for material characterization. This process is summarized in Figure 1. Methods A linear
background subtraction is performed over the peak ranges 1300–1650 and 650–
1205 eV for silicon and germanium respectively, which is necessary as the large
background distorts the true peak intensity. Cross section TEM lamellae were
prepared by EAG Labs using an in-situ focused ion beam (FIB) lift out technique. For
protection the samples were coated with local e-beam and ion-beam platinum prior to
FIB milling. EBSD measurements were also performed by EAG Labs. The sample
surface was etched with a gallium ion beam prior to EBSD imaging. The SEM images
were collected using a Zeiss NVision 40 FIB System. In conclusion, the simple method described here for fabrication of
localized SGOI structures could act as the blueprint for the coexist-
ence of next generation SiGe electronic and photonic devices on the
same wafer. The SiGe composition engineering that produces single
crystal, defect free, SiGe layers could enable the fabrication of a
multitude of devices requiring different compositions of SiGe on
the same wafer in a single deposition step and a single anneal step. Thanks to this methodology the composition of SiGe can now be
varied according to the anneal temperature and/or the material
structure enabling alloy composition engineering dictated by struc-
tural design and not by the deposition or growth mechanism. This
method leads to a simplified (single deposition), design enabled fab-
rication process for the integration of a plethora of electronic and
CMOS compatible photonic devices on a standard Si substrate pro-
viding a path for the seamless integration of electronics and photo-
nics at a low cost. 1. Liu, J. et al. Waveguide-integrated, ultralow-energy GeSi electro-absorption
modulators. Nat. Photon. 2, 433–437 (2008). 2. Pillarisetty, R. Academic and industry research progress in germanium
nanodevices. Nature 479, 324–328 (2011). 3. Bakkers, E. P. A. M. et al. Epitaxial growth of InP nanowires on germanium. Nat. Mater. 3, 769–773 (2004). 4. Liu, H. et al. Long-wavelength InAs/GaAs quantum-dot laser diode
monolithically grown on Ge substrate. Nat. Photon. 5, 416–419 (2011 5. Lever, L. et al. Modulation of the absorption coefficient at 1.3 mm in Ge/SiGe
multiple quantum well heterostructures on silicon. Opt. Lett. 36, 4158–4160
(2011). 6. Chaisakul, P. et al. Integrated germanium optical interconnects on silicon
substrates. Nat. Photon. 8, 482–488 (2014). 7. Littlejohns, C. G. et al. 50 Gb/s silicon photonics receiver with low insertion loss. IEEE Photon. Tech. Lett. 26, 714–717 (2014). 8. Thomson, D. J. Methods et al. Silicon carrier depletion modulator with 10 Gbit/s driver
realized in high-performance photonic BiCMOS. Laser & Photon. Rev. 8, 180–187
(2014). 9. Pitera, A. J. et al. Coplanar integration of lattice-mismatched semiconductors with
silicon by wafer bonding Ge / Si12xGex / Si virtual substrates. J. Electrochem. Soc. 151, G443–G447 (2004). 10. Taraschi, G., Pitera, A. J. & Fitzgerald, E. A. Strained Si, SiGe, and Ge on-insulator
review of wafer bonding fabrication techniques. Solid-State Electron. 48,
1297–1305 (2004). Figure 5 | SEM image of RMG SiGe with poly-Si seed. The poly-Si seed
deposited on a thick (2 mm) SiO2 layer does not have the heat sinking
capabilities of a bulk Si seed resulting in a slower regrowth front
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8288 | DOI 10 1038/
08288
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Southampton. All fabrication was carried out at the Southampton Nanofabrication Centre. The CVD SiGe-on-Si samples for SIMS and Raman analysis were provided by IHP. g
The research leading to these results was funded by the UK Engineering and Physical
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‘‘HERMES’’, as well as the ‘‘Adventure for Research’’ fund provided by the University of
Southampton. All fabrication was carried out at the Southampton Nanofabrication Centre. The CVD SiGe-on-Si samples for SIMS and Raman analysis were provided by IHP. g
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Exploring sensorimotor performance and user experience within a virtual reality golf putting simulator
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Abstract In light of recent advances in technology, there has been growing interest in virtual reality (VR) simulations for training
purposes in a range of high-performance environments, from sport to nuclear decommissioning. For a VR simulation to elicit
effective transfer of training to the real-world, it must provide a sufficient level of validity, that is, it must be representative
of the real-world skill. In order to develop the most effective simulations, assessments of validity should be carried out prior
to implementing simulations in training. The aim of this work was to test elements of the physical fidelity, psychological
fidelity and construct validity of a VR golf putting simulation. Self-report measures of task load and presence in the simula-
tion were taken following real and simulated golf putting to assess psychological and physical fidelity. The performance of
novice and expert golfers in the simulation was also compared as an initial test of construct validity. Participants reported
a high degree of presence in the simulation, and there was little difference between real and virtual putting in terms of task
demands. Experts performed significantly better in the simulation than novices (p = .001, d = 1.23), and there was a significant
relationship between performance on the real and virtual tasks (r = .46, p = .004). The results indicated that the simulation
exhibited an acceptable degree of construct validity and psychological fidelity. However, some differences between the real
and virtual tasks emerged, suggesting further validation work is required. Keywords VR · Sport · Training · Construct validity · Simulation 1
School of Sport and Health Sciences, University of Exeter,
St Luke’s Campus, Exeter EX1 2LU, UK
2
School of Psychology, University of Leeds, Leeds LS2 9JZ,
UK
3
Centre for Immersive Technologies, University of Leeds,
Leeds LS2 9JZ, UK
4
Bradford Teaching Hospitals NHS Foundation Trust,
Bradford, West Yorkshire, UK
5
National Centre for Optics, Vision and Eye Care, University
of South-Eastern Norway, Hasbergs vei 36, 3616 Kongsberg,
Norway David J. Harris1 · Gavin Buckingham1 · Mark R. Wilson1 · Jack Brookes2 · Faisal Mushtaq2,3 ·
Mark Mon‑Williams2,3,4,5 · Samuel J. Vine1 David J. Harris1 · Gavin Buckingham1 · Mark R. Wilson1 · Jack Brookes2 · Faisal Mushtaq2,3
Mark Mon‑Williams2,3,4,5 · Samuel J. Vine1 Received: 3 July 2019 / Accepted: 10 October 2020 / Published online: 21 October 2020
© The Author(s) 2020 5
National Centre for Optics, Vision and Eye Care, University
of South-Eastern Norway, Hasbergs vei 36, 3616 Kongsberg,
Norway 1
School of Sport and Health Sciences, University of Exeter,
St Luke’s Campus, Exeter EX1 2LU, UK Virtual Reality (2021) 25:647–654
https://doi.org/10.1007/s10055-020-00480-4 Virtual Reality (2021) 25:647–654
https://doi.org/10.1007/s10055-020-00480-4 ORIGINAL ARTICLE ORIGINAL ARTICLE 1 Introduction 2018), as well as surgery (Gurusamy et al. 2008) and reha-
bilitation (Adamovich et al. 2009). This often involves the
generation of an immersive computer-simulation which the
user can interact with and manipulate (Burdea and Coif-
fet 2003; Wann and Mon-Williams 1996). Consumer grade The use of immersive training is a growing area of interest
in many sectors. Virtual reality (VR) training has already
been applied to sporting skills (Gray 2019; Neumann et al. * David J. Harris
D.J.Harris@exeter.ac.uk
Gavin Buckingham
G.Buckingham@exeter.ac.uk
Mark R. Wilson
Mark.Wilson@exeter.ac.uk
Jack Brookes
ed11jb@leeds.ac.uk
Faisal Mushtaq
F.Mushtaq@leeds.ac.uk
Mark Mon‑Williams
M.Mon‑Williams@leeds.ac.uk
Samuel J. Vine
S.J.Vine@exeter.ac.uk 3
Centre for Immersive Technologies, University of Leeds,
Leeds LS2 9JZ, UK 4
Bradford Teaching Hospitals NHS Foundation Trust,
Bradford, West Yorkshire, UK (0123
1 23456789)
3 Virtual Reality (2021) 25:647–654 648 Table 1 Types of fidelity and validity
Type
Description
How to test
Physical fidelity (or face validity)
Is there a high degree of detail and realism pro-
vided by the physical elements of the simulation? User reports of realism and experiences of immer-
sion
Construct validity
Does the simulation provide an accurate represen-
tation of real task performance? Ability of the simulation to distinguish real-world
experts form novices
Emotional/affective fidelity
Does the simulation elicit emotional responses,
such as fear or enjoyment, which are similar to
the real task? Self-reported experiences of users, or online
monitoring of psychophysiology
Ergonomic and biomechanical fidelity
Does the simulation demand/allow realistic motor
movements? Comparison of movement amplitude and speed or
joint coordination between real and simulated
task
Psychological fidelity
Does the simulation replicate the perceptual-cogni-
tive demands of the real task? Measurement of mental effort, gaze behaviour or
neural activity Table 1 Types of fidelity and validity VR equipment that delivers such interactive and immersive
environments is becoming increasingly affordable and port-
able, thus providing new possibilities for training. For these
training approaches to enable effective transfer to real-world
performance, simulations must be representative of the real
skills that they intend to train and accurately represent real
performance (Bright et al. 2012; Gray 2019). Therefore,
more rigorous testing of the validity and fidelity of environ-
ments is required before they can be widely adopted in sports
training (Harris et al. 2020). Here, we explore an evidence-
based approach to simulation validation by testing the fidel-
ity and validity of a golf putting simulator. 1 Introduction for juggling and table tennis with low fidelity 2D environ-
ments (Lammfromm and Gopher 2011; Todorov et al. 1997). Gray (2019) suggests that, with regards to training, psycho-
logical fidelity (the extent to which the environment repli-
cates the perceptual-cognitive demands of the real task) is
more important than physical fidelity. High psychological
fidelity means athletes are more likely to utilise similar per-
ceptual information to control their actions, display similar
gaze behaviour, and experience a similar level of cognitive
demand to the real-world task. These additional elements are
likely to facilitate transfer (Gray 2019; Harris et al. 2020). Therefore, in order to develop more effective simulations,
more attention must be paid to testing a range of validity
metrics. In the sporting domain, the existing (but sparse) research
has largely supported the effectiveness of VR training for
visuomotor skills such as baseball batting, darts and table
tennis (Gray 2017; Lammfromm and Gopher 2011; Tirp
et al. 2015; Todorov et al. 1997). However, it remains
unclear which facets of a simulation determine training
effectiveness. Fidelity and validity refer to particular quali-
ties of the simulation that contribute to training success
(Gray 2019). Fidelity refers to how well a simulation recon-
structs the real-world environment, both in terms of appear-
ance but also the emotional states, cognitions and behaviours
it elicits from its users. Meanwhile, validity broadly relates
to accurate measurement or reproduction of real task perfor-
mance. This is not to say the simulation is the same, but that
it captures key features of the task (Stoffregen et al. 2003). While a high degree of physical fidelity1 (how a simulation
looks, feels and sounds) is useful for immersing the user and
achieving engagement (Ijsselsteijn et al. 2004), skills can
still be learned in environments with low physical fidelity. For instance, positive real-world transfer has been observed First, a valid simulation, which is likely to produce posi-
tive real-world transfer, should have good physical fidelity
to provide an immersive experience and elicit user engage-
ment (Bowman and McMahan 2007; Ijsselsteijn et al. 2004). Second, it should display construct validity, that is, it should
provide a good representation of the task it intends to train,
and accurately reflect performance. For instance, for a simu-
lation to be a valid representation of a sporting skill, indi-
viduals with real-world expertise should also be experts in
the simulation. 1 The term face validity is sometimes used to refer to the physical
accuracy of a simulation, particularly in the surgical training litera-
ture (e.g. Bright et al. 2012). 2 Presence refers to the psychological response of feeling ‘in the sim-
ulation’. Presence is linked to immersion, which is the objective level
of sensory fidelity provided by the VR system (Slater 2003), but the
terms are sometimes used interchangeably. 1 Introduction This kind of expert versus novice discrimina-
tion has been widely used for validating surgical simulations
(Bright et al. 2012; Sweet et al. 2004), as it indicates whether
the simulation has captured some important features of task
performance. Finally, the perceptual-cognitive demands of
the task should also resemble the real task (i.e. psychological
fidelity). Ideally, measures such as gaze behaviour and men-
tal effort should be employed to compare the psychological
demands of the virtual and real tasks. Additional types of
fidelity that could contribute to successful design include
ergonomic/biomechanical and affective/emotional fidelity
(see summary of types of fidelity in Table 1). i
Consequently, we aimed to assess the validity of a golf
putting simulation in a variety of ways. Following the 1 3 649 Virtual Reality (2021) 25:647–654 Fig. 1 The VR putting task
(left) and real-world putting
task (right) amework for the testing and
ments (Harris et al. 2020) we
ments of fidelity and validity
as golf putting, we assessed whether distance was perceived
to be similar between the real and VR tasks. Fig. 1 The VR putting task
(left) and real-world putting
task (right) as golf putting, we assessed whether distance was perceived
to be similar between the real and VR tasks. recommendations of recent framework for the testing and
validation of simulated environments (Harris et al. 2020) we
aimed to assess a number of elements of fidelity and validity
of the VR environment. Firstly, participants were asked to
report feelings of presence2 to ensure that the simulation was
of sufficiently high physical fidelity to immerse the user in
the task (Slater 2003). Secondly, as in medical simulation
validation we assessed construct validity by comparing the
performance of novices and experts in the simulation (Bright
et al. 2012; Sweet et al. 2004). If real-world expertise corre-
sponds to expertise in the simulation, it would provide initial
evidence that the simulation is a good representation of the
real task. It was expected that expert golfers would exhibit
significantly better performance in both real and simulated
putting, and that there would be a correspondence between
real-world and VR performance. Thirdly, to address aspects
of psychological fidelity, that is the perceptual and cognitive
demands of the simulation, we explored whether the cogni-
tive demands of the simulation were similar to the real-world
using a self-report measure of task load (the SIM-TLX; Har-
ris et al. 2019b). 2.1 Participants Thirty-six participants took part in the study; 18 expert ama-
teur golfers (11 male, mean age = 29.2 years, SD = 13.7)
and 18 novice golfers (10 male, mean age = 21.9 years,
SD = 1.8). Expert golfers were recruited from three com-
petitive golf teams (University of Exeter Golf Club, Exeter
Golf and Country Club, and Devon Golf men’s first team). All expert golfers were of a high level and held active cat-
egory one handicaps (≤ 5.0), with an average handicap of
1.7 (SD = 2.5). Novice golfers were recruited from under-
graduate sport science students at the University of Exeter
(mean age = 21.94 years, SD = 1.83). ‘Novice golfers’ were
defined as individuals with no official golf handicaps or prior
formal golf putting experience (as in Moore et al. 2012). Participants were provided with details of the study before
attending testing, and gave written consent before testing
began. Ethical approval was obtained from the University
Ethics Committee prior to data collection. 1 Introduction Additionally, we compared perceived dis-
tance to the hole between real and VR tasks. Concerns have
been raised that stereoscopic viewing in head-mounted dis-
plays can influence the performance of visuomotor skills due
to the conflict between cues to depth (Bingham et al. 2001;
Harris et al. 2019a). Additionally, some early three degrees
of freedom VR systems induced a perception of ‘flatness’
(Willemsen et al. 2008). As processing of distance and depth
information is important for performing an aiming skill such recommendations of recent framework for the testing and
validation of simulated environments (Harris et al. 2020) we
aimed to assess a number of elements of fidelity and validity
of the VR environment. Firstly, participants were asked to
report feelings of presence2 to ensure that the simulation was
of sufficiently high physical fidelity to immerse the user in
the task (Slater 2003). Secondly, as in medical simulation
validation we assessed construct validity by comparing the
performance of novices and experts in the simulation (Bright
et al. 2012; Sweet et al. 2004). If real-world expertise corre-
sponds to expertise in the simulation, it would provide initial
evidence that the simulation is a good representation of the
real task. It was expected that expert golfers would exhibit
significantly better performance in both real and simulated
putting, and that there would be a correspondence between
real-world and VR performance. Thirdly, to address aspects
of psychological fidelity, that is the perceptual and cognitive
demands of the simulation, we explored whether the cogni-
tive demands of the simulation were similar to the real-world
using a self-report measure of task load (the SIM-TLX; Har-
ris et al. 2019b). Additionally, we compared perceived dis-
tance to the hole between real and VR tasks. Concerns have
been raised that stereoscopic viewing in head-mounted dis-
plays can influence the performance of visuomotor skills due
to the conflict between cues to depth (Bingham et al. 2001;
Harris et al. 2019a). Additionally, some early three degrees
of freedom VR systems induced a perception of ‘flatness’
(Willemsen et al. 2008). As processing of distance and depth
information is important for performing an aiming skill such Fig. 1 The VR putting task
(left) and real-world putting
task (right) 2.2.2 Real‑world golf task Real-world putts were taken from 10ft (3.05 m) to a tar-
get the size of a regulation hole (diameter 10.80 cm) on an
indoor artificial putting green. As in the simulation, the hole
was covered so it remained visible but the ball would not
drop in. Participants used a standard putter (Cleveland Clas-
sic Collection HB 1 putter), and regulation size (4.27 cm
diameter) white golf balls. 2.3.1 Presence questionnaire Expert participants were asked to rate their sense of pres-
ence—the feeling that they were really in the virtual
world—using questions from the Presence Questionnaire
(Slater et al. 1995). These questions relate to the experi-
ence of being in the virtual environment, such as ‘To what
extent were there times during the experience when the vir-
tual environment was the reality for you?’ There are three
questions in total and responses are given on a seven-point
Likert scale. Participants attended the lab on one occasion for approxi-
mately 20 min. Participants were given just 3 familiarisation
putts on each of the real and VR tasks to avoid any learn-
ing effects. They then completed 10 real-world putts and 10
putts in VR, which was done in a counterbalanced order to
account for order effects. Participants were instructed to land
the ball as close to the ‘hole’ as possible. After each of the
baseline assessments, participants were asked to estimate
the distance to the hole. Following the VR putts, experts
also answered the Presence Questionnaire and completed
the SIM-TLX. 2.3.4 Simulation task load index (SIM‑TLX) The SIM-TLX (Harris et al. 2019a) assesses workload dur-
ing simulated tasks using a two-part evaluation. Following
task completion participants rated the level of demand they
had experienced on nine bipolar scales, reflecting different
workload dimensions: mental demands; physical demands;
temporal demands; frustration; task complexity; situational
stress; distractions; perceptual strain; and task control. Rat-
ings were made on a visual analogue scale anchored between
‘very low’ and ‘very high’ (see supplementary materials). Participants also rated the relative importance of each work-
load dimension for the task, by making a series of 45 pair-
wise comparisons between dimensions. A workload score
for each dimension is calculated based on the product of
the visual analogue scores and the weighting dimension. An
overall workload score is obtained from the sum of all nine
dimensions (cf. the NASA-TLX; Hart and Staveland 1988). 2.3.3 Perceived distance graphics card (NVIDIA Inc., Santa Clara, CA). A Vive
tracker sensor was attached to the head of a real putter to
allow tracking and create the VR putter. Auditory feedback
(which sounded like a club striking a ball) was provided
on contact with the ball, but no additional haptic feedback
was provided. The ball was matched in size and weight to
a real golf ball. Participants putted from 10 ft (3.05 m) to
a target the same size and shape (diameter 10.80 cm) as
a standard golf hole. The game incorporated background
audio (wind in the trees and bird noises) to simulate the
real-world and enhance immersion. Following both VR and real-world baseline putts partici-
pants were asked to make a verbal estimate, in feet, of the
distance between the hole and the putting location. The esti-
mate was recorded by the experimenter. 2.2.1 VR golf task The VR golf task (see Fig. 1) was developed using the
gaming engine Unity 2018.2.10.f1 and the Unity Experi-
ment Framework (Brookes et al. 2019). The simulation
was presented using an HTC Vive HMD (HTC Inc., Taoy-
uan City, Taiwan), and a 3xs laptop (Scan Computers, Bol-
ton, UK) with an i7 processer and GeForce GTX 1080 1 1 650 Virtual Reality (2021) 25:647–654 2.5 Statistical analysis Putting performance in both the real-world and VR was
operationalized as radial error of the ball from the hole (i.e. the two-dimensional Euclidean distance between the top of
the ball and the edge of the hole; in cm), as in Walters-
Symons et al. (2018). In the real-world condition the dis-
tance was measured with a tape measure following each
attempt and was recorded automatically by the simulation
in the VR condition. If the ball landed on top of the hole, a
score of zero was recorded. On real-world trials where the
ball hit the boundary of the putting green (90 cm behind the
hole), the largest possible error was recorded (90 cm) (as in
Moore et al. 2012). Statistical analysis was performed in JASP (v0.9.2; JASP
team 2018). Data were checked for homogeneity of variance
(Levene’s test), and skewness and kurtosis. Performance
data exceeding 3 standard deviations from the mean were
excluded (three data points for VR putting and zero data
points for real-world putting). A 2 (Group: Expert vs Nov-
ice) × 2 (Task: real-world vs VR) mixed ANOVA was used
to compare both putting accuracy and distance estimation for
novice and expert groups between VR and real-world tasks. A MANOVA was run on overall TLX questionnaire scores,
with follow-up t-tests to examine individual scales. Cohen’s 1 3 Results A 2 (group) x 2 (task) mixed ANOVA showed that there was
no difference in perceived putting distance between real-
world and VR tasks, F(1,34) = 0.07, p = .79, ηp
2 = .002. There
was, however, a significant effect of group, F(1,34) = 16.97,
p < .001, ηp
2 = .330, with novices estimating the hole to be
closer than their more experienced golfing counterparts
(see Fig. 4). There was no interaction effect, F(1,34) = 0.18,
p = .67, ηp
2 = .005. 3 3 Virtual Reality (2021) 25:647–654 651 5
10
15
20
0
1
2
3
4
Frequency
Presence score
Fig. 2 Distribution plot of self-reported presence during the VR sim-
ulation the VR environment. The distribution plot below (Fig. 2)
shows, however, that there was a high degree of variation
across participants. 3.2 Putting performance To compare group differences in real-world and VR task
performance a 2 (task) x 2 (group) ANOVA was run on
radial error scores. A main effect of task indicated that
radial error scores were significantly higher in the VR task,
F(1,34) = 25.29, p < .001, ηp
2 = .43. There was also a main
effect of group, with experts displaying lower radial errors,
F(1,34) = 33.06, p < .001, ηp
2 = .49. There was no interac-
tion effect, F(1,34) = 0.25, p = .62, ηp
2 = .01. As expected,
Bonferroni-Holm corrected t-tests showed that, compared
to novice golfers (M = 51.6 cm, SD = 50.9), expert golf-
ers (M = 25.5 cm, SD = 23.4) displayed significantly lower
radial errors when putting in the real-world, t(34) = 6.84,
p < .001, d = 2.28. Similarly, compared to novice golfers
(M = 74.2 cm, SD = 29.3), expert golfers (M = 44.1 cm,
SD = 18.5) displayed significantly lower radial errors when
putting in VR, t(34) = 3.68, p = .001, d = 1.23 (Fig. 3). Over-
all there was a significant positive relationship between per-
formance on the real and virtual tasks, r(35) = .46, p = .004. Fig. 2 Distribution plot of self-reported presence during the VR sim-
ulation d effect sizes were calculated for all t-tests, and partial eta
squared for all F-tests. All data are available through the
Open Science Framework (https://osf.io/dchgz/). 3.1 Presence 4
illustrates that while the expert group were accurate across
both conditions, admittedly with some variability, novices
consistently underestimated distance Underestimation of
Fig. 4 Estimated putting
distances for novice and expert
golfers in a real-world and b
VR conditions. Individual data
are provided as ‘dots’ and error
bars show standard error of the
mean. The dashed horizontal
line indicates veridical putting
distance timated putting
for novice and expert
a real-world and b
itions. Individual data
ded as ‘dots’ and error
w standard error of the
e dashed horizontal
ates veridical putting 3.4 Task load
A one-way multivariate ANOVA was run to test whether
there was an overall difference in task load (as measured by
the SIM-TLX) between real and virtual putting. There was
an overall effect of group, F(1,34) = 2.75, p = .02, Pillai’s
trace=0 49 Follow-up t-tests with Bonferroni-Holm cor-
the real and virtual tasks, displaying significantly lower
radial errors. We also found an overall positive correlation
between real and virtual performance. The radial errors of
both groups were, however, larger in VR, as was the vari-
ance in performance (see Fig. 3). Additionally, the corre-
lation between real and VR performance was only moder-
ate (r = 46) This highlights that although the simulation
Fig. 4 Estimated putting
distances for novice and expert
golfers in a real-world and b
VR conditions. Individual data
are provided as ‘dots’ and error
bars show standard error of the
mean. The dashed horizontal
line indicates veridical putting
distance the real and virtual tasks, displaying significantly lower
radial errors. We also found an overall positive correlation
between real and virtual performance. The radial errors of
both groups were, however, larger in VR, as was the vari-
ance in performance (see Fig. 3). Additionally, the corre-
lation between real and VR performance was only moder-
ate (r = .46). This highlights that, although the simulation
showed a certain level of construct validity, there remain
important differences between the two tasks. An additional
finding, which further supports the construct validity of the
simulation, was that expert golfers were more accurate at
estimating putting distance in both tasks. It has previously
been demonstrated that golfers are more accurate at esti-
mating egocentric distance (Durgin and Li 2011) and Fig. 4
illustrates that while the expert group were accurate across
both conditions, admittedly with some variability, novices
consistently underestimated distance. 3.1 Presence Underestimation of
distance has previously been reported as a general effect in 4 Discussion Despite previous work extolling the benefits of training
sporting skills in VR (Gray 2017; Lammfromm and Gopher
2011; Tirp et al. 2015), methods for testing the validity of
environments prior to their use are not well established (Har-
ris et al. 2020). For the field of VR training to progress,
rigorous approaches to simulation validation are required
(e.g. Bright et al. 2012; Sweet et al. 2004; Tirp et al. 2015). Consequently, we aimed to apply an evidence-based method
in testing the validity and fidelity of a golf putting simulator. Despite previous work extolling the benefits of training
sporting skills in VR (Gray 2017; Lammfromm and Gopher
2011; Tirp et al. 2015), methods for testing the validity of
environments prior to their use are not well established (Har-
ris et al. 2020). For the field of VR training to progress,
rigorous approaches to simulation validation are required
(e.g. Bright et al. 2012; Sweet et al. 2004; Tirp et al. 2015). Consequently, we aimed to apply an evidence-based method
in testing the validity and fidelity of a golf putting simulator. Overall, evaluation of the simulation indicated a fairly
good correspondence between the real and virtual tasks. Firstly, expert users reported a moderate to high level of
presence in the environment, relative to previous use of this
scale (Usoh et al. 2000), suggesting that the physical fidel-
ity was sufficient to immerse users in the task. Presence is
unlikely to be a major determinant of training efficacy but
is an important first step for user engagement (Bowman and
McMahan 2007; Ijsselsteijn et al. 2004). Fig. 5 Grouped plot of the nine subscales of task load for real-world
and VR tasks. Error bars indicate SEM i
Overall, evaluation of the simulation indicated a fairly
good correspondence between the real and virtual tasks. Firstly, expert users reported a moderate to high level of
presence in the environment, relative to previous use of this
scale (Usoh et al. 2000), suggesting that the physical fidel-
ity was sufficient to immerse users in the task. Presence is
unlikely to be a major determinant of training efficacy but
is an important first step for user engagement (Bowman and
McMahan 2007; Ijsselsteijn et al. 2004). Secondly, the construct validity of the simulation was
supported, as expert golfers outperformed novices in both Fig. 5 Grouped plot of the nine subscales of task load for real-world
and VR tasks. 3.4 Task load A one-way multivariate ANOVA was run to test whether
there was an overall difference in task load (as measured by
the SIM-TLX) between real and virtual putting. There was
an overall effect of group, F(1,34) = 2.75, p = .02, Pillai’s
trace = 0.49. Follow-up t-tests, with Bonferroni-Holm cor-
rection, revealed that the only dimension of task load that
significantly differed between VR and real-world conditions
was perceptual strain (p = .02), which was higher in VR. All
other comparisons were non-significant (p’s > .24). Figure 5
suggests that situational stress and time demands were also
somewhat higher in the VR condition. 3.1 Presence Overall scores on the Presence Questionnaire (completed
by expert golfers only) could potentially range from 0 to 21
(21 indicating the highest level of presence). The mean score
during VR putting was 12.7 (SD = 4.5), above the mid-way
point of the scale, indicating a good level of presence in Fig. 3 Radial error scores for novice and expert golfers in a real-world and b VR conditions. Individual data points are shown overlaid on group-
mean scores, with error bars indicating standard error of the mean. c The positive correlation between real and virtual performance Fig. 3 Radial error scores for novice and expert golfers in a real-world and b VR conditions. Individual data points are shown overlaid on group-
mean scores, with error bars indicating standard error of the mean. c The positive correlation between real and virtual performance 1 3 652 Virtual Reality (2021) 25:647–654 3.4 Task load
A one-way multivariate ANOVA was run to test whether
there was an overall difference in task load (as measured by
the SIM-TLX) between real and virtual putting. There was
an overall effect of group, F(1,34) = 2.75, p = .02, Pillai’s
trace = 0.49. Follow-up t-tests, with Bonferroni-Holm cor-
rection, revealed that the only dimension of task load that
significantly differed between VR and real-world conditions
was perceptual strain (p = .02), which was higher in VR. All
other comparisons were non-significant (p’s > .24). Figure 5
suggests that situational stress and time demands were also
somewhat higher in the VR condition. 4 Discussion
the real and virtual tasks, displaying significantly lower
radial errors. We also found an overall positive correlation
between real and virtual performance. The radial errors of
both groups were, however, larger in VR, as was the vari-
ance in performance (see Fig. 3). Additionally, the corre-
lation between real and VR performance was only moder-
ate (r = .46). This highlights that, although the simulation
showed a certain level of construct validity, there remain
important differences between the two tasks. An additional
finding, which further supports the construct validity of the
simulation, was that expert golfers were more accurate at
estimating putting distance in both tasks. It has previously
been demonstrated that golfers are more accurate at esti-
mating egocentric distance (Durgin and Li 2011) and Fig. 1 3 5 Conclusions Bideau B, Kulpa R, Ménardais S, Fradet L, Multon F, Delamarche P,
Arnaldi B (2003) Real handball goalkeeper vs virtual handball
thrower. Presence Teleoper Virtual Environ 12(4):411–421 In this study, we aimed to adopt an evidence-based approach
to testing the validity of a VR simulation of a sporting skill. Assessment of construct validity clearly indicated that the
putting simulation was sufficiently representative of the real
task to distinguish novice from expert performers. Addition-
ally, the simulation created a good degree of presence in
the virtual environment. Assessments of task load indicated
some differences from the real task, but overall the simula-
tion was a fair representation of real putting. Nevertheless,
this does not mean that the simulation is fully validated,
and further work is still required, such as assessing biome-
chanical fidelity through comparisons of body and putter
movement to determine whether the simulation elicits real-
istic putting mechanics. Future work may seek to investigate
additional sources of fidelity, such as affective and biome-
chanical fidelity (e.g. Bideau et al. 2003, 2004) and their
relationship with transfer of learning. Additionally, greater
fidelity in terms of self-visualization may contribute to more
embodiment and presence in the simulation and could influ-
ence the acquisition of sporting skills (Slater et al. 2009). Bideau B, Multon F, Kulpa R, Fradet L, Arnaldi B (2004) Virtual real-
ity applied to sports: do handball goalkeepers react realistically
to simulated synthetic opponents? In: Proceedings of the 2004
ACM SIGGRAPH international conference on Virtual Reality
continuum and its applications in industry, pp 210–216 Bingham GP, Bradley A, Bailey M, Vinner R (2001) Accommo-
dation, occlusion, and disparity matching are used to guide
reaching: a comparison of actual versus virtual environ-
ments. J Exp Psychol Hum 27(6):1314–1334. https://doi. org/10.1037/0096-1523.27.6.1314 Bowman DA, McMahan RP (2007) Virtual reality: how much immer-
sion is enough? Computer 40:36–43. https://doi.org/10.1109/
MC.2007.257 Bright E, Vine S, Wilson MR, Masters RSW, McGrath JS (2012) Face
validity, construct validity and training benefits of a virtual reality
turp simulator. Int J Surg 10:163–166. https://doi.org/10.1016/j. ijsu.2012.02.012 Brookes J, Warburton M, Alghadier M, Mon-Williams M, Mushtaq
F (2019) Studying human behavior with virtual reality: the
unity experiment framework. Res Methods Behav. https://doi. org/10.3758/s13428-019-01242-0f g
Burdea GC, Coiffet P (2003) Virtual reality technology. Wiley, New
York In general, caution is still required in the use of VR for
training sporting skills. 4 Discussion Error bars indicate SEM 1 3 Virtual Reality (2021) 25:647–654 653 skills and for some purposes—to allow the study and/or
training of sporting skills. head-mounted VR, although this effect does seem attenuated
in newer systems (Interrante et al. 2006). A similar degree
of underestimation by novices was seen in both real and VR
tasks in the present work.i skills and for some purposes—to allow the study and/or
training of sporting skills. Funding This work was supported by a Royal Academy of Engineering
UKIC Fellowship awarded to D Harris. F.M and M.M-W were sup-
ported by Fellowships from the Alan Turing Institute and a Research
Grant from the EPSRC (EP/R031193/1). With regard to assessing psychological fidelity, there was
no difference in perceived putting distance between the two
tasks. As distance estimation plays an important role in the
golf putt, and perceptual information can be distorted in VR
(Willemsen et al. 2008), this is an encouraging result. One
possibility to consider, however, is that participants could
have assumed that distances were similar and aligned their
second estimate with their first. Additionally, self-report
scales were used to assess whether the demands of the VR
task differed from the real task. After accounting for mul-
tiple comparisons, only the degree of perceptual strain was
significantly different between the tasks. Perceptual difficul-
ties in HMDs are not uncommon; users can experience dis-
comfort as a result of rendering lag or multisensory conflict
during locomotion, which may preclude their use by some
individuals (Cobb et al. 1999). Overall, these assessments
largely supported the psychological fidelity of the VR task. Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Adamovich SV, Fluet GG, Tunik E, Merians AS (2009) Sensorimotor
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3:1–5 1 3 3 3
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Inclusive Green Development: A Critical Study in the Present Crisis of India
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Samir Kumar Lobwo, Jacob K Davis, Vijay Daniel Anthony Abstract: The concept of green growth assumes centrality of
socio-economic inclusiveness towards sustainable development in
India. Inclusive green growth currently is a development strategy,
which included both economic growth in addition to sustainable
development. In India, for instance, growth in addition to
development is said to have always existed a part of the planning
process.The study makes a descriptive analysis based on the
available data. It highlights the present status of India exists
comparisons
with
other
emerging
countries. While
acknowledging the importance of development strategies to adopt
the principles of green economy in tune with stage of economic
development, the study points out the critical gaps in addition to
problems faced by India just as well as their possible solutions. However, this is not sufficient. Sustainability can be
said to be dependent on satisfying everyone’s demand
without dumping ecological concerns. And a Green
Economy should be best to achieve the former objective. Economy should be best to achieve the former objective. A green economy currently exists as a clean,
environmentally friendly economy that promotes health,
wealth, in addition to well-being. A green economy’s
existence is dependent on sustainable development – and
this means growing our economies in ways that give
importance to benefit, not sacrifice, social justice in addition
to equity just as well as the environment. Green accounting
currently exists as a type of accounting that tries to factor in
environmental costs into the financial results of operations. Merely, gross domestic product (GDP) ignores the
environment, therefore policymakers need a revised model
that incorporates green accounting. The idea of green
growth has its start in Asia in addition to Pacific field,
range. At the fifth Ministerial Conference on general
condition and Development (MCED) said nothing in
March 2005 in Seoul, 52 governments in addition to
other interested organizations from Asia in addition to
the Pacific agreed to move beyond the able to keep going
development over-blown effects in the art of talking in
addition to go after a footway of "green growth". To do
so, they took up a ministerial Declaration (the Seoul
initiative network on green growth) in addition to a
partwise putting into effect map for able to keep going
development (UNESCAP, 2008). International Journal of Management and Humanities (IJMH)
ISSN: 2394–0913 (Online), Volume-5 Issue-1, September 2020 International Journal of Management and Humanities (IJMH)
ISSN: 2394–0913 (Online), Volume-5 Issue-1, September 2020 International Journal of Management and Humanities (IJMH)
ISSN: 2394–0913 (Online), Volume-5 Issue-1, September 2020 Samir Kumar Lobwo, Jacob K Davis, Vijay Daniel Anthony This attempt to stop
over here started a wider act or power of seeing of green
growth just as a part-wise first move of UNESCAP, at
whatever place it currently has existence viewed just as a
key carefully worked design for doing able to keep going
development just as well just as the time of 1,000 years
from end to end development goals (UNESCAP, 2012). Key
Words:
Green
growth,
Sustainable
development,
UNESCAP. Inclusive Green Development: A Critical Study in
the Present Crisis of India Samir Kumar Lobwo, Jacob K Davis, Vijay Daniel Anthony Published By:
Blue Eyes Intelligence Engineering
& Sciences Publication
© Copyright: All rights reserved. I.INTRODUCTION In developing countries, green growth in addition to
sustainable measures have not existed successfully due to
lack of basic development priorities and lack of fund flow
from the developed world. India is currently facing the
problem of co-existence of the conventional economic
growth strategy along with addition to piecemeal efforts to
make the economy ready to mitigate in addition to adapt to
the climate change issues. Undeniably, 'growth' has an
essential part of development planning with the gross
domestic product (GDP), as an indicator for economic
progress in Asian countries. Amartya Sen has argued that
growth-mediated development and supplemented through
social protection policies have led to improved human well-
being with trends such as increase in life expectancy,
reduction in the undernourished population, improvement in
the number of people having access to basic health services
in addition to education. However, several neglected areas
remain that can pose a challenge for inclusive growth and
sustainable development in Asia.We all know that Growth
is needed for Poverty reduction programs and social
protection policies. Green growth according to Lorek et
al is is ‘a political catchword, coined to overcome
reservations of the business sector against all kinds of
‘greenery’,
regardless
of
the
potential
economic
benefits.’ To do this there is a need to bring in
Technological Solutions which will promote more
greenery. According to a Corporates Knights the world’s most
sustainable company is Chr. Hansen Holding, a Danish
bioscience firm that derives over 80% of its revenue
developing natural solutions for preserving foods like yogurt
and milk, protecting crops using natural bacteria instead of
pesticides, and alternatives to antibiotics for animals. It also
has nearly 30% of its business’ board is made up of women,
and its CEO salaries are about 24 times those of an average
worker with the firm—a relatively low ratio for the ranking. Norway has been placed rank 1 in the list of most
sustainable companies by RobecoSAM. It has been placed
first in terms of Environment, social and Governance. It is
closely followed by Sweden, Finland and Denmark ranking
just behind Norway, whilst Switzerland follows in fifth
position. According to Yale Centre for Environmental
Law and Policy which conducts the EPI (Environment
Performance INDEX), Denmark tops the list with an EPI
score of 82.5 followed by
Luxembourg,
Switzerland
and United Kingdom. AUTHOR’S PROFILE: India needs to focus on food security but it should not be
at the cost of supplanting nutritional security. Richa Kumar
(2019) has brought about how through green revolution in
India achieved self-sufficiency in production of two cereals
but on the other hand it also involved heavy imports of rock
phosphate for fertilizers and petroleum for tractors. Added
to that, India heavily imports pulses and oilseeds from
Indonesia and Myanmar which could have been avoided by
focusing equally on varied crop varieties instead of just two. Agriculture should focus on providing new improved
healthy and nutritious food rather than being obsessed with
productivity and technology. Dr Samir Kumar Lobwo, was Associate
Professor, Department of Commerce, St. Xavier’s
College
(Autonomous),
Kolkata
and
Guest
Faculty, Department of Commerce, University of
Kolkata. D. Lobwo completed both his masters
and Ph.D in Commerce from University of
Calcutta. He was a Cost Accountant and he
specialized in Reporting Standards. Dr. Lobwo
was an excellent teacher, a prolific writer and a
very God-fearing Man. He was very humble and
loved by all. Dr. Lobwo left for his heavenly abode on 20th July 2020. Dr Samir Kumar Lobwo, was Associate
Professor, Department of Commerce, St. Xavier’s
College
(Autonomous),
Kolkata
and
Guest
Faculty, Department of Commerce, University of
Kolkata. D. Lobwo completed both his masters
and Ph.D in Commerce from University of
Calcutta. He was a Cost Accountant and he
specialized in Reporting Standards. Dr. Lobwo
was an excellent teacher, a prolific writer and a
very God-fearing Man. He was very humble and
wo left for his heavenly abode on 20th July 2020. P
U Professor. Jacob Davis K, Experienced Assistant
Professor with a demonstrated history of working
in the higher education industry. Skilled in
Data Analysis, Public Speaking, Administration,
Research, and Management. Strong education
professional with a Master of Philosophy - MPhil
focused in Business/Commerce, General from
University of Madras. The European Commission 2018, has said how
interaction between government, Industry, Academia and
civil participants will bring about more structural changes
than any single stakeholder could do it alone. And it is
pertinent that the ideas generated are user-oriented and
focused on involving much citizen participation. In the aftermath of the Covid crises there has been an
effort to push forward a new paradigm of Finance namely
Sustainable
Finance
and
sustainable
Investing. AUTHOR’S PROFILE: Sustainability in Finance is needed to achieve long term
financial success; besides it also showcases a robust
Business Model. The goal should be to promote digital
technologies which are human-centric, labour -augmenting
with proper regulation in areas of competition and data
security which could open up new areas of financial
investment. This would need a multi-country effort with the
United Nations leading the way. (Financing for Sustainable
Development Report 2020 Overview) Mr. Vijay daniel Anthony, teaches undergraduate
Economics at Loyola College, Department of
Economics. Published By:
Blue Eyes Intelligence Engineering
& Sciences Publication
© Copyright: All rights reserved. Inclusive Green Development: A Critical Study in the Present Crisis of India It basis its analysis on 32 performance indicators across
11 issue categories. have economic costs to implement in the short term. Thus, we may face a trade-off with economic growth and
environmental protection. However, over the long term,
job creation, poverty alleviation and increased efficiency
along with an improved lifestyle which promotes
environmental protection will go a long way to decrease
market failures and sub-optimal outcomes. g
The case of Malaysia stands as a classic case how
deliberate government intervention has paid off in
controlling an environmental crisis. In Malaysia’s case the
problem was posed due to Palm Oil Mills discharging
untreated effluents into water bodies which lead to a
massive demand for BOD (Biological Oxygen Demand)
load which is measure of the amount of pollution taking
place of water bodies. After government intervention, the
result was an overall reduction in average discharge and a
massive recycling exercise where the waste sludge was
recycled and used as an animal feed for pigs and poultry. Other byproducts like methane was recycled and used for
electricity generation. In addition, other byproducts were
used in production of pulp and paper from oil palm fronds,
oil palm lumber was used to make blackboards and oil palm
trunks were used in furniture production. REFERENCES: 1. Gupta, Anil K., K. K. Patel, A. R. Pastakia, and P. G. Vijaya Sherry
Chand. 1995. “Building upon local creativity and entrepreneurship
in vulnerable environments”. In V. Titi and N. Singh (eds.)
Empowerment for Sustainable Development: Towards Operational
Strategies. International
Institute
for
Sustainable
Development. pp.112–37. 2. Lau, A. K. W. and R. C. M. Yam. 2005. “A case study of product
modularization on supply chain design and coordination in Hong
Kong
and
China.” Journal
of
Manufacturing
Technology
Management 16(4):432–46. 3. Li, W. D., S. K. Ong, A. Y. C. Nee, and C. McMahon (eds.). 2007. Collaborative Product Design and Manufacturing Methodologies
and Applications. Springer Series in Advanced Manufacturing XIV. Available
at: http://www.springer.com/engineering/mechanical+eng/book/978
–1-84628-801-2Google Scholar Coming to India one realizes that Make in India Scheme
should not only try to bring about improvement in
manufacturing and utilization of labour to more productive
sectors but should ensure that it promotes green
development which will bring about the aspect of
sustainability. Kummitha (2019) stresses that Smart Cities
Mission (SCM) could be integrated with Make in India
which would yield much dividends. at: http://www.springer.com/engineering/mechanical+eng/book/978
–1-84628-801-2Google Scholar g
4. Piller, Frank T. 2008. “Interactive value creation with users and
customers.” In A.S. Huff (ed.). Leading Open Innovation. Munich:
Peter
Pribilla
Foundation. pp. 16–24. Available
at: http://www.mass-customization.de/download/piller_2008-
pribilla.pdf. Mr. Vijay daniel Anthony, teaches undergraduate
Economics at Loyola College, Department of
Economics. Revised Manuscript Received on September 13, 2020. *Corresponding Author By Late Dr Samir Kumar Lobwo*, Associate Professor, Department
of Commerce (Morning), St. Xavier’s College (Autonomous), Kolkata and
Guest Faculty, Department of Commerce, University of Calcutta Mr. Jacob K Davis, Assistant Professor, Department of Corporate
Secretaryship (Shift-2), Loyola College (Autonomous), Chennai Mr. Vijay Daniel Anthony, Assistant Professor, Department of
Economics (Shift-2), Loyola College (Autonomous), Chennai Email :
vijaydaniel@loyolacollege.edu g g © The Authors. Published by Blue Eyes Intelligence Engineering and
Sciences Publication (BEIESP). This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Published By:
Blue Eyes Intelligence Engineering
& Sciences Publication
© Copyright: All rights reserved. Retrieval Number:100.1/ijmh.A1137095120
DOI:10.35940/ijmh.A1137.095120
Journal Website: www.ijmh.org Retrieval Number:100.1/ijmh.A1137095120
DOI:10.35940/ijmh.A1137.095120
Journal Website: www.ijmh.org 57 II.CONCLUSION In concluding, one realizes that green policies lead to a
better environment. But it is true that these policies will Published By:
Blue Eyes Intelligence Engineering
& Sciences Publication
© Copyright: All rights reserved. Published By:
Blue Eyes Intelligence Engineering
& Sciences Publication
© Copyright: All rights reserved. Retrieval Number:100.1/ijmh.A1137095120
DOI:10.35940/ijmh.A1137.095120
Journal Website: www.ijmh.org 58
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Subarrays of phased-array antennas for multiple-input multiple-output radar applications
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International Journal of Informatics and Communication Technology/International Journal of Informatics and Communication Technology (IJ-ICT)
| 2,022
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cc-by-sa
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International Journal of Informatics and Communication Technology (IJ-ICT)
Vol. 11, No. 3, December 2022, pp. 218~228
ISSN: 2252-8776, DOI: 10.11591/ijict.v11i3.pp218-228 International Journal of Informatics and Communication Technology (IJ-ICT)
Vol. 11, No. 3, December 2022, pp. 218~228
ISSN: 2252-8776, DOI: 10.11591/ijict.v11i3.pp218-228 218 218 Keywords: Amount of detected targets
Angular resolution
Detection probability
SNR detector
Subarray MIMO radar
Virtual array This is an open access article under the CC BY-SA license. Corresponding Author:
Syahfrizal Tahcfulloh
Department of Electrical Engineering, Universitas Borneo Tarakan
No.1, Jl. Amal Lama No. Kel, Pantai Amal, Tarakan Tim., Kota Tarakan, Kalimantan Utara, Indonesia
Email: rizalubt@gmail.com Subarrays of phased-array antennas for multiple-input
multiple-output radar applications Syahfrizal Tahcfulloh1, Muttaqin Hardiwansyah2
1Department of Electrical Engineering, Universitas Borneo Tarakan, Tarakan, Indonesia
2Department of Electrical Engineering, Universitas Trunojoyo Madura, Bangkalan, Indonesia Article Info
Article history:
Received Dec 24, 2021
Revised Jul 24, 2022
Accepted Aug 9, 2022 ABSTRACT The subarray MIMO radar (SMIMO) is a multiple-input multiple-output
(MIMO) radar with elements in the form of a sub-array that acts as a phased
array (PAR), so it combines at the same time the key advantage of the PAR
radar, which is high directional gain to increase target range, and the key
advantage of the MIMO radar, i.e., high diversity gains to increase the
maximum number of detected targets. Different schemes for the number of
antenna elements in the transceiver zones, such as uniform and/or variable,
overlapped and non-overlapped, significantly determine the performance of
radars as virtual arrays (VARs), maximum number of detected targets,
accuracy of target angle, detection resolution, SNR detection, and detection
probability. Performance is also compared with the PAR, the MIMO, and the
phased MIMO radars (PMIMO). The SMIMO radar offers great versatility
for radar applications, being able to adapt to different shapes of the multiple
targets to be detected and their environment. For example, for a transmit-
receive with an antenna element number, i.e., M = N = 8, the range of the
number of detected targets for the SMIMO radar is flexible compared to the
other radars. On the other hand, the proposed radar's signal-to-noise ratio
(SNR) detection performance and detection probability (K = 5, L = 3) are
both 1,999 and above 90%, which are better than other radars. Article history:
Received Dec 24, 2021
Revised Jul 24, 2022
Accepted Aug 9, 2022 Corresponding Author:
Syahfrizal Tahcfulloh
Department of Electrical Engineering, Universitas Borneo Tarakan
No.1, Jl. Amal Lama No. Kel, Pantai Amal, Tarakan Tim., Kota Tarakan, Kalimantan Utara, Indonesia
Email: rizalubt@gmail.com 1.
INTRODUCTION Along with the development of multi-antenna systems on radar technology that detects multiple
targets at a long distance, it has a detection function and a flexible response to the target state and its
surroundings, supported by high detection accuracy and angle resolution. One of the most important factors
in radar performance is optimal range and tracking angle [1] and multi-target detection [2]. High accuracy
enhances the ability to detect targets, and high angle resolution increases the number of detected targets. Its
implementation requires a radar with high detection accuracy and angle resolution for multiple target
detection, especially as an automotive radar [3]. The phased array (PAR) and multiple-input multiple-output
(MIMO) radars are two common types of multi-antenna radar systems. This array on the radar system is
arranged in order to get a highdirectivity mainlobe [4], [5]. The PAR radar is that all antenna elements are fed
by adjusting the phase difference to provide higher directional gain in certain directions only [6], [7], so that
the number of detected targets is limited by the small resolution of the resulting angle. The number of Journal homepage: http://ijict.iaescore.com 219 Int J Inf & Commun Technol ISSN: 2252-8776 detected targets is increased by applying the MIMO radar because it exploits all orthogonal transmission
signals to obtain multiple echo signals reflected by multitarget [8]–[11], but has a low directional gain. Besides, the MIMO system concept has been known to have the advantage of increasing the channel capacity
[12], which is directly proportional to the number of detected targets. detected targets is increased by applying the MIMO radar because it exploits all orthogonal transmission
signals to obtain multiple echo signals reflected by multitarget [8]–[11], but has a low directional gain. Besides, the MIMO system concept has been known to have the advantage of increasing the channel capacity
[12], which is directly proportional to the number of detected targets. The troubles skilled via way of means of the PAR and the MIMO radars are triumph over via way of
means of the Phased MIMO radar (PMIMO) [13]–[16] via way of means of forming overlapped subarrays
(SAs) at the transmit array side (Tx) in order that this radar can concurrently take gain of the primary gain of
the PAR radar, specifically excessive directional advantage and the primary gain of the MIMO radar is
excessive range advantage to growth the range of goal detection and range. 2.1. System model The multi-antenna uniform linear array (ULA) radar system in Tx-Rx array consists of a total of M
and N elements, respectively. The distances between the antenna elements of Tx and Rx array are dM and dN,
respectively. The transmitted signal has a non-dispersive and narrowband propagation. As shown in Figure 1,
the Tx and Rx arrays are divided into the numeral of SAs, i.e. K and L. These SAs may follow a combination
of overlapped and/or non-overlapped, and the numeral of elements may vary. The SA numbers for Tx and Rx
are 1 ≤ K ≤ M and 1 ≤ L ≤ N, respectively. p
y
Since each SA of both the Tx array and the Rx array acts as an element of the MIMO radar array
that acts as a directional gain controller like a PAR radar, the subarray acts as an orthogonal Tx-Rx element
of the SMIMO radar and independent of each other. The configuration of these sub-arrays promises a
combination of directional gain and diversity gain that can be used simultaneously with both the MIMO and
the PAR radars. Figure 1 shows some configurations (such as overlapped and/or non-overlapped) of various SAs
within a Tx-Rx array on a SMIMO radar, and the amount of elements in each subarray that are equal and/or
variable. Figure 1(a) shows an proposed radar scheme called a SA in which the SA terms overlap and the
numeral of elements in each sub-array is equal (OES), or K = 3 and L = 4 overlap in Tx and Rx. This
configuration detects multiple targets by generating three quadrature signals transmitted by three SAs on Tx
and received by four SAs operating independently on Rx. Figure 1(b) shows the conditions between the sub-
arrays on the overlapped SMIMO radars, where the amount of elements in each sub-array changes or the
overlapping unequal SAs (OUS) with K = 3 for Tx and L = 4 for Rx. Three orthogonal signals transmitted by
the three SAs of Tx and received by the four SAs of Rx. The difference between the OES configuration and
the OUS configuration is that the radiation shape (beamforming) of the SAs in the OES configuration is the
same for Tx and Rx of each SA, but different for OUS. This is because the OUS configuration has a variable
amount of antenna elements, and a SA with many elements produces higher directivity. 1.
INTRODUCTION Furthermore, this radar is referred
to as PMIMO_Tx. In the [17]–[19] study of Full PMIMO radar (FPMIMO), using SAs that overlap both Tx and Rx
actually yielded the maximum amount of detected targets and angle resolution. It turned out to increase. The
amount of SA elements in Tx-Rx array is the same. This radar is also known as the PMIMO_Tx_Rx. To achieve generalization and flexibility of multi-target detection and high angle resolution, this
paper introduces the Subarray MIMO radar (SMIMO), which allows SAs to have the following conditions: a)
the numeral of SA elements varies and or uniform and b) overlapped and or non-overlapped, on Tx-Rx so
that it can form a PAR, MIMO, PMIMO_Tx, PMIMO_Tx_Rx radar configuration, and other radar
variations. SMIMO radar sub-array conditions provide increased degrees of freedoms (DoFs) compared to
other radar types where traditional radars cannot provide detection accuracy, high angle resolution, high SNR
detection, and high detection probability to adapt to current environmental conditions. While using the
SMIMO radar, all you have to do is adjust the configuration and number of Tx-Rx SAs. The proposed radar performance is expressed in terms of the number of VAs (NVAR), the maximum
amount of detected targets (max), the angle resolution, the maximum complex amplitude of the target return,
the SNR detection (), and the probability detection (Pd). All of these have not been explained in detail in
previous studies and have not been shown comprehensively. The results of this paper show that the SMIMO
radar is highly flexible in various configurations by adjusting the amount of SA elements, the amount of SAs,
overlapped/non-overlapped, and target detection conditions at that time. The structure of this paper is as follows. In section 2 shows the SMIMO radar system and signal
model. The formulas used to calculate NVAR, max, the magnitude of the complex amplitude, , and Pd are
described in section 3. Section 4 presents the results and considerations in the form of numerical calculations
and simulations of the SMIMO radar performance parameters, as well as their analysis and verification. Finally, section 5 contains the conclusions of this research. 2.1. System model Subarrays of phased-array antennas for multiple-input multiple-output … (Syahfrizal Tahcfulloh) 220 ISSN: 2252-8776 The configurations of the proposed radar SAs in Figure 1(c) do not overlap, and each SA has a
uniform number of elements (NOES), or Tx has K sub-arrays and Rx has L sub-arrays. In this configuration,
for multi-target capture, K orthogonal signals are generated that are sent by the Tx array and received by the
L independent SAs in the Rx array. Figure 1(d) shows the non-overlapped schemes between the SAs, where
the numeral of elements in each sub-array varies or is called a non-overlapping unequal SAs (NOUS), where
Tx has K SAs and Rx has L SAs. The difference between the NOES configuration and the NOUS
configuration is that the sub-arrays with the NOES scheme have the same radial shape for Tx and Rx of each
SA, whereas the radial shape for NOUS is different. This is because the NOUS scheme has a variable amount
of antenna elements, as described in Figure 1(c). The distinction among overlapped SA (OS) and non-overlapped SA (NOS) is special in Section 4.1,
i.e. concerning the dimensions of the virtual array (VAR) generated and the sign transmitted through the SAs
is semi-orthogonal. This immediately impacts the accuracy and backbone of the detection attitude of the
target. (a)
(b)
(c)
(d)
Figure 1. The configuration of the Tx-Rx antenna array on an SMIMO radar with SA is a subarray with the
following conditions: (a) overlapped-equal number (OES), (b) overlapped-unequal number (OUS), (c) non-
overlapped-equal number (NOES), and (d) non-overlapped-unequal number (NOUS) (a) (b) (a (b) (a) (b)
(d) (a)
(c) (d) (c) Figure 1. The configuration of the Tx-Rx antenna array on an SMIMO radar with SA is a subarray with the
following conditions: (a) overlapped-equal number (OES), (b) overlapped-unequal number (OUS), (c) non-
overlapped-equal number (NOES), and (d) non-overlapped-unequal number (NOUS) 2.2. Signal model When the Tx coherent vector and the Tx diversity vector are defined as K-elements, respectively: 𝒄(𝜃) = [𝒘1
𝐻𝒂1(𝜃)
𝒘2
𝐻𝒂2(𝜃)
⋯
𝒘𝐾
𝐻𝒂𝐾(𝜃)]𝑇
(3) 𝒄(𝜃) = [𝒘1
𝐻𝒂1(𝜃)
𝒘2
𝐻𝒂2(𝜃)
⋯
𝒘𝐾
𝐻𝒂𝐾(𝜃)]𝑇 (3) and and 𝒅(𝜃) = [𝑒−𝑗2𝜋𝑓𝜏1(𝜃)
𝑒−𝑗2𝜋𝑓𝜏2(𝜃)
⋯
𝑒−𝑗2𝜋𝑓𝜏𝐾(𝜃)]𝑇
(4)
So (2) can be stated briefly as
𝒓(𝑡, 𝜃) = √𝑀/𝐾𝛽(𝜃)(𝒄(𝜃) ∘𝒅(𝜃))𝑇𝝍(𝑡)
(5) 𝒅(𝜃) = [𝑒−𝑗2𝜋𝑓𝜏1(𝜃)
𝑒−𝑗2𝜋𝑓𝜏2(𝜃)
⋯
𝑒−𝑗2𝜋𝑓𝜏𝐾(𝜃)]𝑇 (4) So (2) can be stated briefly as 𝒓(𝑡, 𝜃) = √𝑀/𝐾𝛽(𝜃)(𝒄(𝜃) ∘𝒅(𝜃))𝑇𝝍(𝑡) (5) where ()T denotes the transpose operator, (t) = [1(t) ... K(t)]T and denotes the operator of Hadamard
multiplication. If it is assumed that there are T targets in the direction {t} with t = 1, 2, 3, ...., T, then the
complex of signal received vector by the sub-array L at Rx is where ()T denotes the transpose operator, (t) = [1(t) ... K(t)]T and denotes the operator of Hadamard
multiplication. If it is assumed that there are T targets in the direction {t} with t = 1, 2, 3, ...., T, then the
complex of signal received vector by the sub-array L at Rx is 𝒚(𝑡) = √𝑀/𝐾∑
𝛽𝑡(𝜃𝑡)[𝒆(𝜃𝑡) ∘𝒇(𝜃𝑡)]
𝑇
𝑡=1
[𝒄(𝜃𝑡) ∘𝒅(𝜃𝑡)]𝑇𝝍(𝑡) + 𝒏(𝑡)
(6) (6) with with 𝒆(𝜃) = [𝒗1
𝐻𝒃1(𝜃)
𝒗2
𝐻𝒃2(𝜃)
⋯
𝒗𝐿
𝐻𝒃𝐿(𝜃)]𝑇 and 𝒇(𝜃) = [𝑒−𝑗2𝜋𝑓𝜏1(𝜃)
𝑒−𝑗2𝜋𝑓𝜏2(𝜃)
⋯
𝑒−𝑗2𝜋𝑓𝜏𝐿(𝜃)]𝑇
(8) (8) (8) where bl() denotes the Rx steering vector with N elements in the k-th sub-array and vl is the complex weight
vector with N-unit elements in the l-th sub-array. Meanwhile e() and f() are the Rx coherent vectors and
the Rx diversity vectors with L element and n(t) denotes the N element vectors of zero mean white Gaussian
noise. 2.2. Signal model The SMIMO radar block diagram is an adaptation of the PMIMO_Tx_Rx radar block diagram
described by [18] see Figure 2. The difference lies in the numeral of elements of the SA and the state of the
SA, i.e. a non-overlapping number. Tx subpartitions transmit orthogonal signals simultaneously to multiple
targets. The k-th SA of the Tx network sends signals k(t) independently of each other. For M elements in the
Tx network, transmit the baseband signal vector through the k-th SA with k = 1, 2, ..., M, i.e. 𝒙𝑘(𝑡) = √𝑀/𝐾𝜙𝑘(𝑡)𝒘𝑘
∗
(1) 𝒙𝑘(𝑡) = √𝑀/𝐾𝜙𝑘(𝑡)𝒘𝑘
∗ (1) (1) where ()* denotes the operator of complex conjugate and (M/K) is the normalized power factor, ensuring that
the energy transmitted by the proposed radar in a pulse is M. Meanwhile wk denotes a complex weight vector
of M unit elements perpendicular to the k-th SA. where ()* denotes the operator of complex conjugate and (M/K) is the normalized power factor, ensuring that
the energy transmitted by the proposed radar in a pulse is M. Meanwhile wk denotes a complex weight vector
of M unit elements perpendicular to the k-th SA. The reflected signal with a reflectance coefficient of () from a target in the distant field is
expressed as follows: 𝑟(𝑡, 𝜃) = √𝑀𝐾
⁄
𝛽(𝜃) ∑
𝑤𝑘
𝐻𝑎𝑘(𝜃)𝑒−𝑗2𝜋𝑓𝜏𝑘(𝜃)𝜙𝑘(𝑡)
𝐾
𝑘=1 𝑟(𝑡, 𝜃) = √𝑀𝐾
⁄
𝛽(𝜃) ∑
𝑤𝑘
𝐻𝑎𝑘(𝜃)𝑒−𝑗2𝜋𝑓𝜏𝑘(𝜃)𝜙𝑘(𝑡)
𝐾
𝑘=1
(2) (2) 221
22 ISSN: 2252-8776 Int J Inf & Commun Technol where (.)H is the Hermitian displacement operator, ak() denotes the Tx direction vector with M elements in
the k-th SA, f denotes the carrier signal frequency, k() denotes the corresponding delay time of the first
element of the k-th SA to the first element of the first SA. Wh
th T
h
t
t
d th T di
it
t
d fi
d
K l
t
ti
l where (.)H is the Hermitian displacement operator, ak() denotes the Tx direction vector with M elements in
the k-th SA, f denotes the carrier signal frequency, k() denotes the corresponding delay time of the first
element of the k-th SA to the first element of the first SA. 3.
VIRTUAL ARRAY, MAXIMUM AMOUNT OF DETECTED TARGETS, COMPLEX
AMPLITUDE OF ECHO SIGNAL, SNR DETECTION, AND DETECTION PROBABILITY
FOR SMIMO RADAR The advantage of orthogonally transmitted MIMO radar over the PAR radar is the formation of
VAR [11]. Target detection, angular accuracy, detection angle resolution, and maximum numeral of detected
targets are primarily determined by the VAR of the PAR and MIMO radar [11] and the FPMIMO radar [19]
with evenly overlapping SAs. y
pp g
VAR calculations at the MIMO, the PAR, the PMIMO_Tx_Rx, and the PMIMO_Tx radars had
been supplied through [19], at (14)-(17). For the most wide variety of detectable goals the MIMO and the
PAR radars had been mentioned through [20], whilst for the PMIMO_Tx_Rx and the PMIMO_Tx radars it's
been mentioned through [19]. The calculation of the angular decision acquired through the complicated
amplitude of the goal echo sign at the PMIMO_Tx_Rx, the MIMO, the PMIMO_Tx, and the PAR radars has
been defined through [19]. For the SNR detector on the proposed radar is an extension of the SNR detector
on the MIMO radar by [21]. Meanwhile, to determine the detection probability on the SMIMO radar, we
adapt Pd from the FPMIMO radar by [19]. The calculation of VAR, the maximum amount of targets that can be detected, and the complex
amplitude of the target return signal on the proposed radar is an extension of the study in [19]. The number of
both overlapped and non-overlapped SAs, and/or the number of SMIMO radar VARs that vary uniformly or
have a number of Tx and Rx SAs, is defined by K and L, respectively. 3.
VIRTUAL ARRAY, MAXIMUM AMOUNT OF DETECTED TARGETS, COMPLEX
AMPLITUDE OF ECHO SIGNAL, SNR DETECTION, AND DETECTION PROBABILITY
FOR SMIMO RADAR (9) 𝐸∈[𝐾+ 𝐿−1, 𝐾𝐿] To calculate the maximum amount of detected targets on the proposed radar is (10) To calculate the maximum amount of detected targets on the proposed radar is (10) Subarrays of phased-array antennas for multiple-input multiple-output … (Syahfrizal Tahcfulloh)
To calculate the maximum amount of detected targets on the proposed radar is (10) Subarrays of phased-array antennas for multiple-input multiple-output … (Syahfrizal Tahcfull
222 222 ISSN: 2252-8776 𝑇𝑚𝑎𝑥∈[
𝐾+𝐿−2
2
,
𝐾𝐿+1
2 )
(10)
The complex amplitude of the SMIMO radar target return signal is expressed by (11) 𝑇𝑚𝑎𝑥∈[
𝐾+𝐿−2
2
,
𝐾𝐿+1
2 ) 𝑇𝑚𝑎𝑥∈[
𝐾+𝐿−2
2
,
𝐾𝐿+1
2 )
(10) 𝑇𝑚𝑎𝑥∈[
𝐾+𝐿−2
2
,
𝐾𝐿+1
2 ) (10) The complex amplitude of the SMIMO radar target return signal is expressed by (11) The complex amplitude of the SMIMO radar target return signal is expressed by (11) The complex amplitude of the SMIMO radar target return signal is expressed by (11) 𝛽̑(𝜃) =
√𝑀/𝐾∑
(𝒆(𝜃)∘𝒇(𝜃))𝐻𝑹̂𝒚𝝍(𝒄(𝜃)∘𝒅(𝜃))∗
𝑄
𝑞=1
‖𝒆(𝜃)∘𝒇(𝜃)‖2(𝒄(𝜃)∘𝒅(𝜃))𝑇𝑹̂𝝍𝝍(𝒄(𝜃)∘𝒅(𝜃))∗
(11) 𝛽̑(𝜃) =
√𝑀/𝐾∑
(𝒆(𝜃)∘𝒇(𝜃))𝐻𝑹̂𝒚𝝍(𝒄(𝜃)∘𝒅(𝜃))∗
𝑄
𝑞=1
‖𝒆(𝜃)∘𝒇(𝜃)‖2(𝒄(𝜃)∘𝒅(𝜃))𝑇𝑹̂𝝍𝝍(𝒄(𝜃)∘𝒅(𝜃))∗ (11) with (12) and (13) 𝑹̂𝒚𝝍= (1/𝑄) ∑
𝒚(𝑞)𝝍𝐻(𝑞)
𝑄
𝑞=1
(12)
𝑹̂𝝍𝝍= (1/𝑄) ∑
𝝍(𝑞)𝝍𝐻(𝑞)
𝑄
𝑞=1
= 𝑰𝑀×𝑀
(13) 𝑹̂𝒚𝝍= (1/𝑄) ∑
𝒚(𝑞)𝝍𝐻(𝑞)
𝑄
𝑞=1
𝑹̂𝝍𝝍= (1/𝑄) ∑
𝝍(𝑞)𝝍𝐻(𝑞)
𝑄
𝑞=1
= (12) (13) where ‖⋅‖ is the Euclidean norm operator, 𝛽̂(𝜃) denotes the least squares (LS) estimate of the coeficient
reflectivity of radar or the magnitude of the target's complex amplitude in the direction , and q = 1, 2, 3, ...,
Q denotes the sample data index. The amount of detected targets can be determined from the spatial spectral
peaks in the LS estimate. where ‖⋅‖ is the Euclidean norm operator, 𝛽̂(𝜃) denotes the least squares (LS) estimate of the coeficient
reflectivity of radar or the magnitude of the target's complex amplitude in the direction , and q = 1, 2, 3, ...,
Q denotes the sample data index. The amount of detected targets can be determined from the spatial spectral
peaks in the LS estimate. p
Through the same approach by [19] and based on the SNR detector by [21], to calculate the SNR
detector on the proposed radar is expressed by =
2(𝑀/𝐾)2𝑀𝐾
2𝑁𝐿
2𝐾2𝐿2(𝑆𝑁𝑅)2
(𝑀/𝐾)2𝑀𝐾
2𝑁𝐿
2𝐾2𝐿2(𝑆𝑁𝑅)2+2(𝑀/𝐾)𝑀𝐾𝑁𝐿𝐾𝐿(𝑆𝑁𝑅)+2 =
2(𝑀/𝐾)2𝑀𝐾
2𝑁𝐿
2𝐾2𝐿2(𝑆𝑁𝑅)2
(𝑀/𝐾)2𝑀𝐾
2𝑁𝐿
2𝐾2𝐿2(𝑆𝑁𝑅)2+2(𝑀/𝐾)𝑀𝐾𝑁𝐿𝐾𝐿(𝑆𝑁𝑅)+2 (14) here MK and NL are M – K + 1 and N – L + 1, respectively. where MK and NL are M – K + 1 and N – L + 1, respectively. Meanwhile, to determine the detection probability on the SMIMO radar by developing Pd in [19] for
a certain false alarm probability (Pfa) and the SNR is expressed by 𝑃𝑑= exp (
𝐾ln(𝑃𝑓𝑎)
𝐾+𝑀𝑀𝐾𝑁𝐿𝐾𝐿(𝑆𝑁𝑅)) (15) 4.
RESULTS AND DISCUSSION
4.1. Virtual array y
Calculating VAR (NVAR) with SMIMO radar allows you to apply the concept of VAR with the
MIMO radar. That is, antenna-shaped elements can be used to radiate orthogonal signals and extend to sub-
array-shaped elements that are not mutually exclusive overlaps or NOES. VAR is the result of the
convolution of the Tx array and Rx [11]. To determine the VAR, the subarray is considered a single antenna
in the site center and emits orthogonal signals according to the scheme shown in [11]. Based on a comparison
with PMIMO_Tx with 4 OES, the following is an example of VAR calculation for proposed radar with
(M = N = 8) and 4 NOES (2 elements per subarray). 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 1 0 1 0 1 0 1}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {1 1 1 1 1 1 1 1};
𝑉𝐴𝑅= {1 1 2 2 3 3 4 4 3 3 2 2 1 1 0}
(16) (16) Since there are four NOES, there is one compelling element position that is displayed in different font colors. The Rx array is worth one in the position of eight elements. The VAR of the SMIMO radar using NOES is
14, which is near to the VAR amount of the MIMO radar of 15. For comparison of SMIMO radar using 2
NOES (numeral of elements per 4 SAs). 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 0 0 1 0 0 0 1 0}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {1 1 1 1 1 1 1 1};
𝑉𝐴𝑅= {0 1 1 1 1 2 2 2 2 1 1 1 1 0 0}
(17) 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 1 1 1 1 0 0}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {1 1 1 1 1 1 1 1};
𝑉𝐴= {0 1 1 1 1 2 2 2 2 1 1 1 1 0 0} (18) Since there are 4 SAs per SA 5, the element in the center of each SA is worth 1, shown in different
font colors, and the Rx array is worth 1. There are 8 element positions. The PMIMO radar has an VAR
number of 11, which is between two extreme configurations, slightly higher than the PAR radar, i.e. 8, and
lower than the MIMO radar, i.e. 15. To compare the amount of VARs for proposed radar using OES and NOES, Table 1 shows the
numeral of OES or NOES type SAs, the amount of elements per SA, the amount of OES and NOES, and the
amount of VARs. The numeral of overlapped locations on the SMIMO-OES radar clearly affects the VAR
because there are multiple elements between the shared SAs and the signals are not perfectly orthogonal. This is shown in the configuration of two OESs, with six element positions in the O state, where NVAR is close
to NVAR on the PAR radar i.e. 9. Meanwhile, if the position of the element in state O is small (like the scheme
of 7 OES with 1 element in state O), NVAR will be one or more of NVAR in the MIMO radar. The OES-based
PMIMO_Tx radars generally have a higher SINR [13] due to the large amount of OES elements, but have a
higher of the directional gain, such as the PAR radars [22], [23]. Therefore, a compromise must be made
between NVAR and the amount of OES, which is proportional to the angle resolution, angle accuracy, and the
numeral of detected targets, to conform to the situations of the desired target of radar. Table 1. 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 1 1 1 1 0 0}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {1 1 1 1 1 1 1 1};
𝑉𝐴= {0 1 1 1 1 2 2 2 2 1 1 1 1 0 0} Comparison of the SMIMO radar with NOES and OES for M = N = 8
Amount of
SAs
Amount of elements
per SA
Amount of overlapped locations
between SAs
Amount of non-overlapped locations
between SAs
NVAR
NOES
2
4
0
2
13
4
2
0
4
14
8
1
0
8
15
OES
2
7
6
2
9
3
6
5
2
10
4
5
4
2
11
5
4
3
2
12
6
3
2
2
13
7
2
1
2
14 During the calculation of NVAR on the FPMIMO / PMIMO_Tx_Rx radar reported by [18] and [19],
Tx and Rx are OES, and according to [11] each signal is emitted from the midpoint of the phase or the
position of the antenna element. The Tx subarray with MK has a central element value of 1 and the other
elements 0. The same conditions apply to Rx SAs with many elements. That is, NL. For example, if you
calculate NVAR with K = L = 4 and then MK = NL is 5, the array will be Tx, Rx, and VAR. 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 1 1 1 1 0 0}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {0 1 1 1 1 0 0};
𝑉𝐴𝑅= {0 0 0 0 1 2 3 4 3 2 1 0 0 0 0}
(19) (19) In case there are 4 SAs in Tx and Rx array, the arrangement of the elements is alike. The NVAR of the
PMIMO_Tx_Rx radar is 7 because out of the 15 slots in the VAR element with a value greater than 1, only 7
are available. The following is a table showing the result NVAR of all configurations formed for K = L = 8 using (9)
as shown in Table 2. Table 2 shows that the NVAR in the SA configuration of proposed radars such as
PMIMO_Tx and PMIMO_Tx_Rx is intermediate between the PAR and the NVAR of the MIMO radar and
gives the NVAR,PAR < NVAR,SMIMO < NVAR,MIMO. These results also show the proposed radar with the numeral of
Tx and Rx SAs. K and L are common forms of radar that emit many orthogonal signals. Special conditions
for proposed radar are the PAR or SMIMO OES radar (M = 1, N = 8), the MIMO or SMIMO NOES
(M = N = 8), and the PMIMO_Tx or SMIMO OES (1 < K < M, L = 8 ). 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 0 0 1 0 0 0 1 0}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {1 1 1 1 1 1 1 1};
𝑉𝐴𝑅= {0 1 1 1 1 2 2 2 2 1 1 1 1 0 0}
(17) (17) he VAR variety for the SMIMO radar with 2 NOES is 13. then the VAR variety for the SMIMO radar with 2 NOES is 13. y
For the PMIMO_Tx radar pronounced by [13] which makes use of a radiation detail withinside the
shape of Tx OES, in accordance to [11] every waveform is radiated from the midpoint of the segment or the Int J Inf & Commun Technol, Vol. 11, No. 3, December 2022: 218-228 223 ISSN: 2252-8776 Int J Inf & Commun Technol vicinity of the antenna detail withinside the SA with MK the maximum middle detail can be 1 at the same
time as the others are 0. Here are a few examples of VAR calculations for the PMIMO_Tx radar with the
same amount of Tx-Rx antenna elements. For example, the configuration of K is four then MK is 5. The shape
of Tx-Rx array and VAR are vicinity of the antenna detail withinside the SA with MK the maximum middle detail can be 1 at the same
time as the others are 0. Here are a few examples of VAR calculations for the PMIMO_Tx radar with the
same amount of Tx-Rx antenna elements. For example, the configuration of K is four then MK is 5. The shape
of Tx-Rx array and VAR are 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 1 1 1 1 0 0}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {1 1 1 1 1 1 1 1};
𝑉𝐴= {0 1 1 1 1 2 2 2 2 1 1 1 1 0 0}
(18) 4.2. Angle resolution The formation of the VAR is due to the Tx array transmitting the quadrature signal, thus forming a
more focused radiation or beam ray. This happens because a large array is formed in the VAR. The output
beam scheme is the product of the Tx and the Rx beam scheme. The Tx beam ray is the power pattern
radiated from the Tx antenna in all directions of the target. Similarly, the Rx beam ray is the received power
pattern from the target as the arrival angle function of the return signal. If the Rx uses only one antenna, it is
difficult to determine the arrival of angle as it will be difficult to distinguish between strong signals from the
top of the beam and weak signals in the sidelobes. However, if Rx is an array, this does not happen and the
beam can be digitally directed to the reach angle of the echo signal. Figure 2(a) shows the MIMO radar beam schemes for a two-element Tx array and a three-element
Rx array. The spacing between the Tx and the Rx array elements is 0.5λ. Here, λ is wavelenght a carrier
signal. Its output beam ray is similar to the Rx beam ray. However, if you increase the spacing among the
elements of the Tx array to 3 times dN, the resulting beam scheme is shown in Figure 2(b). The resulting
beam pattern has a narrower beamwidth. Also, the grating lobes that appear in the beam scheme do not seem
to appear in the output beam ray. The output beam ray in Figure 2(c) is similar to Figure 2(b), with the
difference that the spacing between the elements of Tx is 0.5λ, while the spacing between the elements of Rx
is twice dM. Therefore, the beam ray can also be represented as a pattern from a 2 × 3 = 6 element Rx array,
or the output beam ray is a VAR beam scheme as shown in Figure 2(d). This shows that calculating the
correct geometry between the correct element of the Tx array and Rx improves the angle resolution of the
radar system without adding the element of antenna to the array. For PAR transmit signals, a high gain beam is obtained and the SNR is increased M-fold, which
does not occur for quadrature signal transmissions. 𝑇𝑥 𝐴𝑟𝑟𝑎𝑦= {0 1 1 1 1 0 0}; 𝑅𝑥 𝐴𝑟𝑟𝑎𝑦= {1 1 1 1 1 1 1 1};
𝑉𝐴= {0 1 1 1 1 2 2 2 2 1 1 1 1 0 0} However, if dM and dN are not equal with d and are greater than d, the resulting NVAR will be greater
than those tabulated in Table 2. Similarly, for the magnitudes dM = LdN and dN = d, the maximum value of
NVAR becomes KL. For example, if K = L = 8, the maximum NVARs for the PAR, the MIMO, the PMIMO_Tx, Subarrays of phased-array antennas for multiple-input multiple-output … (Syahfrizal Tahcfullo 224 ISSN: 2252-8776 and the PMIMO_Tx_Rx radars are 8, 64, 32, and 16, respectively. However, in reality, M = N, which is
already safe. For example, it is not possible programmatically to set the spacing between the elements of Tx
and Rx in order to get the uppercase NVAR. The antennas on the Tx and Rx arrays are almost always in a
specific location from the beginning. and the PMIMO_Tx_Rx radars are 8, 64, 32, and 16, respectively. However, in reality, M = N, which is
already safe. For example, it is not possible programmatically to set the spacing between the elements of Tx
and Rx in order to get the uppercase NVAR. The antennas on the Tx and Rx arrays are almost always in a
specific location from the beginning. Table 2. The value of NVAR is of the array Tx-Rx with K = L = 8 and dM = dN is d
NVAR
L
1
2
3
4
5
6
7
8
K
1
1
2
3
4
5
6
7
8
2
2
3
4
5
6
7
8
9
3
3
4
5
6
7
8
9
10
4
4
5
6
7
8
9
10
11
5
5
6
7
8
9
10
11
12
6
6
7
8
9
10
11
12
13
7
7
8
9
10
11
12
13
14
8
8
9
10
11
12
13
14
15 Table 2. The value of NVAR is of the array Tx-Rx with K = L = 8 and dM = dN is d
L Int J Inf & Commun Technol, Vol. 11, No. 3, December 2022: 218-228 4.2. Angle resolution Therefore, it is necessary to find a compromise between
signal transmission with directional amplification and quadrature signal transmission in order to obtain
angular resolution that meets the target conditions. This guarantees a compromise between the two extreme
configurations and supports the application of the proposed radar concept, which can simultaneously provide
SNR, angle resolution, maximum amount of detected targets, and beam schemes. The use of SAs is a
compromise between Tx-Rx gain, SINR, and maximum range, as described in [22] and [23]. There is capacity for the usage of SAs with extraordinary variety of elements (unequal subarray,
US) to triumph over the restrictions of identical subarray (ES). Especially if it's far adjusted to the situations
for detecting more than one objectives due to the fact usually with excessive directional benefit it's far
appropriate for objectives which have an extended variety and weak or small radar cross section (RCS). If the
goal situations range i.e. varies in variety, RCS, etc., then using a aggregate of various SA schemes (OS/NOS
and ES/US) may be found out as an example in a single time and one Tx array may be found out as a non-
overlapped identical SA (NOES), overlapped identical SA (OES), non-overlapped unequal SA (NOUS), or
overlapped unequal SA (OUS). However, there additionally wishes to be technical issues in its
implementation which decide whether or not it may be implemented practically, particularly to the waveform
generator in Tx and sign processor in Rx for the utility of the SA methods. Int J Inf & Commun Technol, Vol. 11, No. 3, December 2022: 218-228 ISSN: 2252-8776
225
225 225 Int J Inf & Commun Technol . (a)
(b)
(c)
(d)
Figure 2. 4.2. Angle resolution The MIMO radar beampatterns with M = 2, N = 3, and d = 0.5λ for: (a) dM = dN is d,
(b) dM = 3d, dN = d, (c) dM = d, dN = 2, and (d) VAR and the whole sample
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
Tx pattern
Rx pattern
Overall pattern
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
Tx pattern
Rx pattern
Overall pattern
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
Tx pattern
Rx pattern
Overall pattern
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
VA pattern
Overall pattern (b)
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
Tx pattern
Rx pattern
Overall pattern . (a)
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
Tx pattern
Rx pattern
Overall pattern (b) (a) (d)
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
VA pattern
Overall pattern (c)
-80
-60
-40
-20
0
20
40
60
80
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Angle, (deg)
Power (dB)
Tx pattern
Rx pattern
Overall pattern (d) (c) Figure 2. The MIMO radar beampatterns with M = 2, N = 3, and d = 0.5λ for: (a) dM = dN is d,
(b) dM = 3d, dN = d, (c) dM = d, dN = 2, and (d) VAR and the whole sample 4.3. Relationship between VAR and maximum amount of discoverable targets 4.3. Relationship between VAR and maximum amount of discoverable targets
Section 4.1 explained the relationship between the numerical calculation of VAR using (9) reported
in [20]. Since VAR is proportional to angle resolution and accuracy, it is also proportional to the calculation
of the maximum amount of detected targets (max). In calculating the maximum numeral of the MIMO radar
targets formulated in [20], at (22), it is also useful to calculate the maximum amount of detected targets for
PAR, PMIMO_Tx, and PMIMO_Tx_Rx radars i.e. special conditions for proposed radar. Radar uses (10). To
demonstrate this, (11) is used to obtain numerical results as shown in Figure 3. This shows a comparison of
these four parameters for all variations of the proposed radar at K = 4, L = 5, and M = N is about 8. As shown in Figure 3, the MIMO and the PMIMO_Tx radars can detect the four targets indicated by
the red dotted vertical lines. That is, D = {-25o, -10o, 5o, 15o}, but only two the PAR radar and the
PMIMO_Tx_Rx radars can detect the target. = {-25o, -10o}. MIMO radar has the highest detection
accuracy of the four radars. Similarly, it detects the magnitude of the complex amplitude of the return signal
most often obtained by MIMO radar. This is related to the amount of VARs in the largest MIMO radar,
which improves angle detection accuracy and angle resolution. Based on the max equation in (10) of the SMIMO radar, the max value of the PMIMO_Tx and
PMIMO_Tx_Rx radar signal schemes is between the PAR and the max value of the MIMO radar, and they
are related i.e. max,PAR < max,PMIMO < max,MIMO. For example, for Tx-Rx with K = L is 8, get max,PAR,
max,MIMO, max,PMIMO-Tx, and max,PMIMO-Tx-Rx, that is, 4, (7, 32), (5, 16) and (3, 8), respectively. Looking at
Figure 3, the MIMO and the PMIMO radars are within the detectability limits of more than four radars, so
they can properly detect four targets. Subarrays of phased-array antennas for multiple-input multiple-output … (Syahfrizal Tahcfulloh) Subarrays of phased-array antennas for multiple-input multiple-output … (Syahfrizal Tahcfulloh)
226 226 ISSN: 2252-8776 Figure 3. 4.3. Relationship between VAR and maximum amount of discoverable targets The magnitude of the complex amplitude as an assessment of the accuracy and resolution of the
goal attitude with D and () = 1 for numerous radar configurations
-80
-60
-40
-20
0
20
40
60
80
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Angle (deg)
Magnitude of Complex Amplitude
PAR(K=1,L=8)
MIMO(K=L=8)
PMIMO-Tx(K=4,L=8)
PMIMO-Tx-Rx(K=L=4)
(K=L=6) Figure 3. The magnitude of the complex amplitude as an assessment of the accuracy and resolution of the
goal attitude with D and () = 1 for numerous radar configurations 4.4. SNR detector The best performance measure of various detectors is Pd which will be presented in section 4.5. So
comparisons of Pd of various radar systems must be made and selected to determine which one is the best. The comparison is a numerical calculation of a single scalar performance measure such as a function of the
detector's SNR performance [21]. p
[
]
Figure 4 is the result of the evaluation of expression (14) to calculate the SNR detector performance
of the proposed radar. It is assumed that M = N = 8 and the SNR range = {-10:1:15} where the radar
configurations of the PAR, the MIMO, the PMIMO_Tx, and two variations of SMIMO are (K = 1, L = 8),
(K = L = 8), (K = 4, L = 8), (K = 5, L = 3), and (K = 7, L = 6), respectively. It appears that the SNR detector
of the PAR radar is superior to other radar configurations, especially for low SNR, but when the SNR is
above 10 dB, generally the performance of the SNR detector from the proposed radar, namely SMIMO, tends
to reach the same value as the SNR detector from the PAR radar. For example, for an SNR condition of 10
dB, the SNR detector constant value () for PAR, MIMO, PMIMO_Tx radars, and the two proposed radar
variants are 1,998, 1,994, 1,996, 1,999, and 1,995, respectively. The results also show that the variation in the
amount of SAs in Tx-Rx, i.e. K and L, affects the value of where it is evident that the proposed radar
configuration (K = 5, L = 3) has a high value compared to the special configuration of the proposed radar,
namely the PAR radar. Thus, the SMIMO radar has high flexibility to detect multiple targets by adjusting the
numeral of Tx-Rx SAs, i.e. K and L. 5.
CONCLUSION This article described the number of the VARs, the angle resolution, the maximum number of
detectable targets, the magnitude of the complex amplitude, the SNR detector, and the detection probability
of the SMIMO radar return signal under various SA configurations. The number of VARs strongly
determines the angle resolution and the number of detected targets. These are strongly influenced by SA
schemes like OES, OUS, NOES, and NOUS. For the total amount of Tx-Rx array of antennas, or the
proposed radar with M and N, all parameters of SMIMO radar performance are primarily determined by the
number of Tx-Rx SAs, K and L. The maximum number of targets that can be detected, the SNR detector, the
size of the complex amplitude, and the detection probability turn on the number of SAs of the Tx-Rx
proposed radar, where the PAR, the MIMO, the PMIMO_Tx, and the PMIMO_Tx_Rx radars represent
special schemes. Changing the number of specific K and L Tx-Rx SAs (M and N) is adaptive, flexible, and
programmable with the proposed radar, providing multi-target detection capabilities with multi-antenna radar
systems that increase with target and environment. The condition is more reliable and the design planning
radar is easier. 4.5. Detection probability The detection probability for various
SMIMO radar configurations for Pfa variations
-10
-5
0
5
10
15
1.4
1.5
1.6
1.7
1.8
1.9
2
SNR (dB)
PAR(K=1,L=8)
MIMO(K=L=8)
PMIMO-Tx(K=4,L=8)
(K=5,L=3)
(K=7,L=6)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Pfa
Pd
PAR(K=1,L=8)
MIMO(K=L=8)
PMIMO-Tx(K=4,L=8)
(K=L=4) 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Pfa
Pd
PAR(K=1,L=8)
MIMO(K=L=8)
PMIMO-Tx(K=4,L=8)
(K=L=4) Figure 4 SNR detection of various SMIMO radar
-10
-5
0
5
10
15
1.4
1.5
1.6
1.7
1.8
1.9
2
SNR (dB)
PAR(K=1,L=8)
MIMO(K=L=8)
PMIMO-Tx(K=4,L=8)
(K=5,L=3)
(K=7,L=6) Figure 4. SNR detection of various SMIMO radar
configurations for SNR variations Figure 5. The detection probability for various
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Pfa = {0:0.1:1} where the configurations of the PAR, the MIMO, the PMIMO_Tx, and the SMIMO radars
respectively are (K = 1, L = 8), (K = L = 8), (K = 4, L = 8), and (K = L = 4), the results are obtained as
presented in Figure 5. The numerical evaluation results in Figure 5 corroborate the results discussed in
section 4.4 and as reported by [21] namely the Pd performance of the PAR radar outperforms the
performance of other radars. While at a low Pfa of about 10-4, all Pd performances of the SMIMO radar
variation have Pd values above 90%. This shows that detection target is more best for SNR values above
10 dB. It also appears that the variation in the amount of SAs in Tx-Rx namely K and L affects the Pd value. This is evident because the PAR, the MIMO, and the PMIMO_Tx radars are special conditions of the
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Figure 5. The detection probability for various
SMIMO radar configurations for Pfa variations
0
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Pd
PAR(K=1,L=8)
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PMIMO-Tx(K=4,L=8)
(K=L=4) Figure 4. SNR detection of various SMIMO radar
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Applications,” IEEE Access, vol. 8, pp. BIOGRAPHIES OF AUTHORS Syahfrizal Tahcfulloh
received the B.Eng. degree from Universitas Gadjah
Mada, Yogyakarta, Indonesia in 2003, and the M.Eng. and Ph.D. degrees from Institut
Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia in 2010 and 2020, respectively, all
in Electrical Engineering. Currently, he is a Lecturer with the Department of Electrica
Engineering of Universitas Borneo Tarakan. His research interests include array signal
processing and MIMO radar. He is a member of IEEE especially the IEEE Antennas and
Propagation Society. He can be contacted at email: rizalubt@gmail.com. Muttaqin Hardiwansyah
is a Lecturer at the Department of Electrical
Engineering of Universitas Trunojoyo Madura. He is received the B.Eng. degree from
Telecommunication Engineering of Politeknik Elektronika Negeri Surabaya (PENS)
Indonesia in 2011, and the M.Eng. degree from Electrical Engineering of Institut Teknologi
Sepuluh Nopember (ITS). His research interests include MIMO radar and internet of thing
(IoT). He can be contacted at email: muttaqin.hardiwansyah@trunojoyo.ac.id. Syahfrizal Tahcfulloh
received the B.Eng. degree from Universitas Gadjah
Mada, Yogyakarta, Indonesia in 2003, and the M.Eng. and Ph.D. degrees from Institut
Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia in 2010 and 2020, respectively, all
in Electrical Engineering. Currently, he is a Lecturer with the Department of Electrical
Engineering of Universitas Borneo Tarakan. His research interests include array signal
processing and MIMO radar. He is a member of IEEE especially the IEEE Antennas and
Propagation Society. He can be contacted at email: rizalubt@gmail.com. Muttaqin Hardiwansyah
is a Lecturer at the Department of Electrical
Engineering of Universitas Trunojoyo Madura. He is received the B.Eng. degree from
Telecommunication Engineering of Politeknik Elektronika Negeri Surabaya (PENS),
Indonesia in 2011, and the M.Eng. degree from Electrical Engineering of Institut Teknologi
Sepuluh Nopember (ITS). His research interests include MIMO radar and internet of thing
(IoT). He can be contacted at email: muttaqin.hardiwansyah@trunojoyo.ac.id. Muttaqin Hardiwansyah
is a Lecturer at the Department of Electrical
Engineering of Universitas Trunojoyo Madura. He is received the B.Eng. degree from
Telecommunication Engineering of Politeknik Elektronika Negeri Surabaya (PENS),
Indonesia in 2011, and the M.Eng. degree from Electrical Engineering of Institut Teknologi
Sepuluh Nopember (ITS). His research interests include MIMO radar and internet of thing
(IoT). He can be contacted at email: muttaqin.hardiwansyah@trunojoyo.ac.id.
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https://openalex.org/W3095261714
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https://publicatt.unicatt.it/bitstream/10807/167011/1/Social%20Frailty%20in%20the%20COVID-19%20Pandemic%20Era%2c%202020.pdf
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Social Frailty in the COVID-19 Pandemic Era
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Frontiers in psychiatry
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PERSPECTIVE
published: 03 November 2020
doi: 10.3389/fpsyt.2020.577113 PERSPECTIVE Keywords: SARS-CoV-2, coronavirus, social dysfunction, loneliness, immune system, biomarkers, Late-Life
Depression (LLD), Multimorbidity (MM) Social Frailty in the COVID-19
Pandemic Era Madia Lozupone 1*, Maddalena La Montagna 2, Ilaria Di Gioia 2, Rodolfo Sardone 3,
Emanuela Resta 4,5, Antonio Daniele 6,7, Gianluigi Giannelli 3, Antonello Bellomo 2 and
Francesco Panza 3* 1 Neurodegenerative Disease Unit, Department of Basic Medicine, Neuroscience, and Sense Organs, University of Bari Aldo
Moro, Bari, Italy, 2 Psychiatric Unit, Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy,
3 Population Health Unit - “Salus in Apulia Study” - National Institute of Gastroenterology “Saverio de Bellis,” Research
Hospital, Bari, Italy, 4 Department of Cardiac, Thoracic, and Vascular Science, Institute of Respiratory Disease, University of
Bari Aldo Moro, Bari, Italy, 5 Translational Medicine and Management of Health Systems, University of Foggia, Foggia, Italy,
6 Institute of Neurology, Catholic University of Sacred Heart, Rome, Italy, 7 Institute of Neurology, Fondazione Policlinico
Universitario A. Gemelli Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy Special attention and efforts to protect from or reduce health-related outcomes of
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus triggering
coronavirus disease 2019 (COVID-19), should be applied in susceptible populations,
including frail older people. In particular, the early death cases occurred primarily in
older people with a frailty status, possibly due to a weaker immune system fostering
faster progression of the viral infection. Frailty is an age-related multidimensional clinical
condition defined as a non-specific state of vulnerability, identifying older people at
increased risk of falls, institutionalization, hospitalization, disability, dementia, and death. Among frailty phenotypes, social frailty has been least studied. It considers the role of
socioeconomic context as a vulnerability status later in life. COVID-19 does not affect all
populations equally, and social inequalities contribute to drive the spread of infections. It was known that the perception of social isolation, e.g., loneliness, affects mental and
physical health, but the implicated molecular mechanisms, also related to the immune
system, and its associated cognitive and health-related sequelae, are poorly understood. The increasing psychological distress derived by prolonged exposure to stress due to
the lockdown scenario, and the reduced sources of support, contributed to making
heavy demands on personal resources, i.e., self-efficacy and interpersonal variables. So, perceived loneliness may be a factor associated with psychological distress and an
outcome in itself. In the COVID-19 pandemic era, a correct assessment of social frailty
may be essential in terms of the prevention of late-life neuropsychiatric disorders. Edited by:
Miia Kivipelto,
Karolinska Institutet (KI), Sweden
Reviewed by:
Ellen E. Lee,
University of California, San Diego,
United States
José Alberto Avila-Funes,
Instituto Nacional de Ciencias
Médicas y Nutrición Salvador Zubirán
(INCMNSZ), Mexico Edited by:
Miia Kivipelto,
Karolinska Institutet (KI), Sweden Reviewed by:
Ellen E. Lee,
University of California, San Diego,
United States
José Alberto Avila-Funes,
Instituto Nacional de Ciencias
Médicas y Nutrición Salvador Zubirán
(INCMNSZ), Mexico *Correspondence:
Francesco Panza
f_panza@hotmail.com
Madia Lozupone
madia.lozupone@gmail.com *Correspondence:
Francesco Panza
f_panza@hotmail.com
Madia Lozupone
madia.lozupone@gmail.com *Correspondence:
Francesco Panza
f_panza@hotmail.com
Madia Lozupone
madia.lozupone@gmail.com Specialty section:
This article was submitted to
Aging Psychiatry,
a section of the journal
Frontiers in Psychiatry Received: 28 June 2020
Accepted: 07 October 2020
Published: 03 November 2020 INTRODUCTION The
community
healthcare
professionals
consider
relevant
the
assessment
of
subtle
biological,
phenotypic
and functional changes of mental health later in life, since
the duration of the pandemic-related period of isolation
remains uncertain. Neuropsychiatric consequences of brain
damage or disease—i.e., mental disorders—can derive either
from direct effects of infection on the central nervous
system (CNS) or indirectly via the immune response or
otherwise
from
medical
therapy
(3). Neurotropic
and
neuroinvasive effects of coronaviruses have been described
in humans. The present perspective article aims to explore the risk
of social isolation and loneliness sequelae in older frail
adults subjected to isolation measures during the ongoing
COVID-19 pandemic, for both preventative and transmission-
restricting purposes. The immune response in SARS-CoV-2 infection has a
major importance and may induce a hyperinflammatory state
similar to the hemophagocytic lymphohisticytosis, featuring a
transitory condition of increased C-reactive protein, ferritin,
and interleukin-6 levels. Some of the psychiatric multimorbidity
aspects might be explained by the well-described interplay
between inflammation and depression (9, 10). Social participation is an indicator of successful aging and
important determinant of health-related outcomes, including
mortality (4). According to a deficit accumulation approach
to frailty phenotypes, social vulnerability can be measured
as an index of social problems, or “deficits,” such that
the more social deficits one has, the more vulnerability
to adverse outcomes one has. It is important to note
that mental health in the geriatric population requires to
be integrate in the wider context of
the health status
of socially frail individuals, also in view of the possibility of a
weaker immune system permitting faster progression of viral
infection. Fulfillment of basic social needs is necessary to
function adequately and experience social well-being, just
as basic physical needs fulfillment is required to experience
physical well-being. Social frailty could be considered as a
lack of resources to fulfill one’s basic social needs (5). A
correct assessment of social frailty is needed to prevent late-
life neuropsychiatric disorders precipitated by the COVID-
19 pandemic. SOCIAL INEQUALITIES AND MENTAL
HEALTH IN OLDER AGE Regardless of COVID-19 pandemic, social inequalities are
reported in mental disorders: income inequality, low levels
of expected social support and educational attainment affect
social participation, contributing to social exclusion especially in
older age (11). The positive mental effects of abundant, stable
social interactions from a person with a wide social network,
can alleviate stress deriving from negative life events, resulting
in speed recovery from illness, and preserving psychological
health (12). Everyday social environments rife with challenges represent
an exposure to chronic stress whose proxy could be considered
the socioeconomic status (SES) gradient. In the context of
the current COVID-19 pandemic, there is a risk of increasing
inequities in healthcare among vulnerable populations over the
age of 65 and/or with multimorbidity, as potentially at-risk
individuals (13). Furthermore, financial stress, transportation
problems, and housing issues, as well as increased exposure
to crime gained from a lower adolescent SES, may effectively
compromise the benefits of a calm and mature personality on
the stress response pathways, increasing the risk of dementia (14)
(Figure 1). INTRODUCTION Lozupone M, La Montagna M, Di
Gioia I, Sardone R, Resta E, Daniele A,
Giannelli G, Bellomo A and Panza F
(2020) Social Frailty in the COVID-19
Pandemic Era. Data coming from epidemiological studies suggest an association between aging and the risk of
developing life-threatening health problems and mortality related to the spread of severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus implicated in coronavirus disease
2019 (COVID-19) (1). The hierarchical relationship and interlaced time courses of molecular, November 2020 | Volume 11 | Article 577113 Frontiers in Psychiatry | www.frontiersin.org COVID-19 Pandemic and Social Frailty Lozupone et al. phenotypic, and functional aging domains have not yet been
established in humans. Although justified and necessary, the
COVID-19 lockdown will inevitably compromise mental health
of susceptible age strata groups, especially frail older people. To understand the implications of a specific phenotype of the
frailty construct named social frailty on mental health in the
COVID-19 pandemic era, we need a good understanding of
the time and metrics of aging, especially those indicating the
continuum ranging from biological to phenotypic and functional
aging (2). means of a receptor little expressed in the brain, the angiotensin
converting enzyme 2 receptor. Additionally, SARS-CoV-2 can
pass to the brain by means of the cribriform plate nearby to
the olfactory bulb, enabling the virus to reach and affect the
CNS, contributing to neurological tissue damage and to COVID-
19-related morbidity and mortality. In particular, the reported
hyposmia suggests, as shown for SARS-CoV-2, a nasal infection
pathway allowing a possible direct access to the CNS (3). Findings from the study by Helms and colleagues suggested
that the most common neurological features in the COVID-
19 patients were non-focal: confusion, agitation, dysexecutive
syndrome, and diffusely boosted reflexes (7). Preliminary
data suggested that delirium, confusion, agitation, and altered
consciousness, as well as symptoms of depression, anxiety, and
insomnia were also common in patients with COVID-19 (8). The etiology of the neuropsychiatric consequences of COVID-
19 infection is likely multifactorial, including direct effects of
viral infection inside the brain, a procoagulant state inducing
cerebrovascular disease, a physiological impairment in terms
of hypoxia deriving from respiratory failure, the activation of
the immunological cascade, and the indirect effects of medical
interventions, social isolation, the psychological impact deriving
from a novel, severe, potentially fatal illness, concerns about
infecting other people, and social stigma. SARS-CoV-2 AND RELATIONSHIPS WITH
THE CENTRAL NERVOUS SYSTEM The epidemiological criteria about COVID-19 spread, from
China to 229 countries, soared out of control to reach a
pandemic. Globally, at the end of June 2020, there were
9,277,214 confirmed cases of COVID-19 (6), encompassing a
wide clinical spectrum extending from asymptomatic infection,
mild upper respiratory tract illness, to severe viral pneumonia
with respiratory failure. SARS-CoV-2 enters human host cells by November 2020 | Volume 11 | Article 577113 Frontiers in Psychiatry | www.frontiersin.org 2 Lozupone et al. COVID-19 Pandemic and Social Frailty FIGURE 1 | Relationship between coronavirus disease 2019 (COVID-19) lockdown and social resources and direct and indirect effects of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, on psychiatric outcomes in older age. FIGURE 1 | Relationship between coronavirus disease 2019 (COVID-19) lockdown and social resources and direct and indirect effects of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, on psychiatric outcomes in older age. Frontiers in Psychiatry | www.frontiersin.org LONELINESS, SOCIAL ISOLATION, AND
LATE-LIFE NEUROPSYCHIATRIC
DISORDERS DURING THE COVID-19
PANDEMIC: MECHANISMS OF ACTION cardiovascular and neuroendocrine markers of stress, impaired
sleep, and proinflammatory physiological effects, that can cause
neurodegeneration in the hippocampus and in other brain
regions deputed to emotional regulation and cognition (17). Moreover, the amount of loneliness was associated to a greater
brain amyloid-β (Aβ) protein burden after adjustment for
demographic and clinical confounders, inversely Aβ-positive
participants have a risk of 7.5 times higher to suffer from
loneliness. These associations were stronger in apolipoprotein E
ε4 allele carriers (18) (Figure 1). Several variables may explain why the current COVID-19
pandemic causes neuropsychiatric consequences especially in
older age. The wider social impact of the pandemic and the
legislative response, imposing physical distancing measures and
quarantine, are among these reasons (15). The psychobiological
etiological factors underlying anxiety and mood disorders arising
at this social-environmental level should be better understood,
as well as those at the genetic, molecular, or neural-circuitry
level. An increasing body of research is focusing on the late-life
depression–dementia interplay to probe the possible interaction
between depression and aging mechanisms, including the
influence of social determinants on epigenetic mechanisms (16). Furthermore, physical activity and other healthy lifestyles
worldwide were also affected by social distancing and quarantine
restrictions (19). Home-isolation also tends to affect vitamin D
levels by reducing the number of hours spent outdoors. There is
evidence that vitamin D deficiency is linked to impaired immune
function, potentially causing autoimmunity and increased risk
of infections (20). Decreased levels of vitamin D might also
determine a rise in mental health disorders (21). Loneliness or emotional isolation is a subjective, undesirable
experience, resulting from a cognitive mismatch between
the
quantity
and
quality
of
existing
relationships
and
relationship standards. Loneliness has been viewed as a
marker of psychosocial stress, resulting from depression,
bereavement,
and
other
social
disconnection
experiences. The downstream effects of loneliness on neural networks and
systemic health is mediated by stress-related and inflammatory
processes. In other words, great loneliness is linked to elevated It is interesting to note that, according to recent findings,
psychosocial interventions (cognitive behavior therapy and
multiple or combined interventions) are linked to an enhanced
immune
system
function
(proinflammatory
cytokines
or
markers) and may therefore be useful for improving immune-
related health (22, 23). It may be very important to study the
mechanisms of psychosocial interventions and the link to
beneficial effects on the immune system and health, particularly
as related to COVID-19 infection (24). LONELINESS, SOCIAL ISOLATION, AND
LATE-LIFE NEUROPSYCHIATRIC
DISORDERS DURING THE COVID-19
PANDEMIC: MECHANISMS OF ACTION Epigenomics studies November 2020 | Volume 11 | Article 577113 3 COVID-19 Pandemic and Social Frailty Lozupone et al. The biopsychosocial model of frailty may add important
advantages in terms of both assessment and intervention targets. Influenced by a range of variables, it has been defined as a
dynamic state affecting an individual who experiences injuries
in one or more human function fields (physical, psychological,
social), that increases the risk of adverse outcomes (32). Although
different theories on social needs exist, social frailty can be
defined as the continuum of progressive loss of social and general
resources, activities, or abilities serving during the course of
life to fulfill one or more basic social needs. The framework of
social frailty takes into account the various types of social and
general resources (or constraints), social behaviors and activities,
and self-management abilities, utilized for accomplishing (or
affecting) social needs (5). For example, the fulfillment of the
need to love and to be loved, the need to feel that one is doing
the “right” thing according to relevant others and oneself, and
to be part of a group with shared values. Furthermore, the need
to distinguish oneself from others by means of specific talents
or assets. investigated
molecular
mechanisms
by
which
loneliness
exacerbates a wide range of neurodegenerative, psychiatric,
and somatic diseases: Alzheimer’s disease, psychiatric illness,
immune dysfunction, and cancer gene sets seem to constitute
essential targets for future investigations. The expression
of pleiotropic genes at the time of death was found to be
significantly enriched as a function of loneliness, experienced by
a large sample of autopsied participants almost 5 years prior to
death (25) (Figure 1). PSYCHOSOCIAL DETERMINANTS AND
COVID-19 MORTALITY Older age is associated with greater mortality due to COVID-
19. However, the vulnerability to physical comorbidities is not
granted by physiological factors only, but also by psychosocial
factors. Regardless of COVID-19 pandemic, it is known that
different aspects of social relations are measured by social
isolation and loneliness and both are slightly associated with
different health outcomes and also mortality (social isolation to a
greater degree than loneliness) (26). Currently, with the spread of
COVID-19, social connectedness not necessarily is associated to
higher mortality rate among older Italian adults (27). Inversely,
variables associated with social isolation are found to be risk
factors for an increased proportion of mortality in Italian patients
aged 80 years and over. The conclusion could be that social
relationships during a crisis impacting the frailest populations
are a protective factor against increased mortality rates (27). Considering the lack of data on the acute effects of the illness,
in terms of applicability to COVID-19, inferences must be drawn
with care. Furthermore, no data on the post-illness phase have yet
been described, although the higher COVID-19 mortality might
be correlated with poorer psychiatric outcomes at a later date (8). Acute illness is less well-tolerated by frail patients, but the
degree of disease severity and the degree of frailty are each
important (33), particularly in the COVID-19 pandemic era. Most importantly, the type and severity of the presenting illness
are important variables independently associated with the clinical
outcome and ability to fully recover. There are other mediating
factors: female sex (34), smoking (35), and social vulnerability
(36) that also influence the risk related to frailty status. A
recent study conducted in Japanese older adults evidenced
an association between social frailty with both cognitive and
physical functions (37). Further studies are needed to confirm the
hypothesized association between social frailty and cognitive and
physical function. Moreover, compared to physical frailty and
cognitive impairment, social frailty is more strongly associated
with the occurrence of depressive symptoms among community-
dwelling older adults after 4 years of follow-up (38). In other
words, a greater incidence of neuropsychiatric disorders, directly
proportional to the social frailty status, may be expected as late
consequences of the COVID-19 pandemic. However, it should be made clear that physical frailty
and
social
vulnerability
(social
frailty)
are
both
entities
clearly distinct, and that each contributes independently to
mortality (28). Frontiers in Psychiatry | www.frontiersin.org SOCIAL FRAILTY ASSESSMENT TOOLS Frailty is a dynamic process and an intermediate state of aging,
detrimental for health, involving a progressive reduction in
physical, psychological and/or social functions. This condition
has implication for public health linked to its multiple clinical and
social consequences, as well as its dynamic nature also in terms
of prevention (29). Physical frailty components such as a slower
gait, exhibit significant reciprocal relationships with cognition,
and may thus be a transitional step in the progression to late-
life cognitive decline in some older adults. But the vulnerability
of older adults does not appear to be completely explained
by the biological perspective (physical or deficit accumulations
approaches to frailty) (30). Different frailty phenotypes have
been associated with a variety of socioeconomic, behavioral, and
other clinical characteristics, including lower education, lower
income, female gender, unmarried status, obesity, underweight,
multimorbidity, and premorbid disabilities (31). Deficit accumulation model of frailty can be understood to
occur at many levels, from the (sub-)cellular level to tissues,
organisms/complex systems and societies. Deficits can also
accumulate at the tissue level and at the level of complex systems,
i.e., in individual people or animals, which are effectively complex
systems. Of particular relevance to the present discussion, deficits
can also accumulate at levels higher than individuals, e.g., at the
social level, pertaining to social environments and circumstances,
and these are the clinical epiphenomena that we need to
measure. More complex tools to evaluate social frailty basis
beyond symptom scales/health checklists are needed (genetics,
laboratory-based biomarkers, neuroimaging, etc), because social
frailty could be considered a complex clinical phenotype. Mental healthcare clinicians face substantial time challenges,
including limited time available for evaluation for therapy, which November 2020 | Volume 11 | Article 577113 4 COVID-19 Pandemic and Social Frailty Lozupone et al. of subjective and objective evaluations considering both the
rater’s opinion and the subject’s own self-evaluation. The scale
includes important elements of functioning such as personal
satisfaction and self-fulfillment and takes into account social
role performance only peripherally (48). For example, among
the items evaluating self-system, questions about self-concept,
goallessness, meaning in life, self-health concerns are asked to
the subjects; the investigation of interpersonal system implies to
ask about emotional withdrawal, hostility, anxiety etc. Finally,
questions about performances system consist in investigating
lack of satisfying relationships with significant persons, express
need for social contact or friends, lack of satisfaction from
work, expressed need for more leisure activities, financial
insecurities, etc. SOCIAL FRAILTY ASSESSMENT TOOLS does not yet include standardized assessments of social isolation
and loneliness. The question is: what are we measuring to make
interventions? Loneliness, social isolation, social relationships or
all three? Social frailty is usually evaluated by single questions or
items deriving from functional and depressive symptom scales or
health checklists. But each of these instruments measures only
partial aspects deriving from structural or functional aspects of
social relationships, and is based on subjective responses (39). Social frailty has been operazionalized with single questions or
items from functional and depressive symptom scales or health
checklists (40). A shared opinion is that there is heterogeneity
in the definition of social frailty in different studies, and
there is a request of homogeneity and simplification in the
instruments of this assessment. Social contact, participation,
depression, and loneliness characterize different scales of
assessment of different social frailty models (41–44). Also, well-
known instruments for assessing frailty in community-dwelling
older people such as the Tilburg Frailty Indicator (45), aim
to assess physical, psychological, and social frailty and their
health-related outcomes (4). The Social Vulnerability Index, as
a method of quantification of social vulnerability, predicted long-
term mortality in different population-based settings and could
have a role in this context of COVID-19 pandemic (46). The SDRS could be a valid instrument to capture size
(isolation) and quality (loneliness, neuroticism) of social
adjustment in older age. The perception of social dysfunction
was not associated with material deprivation, and this adds
another stratum of complexity in the assessment of health
status in older age. Factorial analysis of SDRS’s twenty-one
items was performed (47) and five factors were identified
for the 21-item SDRS, according to their loads in the
analysis: social isolation; loneliness; feelings of contribution/
uselessness; lack of leisure activities; anxiety for the health. Furthermore, SDRS was correlated to cognitive (apathy, Mini-
Mental State Examination, and Frontal Assessment Battery) and
psychiatric outcomes. Recently, the Social Dysfunction Rating Scale (SDRS) was
validated with a proposed cut-offfor detecting social frailty in
older age, for the purposes of considering possible interventions
to maintain healthy aging (47). SDRS items are a mixture FIGURE 2 | The influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on mental health in older age can impinge on social frailty, a particular
frailty phenotype that has not yet been assigned a full, universally recognized definition. SOCIAL FRAILTY ASSESSMENT TOOLS The mechanisms by which social isolation and loneliness affect mental health
are now under study, also in terms of how best to assess them. FIGURE 2 | The influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on mental health in older age can impinge on social frailty, a particular
frailty phenotype that has not yet been assigned a full, universally recognized definition. The mechanisms by which social isolation and loneliness affect mental health
are now under study, also in terms of how best to assess them. FIGURE 2 | The influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on mental health in older age can impinge on social frailty, a particular
frailty phenotype that has not yet been assigned a full, universally recognized definition. The mechanisms by which social isolation and loneliness affect mental health
are now under study, also in terms of how best to assess them. November 2020 | Volume 11 | Article 577113 Frontiers in Psychiatry | www.frontiersin.org 5 COVID-19 Pandemic and Social Frailty Lozupone et al. (55). More rigorous methods and larger samples of participants
are required in terms of future standpoint for this area
of research. These concepts and findings may help us to develop low cost
methods for screening older persons for mental health outcomes,
and selecting participants for prevention interventions (48). In
the present perspective article, we underline the importance
of early detection and interventions on dysfunctional aspects
of social functions. Social functioning and the SDRS might be
included in a risk index helping to stratify older persons for
biomarker assessment of mental and cognitive health (i.e., late-
life depression or dementia) also in the COVID-19 pandemic era
(49, 50) (Figure 2). The future perspective of old age psychiatry in COVID-
19 pandemic is to cope with the framework of negative
moods, stress and socially mediated traumatic experiences
and adverse developments deriving from social epidemiology
(56). Social determinants may positively modulate the effects
of epigenetic factors on neuropsychiatric disorders in older
age also via the modulation of immune system. In the future,
social incentive exposure—which relies on patient social
and physical activation—could be a potential mechanism
of treatment for different psychiatric disorders, including
late-life
depression
(57). DISCUSSION Loneliness could be tackled with various interventions (51),
broadly divided into two categories, that is, social interventions
and technological interventions. Social interventions applied to
reduce loneliness include befriending, residential and school-
based camps, reminiscence therapy, animal interventions,
gardening, physical activity and technology (52). However, in
older people, loneliness can create serious problems that could
not be alleviated with the social support only (53). Therefore,
particular in older age, other types of interventions are required
such as technological interventions (i.e., digital applications,
online social networks and social robots) to enhance emotional
support and social interaction (54). ACKNOWLEDGMENTS The authors thank the Salus in Apulia Research Team. This manuscript was the result of the research work on
frailty undertaken by the Research Network on Aging team,
Research Networks of National Health Institutes. The authors
thank M. V. Pragnell, B.A., for her precious help as native
English supervisor. AUTHOR CONTRIBUTIONS ML: conceptualization. ML and FP: manuscript writing and
supervision. MLM and ID: drawn the figures. RS, AD, ER, GG,
and MM AB: contributed to the bibliographic search, review,
and editing. DATA AVAILABILITY STATEMENT The original contributions generated for this study are included
in the article/supplementary material, further inquiries can be
directed to the corresponding author/s. The media, such as television and radio, have become
ever more important during the period of social isolation
due to COVID-19 lockdown. However, in this climate, the
level of distress and anxiety can height, because of news
coverage. Internet, electronic communication and, first and
foremost, social media, had offered an extraordinary increase in
connectivity between people and societies, playing a principal
role in forcing the spread of bad news and in deepening
the impact of the worldwide major problems on mental
health, considered an outcome. Thanks to the help of video
calls, older people stay connected during the current crisis. In this way, they widen their social circle and increase the
frequency of contact with existing contacts. However, based
on a recent review, evidence of the efficacy of video call
interventions in reducing older adults’ loneliness, is currently
very uncertain (55), as was the evidence of their effectiveness
as a means of evaluating outcomes of symptoms of depression SOCIAL FRAILTY ASSESSMENT TOOLS Moreover,
social
isolation
and
loneliness is a potentially modifiable risk factor for later
psychiatric multimorbidity that may offer an opportunity
to enhance psychiatric care in new ways by addressing
the underlying causes, and building coalitions to increase
engagement and support by others outside the healthcare
system (50). 5. Bunt S, Steverink N, Olthof J, van der Schans CP, Hobbelen JSM. Social
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34. doi: 10.1007/s10433-017-0414-7 5. Bunt S, Steverink N, Olthof J, van der Schans CP, Hobbelen JSM. Social
frailty in older adults: a scoping review. Eur J Ageing. (2017) 14:323–
34. doi: 10.1007/s10433-017-0414-7
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Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines
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Particle and fibre toxicology
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Open Access Mechanism-based genotoxicity screening of
metal oxide nanoparticles using the ToxTracker
panel of reporter cell lines Mechanism-based genotoxicity screening of
metal oxide nanoparticles using the ToxTracker
panel of reporter cell lines Hanna L Karlsson1*, Anda R Gliga1, Fabienne MGR Calléja2, Cátia SAG Gonçalves2, Inger Odnevall Wallinder3,
Harry Vrieling2, Bengt Fadeel1 and Giel Hendriks2* * Correspondence: Hanna.L.Karlsson@ki.se; g.hendriks@lumc.nl
1Nanosafety & Nanomedicine Laboratory, Division of Molecular Toxicology,
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
2Department of Toxicogenetics, Leiden University Medical Center, Leiden,
the Netherlands
Full list of author information is available at the end of the article © 2014 Karlsson 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 Background: The rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and
reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay
is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses
GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying
mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials
(diesel, carbon nanotubes and quartz). Methods: The metal oxide- and silver nanoparticles were characterized in terms of agglomeration and ion release
in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission
spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by
means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously
determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay,
γ-H2AX and RAD51 foci formation). Results: We show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to
CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although
only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating
additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in
the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of
released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced
reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles. Conclusions: We conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the
identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur
mainly via oxidative stress rather than direct DNA binding with subsequent replication stress. Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Background The exponential increase in the total number of engi-
neered nanoparticles (NPs) for research, development,
and commercialization requires tools for rapid and effi-
cient toxicity screening [1]. Ultimately, such rapid screen-
ing should allow for mechanistic profiling in order to
better inform on hazard identification and to improve risk
assessment [2]. Oxidative stress has been identified as a
major mechanism of toxicity for nanoparticles. The so-
called oxidative stress paradigm describes how increased
levels of reactive oxygen species (ROS) lead to various cel-
lular responses, such as antioxidant response, inflammation
and cytotoxicity, following nanoparticle-cell interactions
[3]. ROS and inflammation can for instance be a conse-
quence of the released toxic metal ions or a reactive par-
ticle surface leading to lysosomal destabilization [4,5]. One
main concern following exposure to inhalable particles is
their potential genotoxicity, which is closely associated with
carcinogenesis. The mechanisms of NP genotoxicity are
still not well understood but oxidative stress and/or direct
interactions with DNA are considered important [6,7]. Dir-
ect DNA interaction could represent a more nano-specific
mechanism due to the fact that small nanoparticles may
reach the nucleus via transportation through the nuclear
pore complexes [8]. However, also larger nanoparticles of
e.g. silver (60 nm) [9], SiO2 (40–70 nm) [10] and CuO
(50–100 nm) [11] have been observed in the nucleus sug-
gesting that larger NPs may get access to the DNA in div-
iding cells when the nuclear membrane disassembles. Here we investigated whether the ToxTracker assay
could be used as a rapid mechanism-based tool for as-
sessing genotoxic effects of NPs. In addition, we explored
particle vs. ion effects in order to identify the particle
properties that determine the reporter cell response. For
this purpose we used a panel of well-characterized nano-
materials including metal oxide NPs (CuO, ZnO, NiO,
CeO2, Fe3O4, TiO2) with a primary particle size <100 nm
and Ag NPs of two specific sizes (10 nm and 40 nm). Ad-
ditionally, the quartz material DQ12 was included as a
poorly soluble benchmark particle, and the results were
compared with diesel particles (standard reference ma-
terial SRM1650b) and multi-walled carbon nanotubes
(MWCNTs). The selection of metal oxide NPs was based
on their various abilities to cause DNA damage and oxida-
tive stress upon exposure of lung epithelial cells [17,18]. Background Results from the ToxTracker reporter assay were com-
pared with conventional assays for genotoxicity assess-
ment (comet assay, γ-H2AX and RAD51 foci formation)
and NP uptake was confirmed by transmission electron
microscopy (TEM). The most commonly used methods for assessing geno-
toxicity of nanomaterials up to date are the comet assay
and the micronucleus (MN) tests [6,12]. However, such
assays are time consuming and give limited information
on the mechanisms of damage, thereby hampering hu-
man hazard assessment. An alternative approach for ra-
pid genotoxicity testing could be reporter cell systems in
which the induction of certain genes can be studied by a
simple readout such as luminescence or fluorescence. These reporter assays indicate the cellular signaling path-
ways that are activated upon exposure, thereby providing
insight into the mechanisms of toxicity. However, only
very few examples of studies using reporter cell lines for
assessing toxicity of NPs have been reported [13]. The
mammalian GreenScreen HC assay that uses a Gadd45α-
GFP (green fluorescent protein) reporter gene in TK6 hu-
man lymphoma cells [14] has been extensively validated
for genotoxicity testing of chemicals. Gadd45α is directly
controlled by the tumor suppressor p53 but also by vari-
ous additional cellular stress-related signaling pathways,
thereby limiting the usability of the GreenScreen HC assay
for identification of the primary mechanism of toxicity of
compounds [15]. Abstract Keywords: Metal oxide nanoparticles, Nanomaterials, ToxTracker, High-throughput screening, Genotoxicity, Oxidative
stress, Reporter cells * Correspondence: Hanna.L.Karlsson@ki.se; g.hendriks@lumc.nl
1Nanosafety & Nanomedicine Laboratory, Division of Molecular Toxicology,
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
2Department of Toxicogenetics, Leiden University Medical Center, Leiden,
the Netherlands
Full list of author information is available at the end of the article Full list of author information is available at the end of the article Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 2 of 14 the biological damage induced by chemicals [16]. The
ToxTracker assay consists of a panel of mouse embry-
onic stem (mES) cell lines that each contains a different
GFP-tagged reporter for a distinct cellular signaling path-
way. The preferential induction of the different reporters
indicates the nature of biological damage and associated
cellular response pathways. The ToxTracker assay can dis-
criminate between the induction of DNA damage via dir-
ect DNA interaction, oxidative stress and general cellular
stress (Figure 1A). The DNA damage-associated Bscl2-
GFP reporter depends on the ATR (ataxia telangiectasia
mutated and Rad3-related)-associated DNA damage sig-
naling pathway and is selectively activated after exposure
to genotoxic agents and the subsequent interference with
DNA replication [16]. The Srxn1-GFP reporter is prefer-
entially induced upon oxidative stress and is part of the
Nrf2 (Nuclear Factor, Erythroid Derived 2, Like 2) antioxi-
dant response pathway. Finally, the Btg2-GFP reporter
gene is controlled by p53 and is activated by various types
of cellular stress. The combination of different fluorescent
reporter cell lines in a single toxicity assay allows not only
for rapid and reliable identification of genotoxic properties
of chemicals but also enables mechanistic understanding
of different modes of toxicity [16]. Characterization of particle solubility, agglomeration
and reactivity ZnO NPs dissolved rapidly
with approximately 40% reduction in total particle mass
already after sonication and centrifugation (0 h), also indi-
cated by a rapid reduction in particle size and scattered
light intensity (kcps) as observed using PCCS measure-
ments (data not shown). Only 20% of the particle mass
remained in solution after 24 h of exposure (see Table 1). CuO NPs also dissolved to a relatively large extent with
time, although significantly slower (2.5% of the total par-
ticle mass after 0 h and 37% after 24 h). Similar kinetics were evident for the Ag NPs that showed a clear size-
dependent metal release with approximately 22% (Ag-10)
and 11% (Ag-40) of the particle mass dissolved after 24 h. The amount of dissolved metals in solution after 24 h was
significantly lower for NiO (5% after 24 h), and even lower
for TiO2 (<0.1%), Fe3O4 and CeO2 (<< 0.01%). NiO NPs
were by far the most efficient particles to generate ROS
acellularly, followed by CuO (though to a significantly
lower extent) whereas the other NPs showed no or only a
slight increase in ROS production. Characterization of particle solubility, agglomeration
and reactivity Metal oxide NPs (CuO, ZnO, NiO, CeO2, Fe3O4, TiO2)
and silver NPs of different sizes (10 nm and 40 nm, citrate
coated) were carefully characterized in terms of primary
size, zeta potential, metal ion release in cell medium,
and acellular ROS generation (Table 1). All tested NPs Recently, we developed an in vitro assay called Tox-
Tracker that can rapidly provide mechanistic insight into Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 3 of 14 A
B
Figure 1 The ToxTracker reporter assay for mechanism-based toxicity testing. (A) The ToxTracker assay consists of a panel of GFP-based
mES cell lines. The GFP reporters indicate activation of the Nrf2-associated antioxidant response, ATR-associated DNA damage response and the
p53 cellular stress response. Induction of the GFP reporters as well as cytotoxicity is determined by flow cytometry. (B) Cellular uptake but no
evidence for nuclear localization of metal oxide NPs in mES cells. Internalization of the NPs after 24 h exposure to 20 μg/mL CuO, 30 μg/mL ZnO,
100 μg/mL NiO, 100 μg/mL CeO2 and 10 μg/mL Ag-10 NPs was determined by means of TEM. NPs were taken up by mES cells and were
localized in endosomal vesicles or free in the cytoplasm (black arrow heads). A
B B A B Figure 1 The ToxTracker reporter assay for mechanism-based toxicity testing. (A) The ToxTracker assay consists of a panel of GFP-based
mES cell lines. The GFP reporters indicate activation of the Nrf2-associated antioxidant response, ATR-associated DNA damage response and the
p53 cellular stress response. Induction of the GFP reporters as well as cytotoxicity is determined by flow cytometry. (B) Cellular uptake but no
evidence for nuclear localization of metal oxide NPs in mES cells. Internalization of the NPs after 24 h exposure to 20 μg/mL CuO, 30 μg/mL ZnO,
100 μg/mL NiO, 100 μg/mL CeO2 and 10 μg/mL Ag-10 NPs was determined by means of TEM. NPs were taken up by mES cells and were
localized in endosomal vesicles or free in the cytoplasm (black arrow heads). agglomerated to various extents in mES cell culture me-
dium with bimodal and trimodal distributions, but all sus-
pensions contained particles sized <100 nm (see Table 1
and Additional file 1: Figure S1). As expected, the NPs
showed a high variation in their ability to dissolve/release
metal ions in the cell medium. Cellular uptake, but no evidence for nuclear localization
of metal oxide NPs Clearly, all the tested metal and metal oxide NPs
analyzed using TEM were internalized by the mES cells,
but no nuclear localization was observed (Figure 1B). A
significant side scatter shift was also observed for all
particles in the study (Additional file 1: Figure S2). Al-
though contributions by possible particles tightly bound
to the outer cell membrane cannot be excluded, this
data support the uptake observed in the TEM analysis. Fewer internalized particles were observed after expos-
ure to ZnO and CuO NPs due to their extra- and intra-
cellular dissolution. To further confirm uptake of CuO
NPs we quantified the metal content after exposure of
mES cells to 20 μg/mL for 4 h by means of atomic ab-
sorption spectroscopy (AAS). The results showed a
metal content of approx. 6.1 pg/cell which is in the same
range as our previous studies on CuO NPs uptake in
A549 and BEAS-2B cells [21]. nanoparticle toxicity testing (Figure 1A), we first investi-
gated the ability of the mES cells to internalize NPs by
means of TEM and by analyzing the side scatter shift by
using flow cytometry. Indeed, NPs may cause genotoxic
effects via indirect mechanisms [19], but without cellular
uptake the applicability of the assay for NP testing will
be limited as in the case for the Ames mutagenicity test
[20]. Clearly, all the tested metal and metal oxide NPs
analyzed using TEM were internalized by the mES cells,
but no nuclear localization was observed (Figure 1B). A
significant side scatter shift was also observed for all
particles in the study (Additional file 1: Figure S2). Al-
though contributions by possible particles tightly bound
to the outer cell membrane cannot be excluded, this
data support the uptake observed in the TEM analysis. Fewer internalized particles were observed after expos-
ure to ZnO and CuO NPs due to their extra- and intra-
cellular dissolution. To further confirm uptake of CuO
NPs we quantified the metal content after exposure of
mES cells to 20 μg/mL for 4 h by means of atomic ab-
sorption spectroscopy (AAS). The results showed a
metal content of approx. 6.1 pg/cell which is in the same
range as our previous studies on CuO NPs uptake in
A549 and BEAS-2B cells [21]. Cellular uptake, but no evidence for nuclear localization
of metal oxide NPs In order to investigate the general applicability of the re-
cently developed mES cell-based ToxTracker assay for Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 4 of 14 Table 1 Physico-chemical characterization of the metal and metal oxide nanoparticles
Particle
Sizea (nm, TEM)
Size cell medium
(approximate peak
max, nm PCCS)
Zeta potential, mV
Dissolved amount of metals in cell medium, wt%
Acellular ROSb
0 h
24 h
CuO
20-40
3 and 500
+31
2.5
37.2
2.3
Fe3O4
20-40
2, 40 and 800
+2
<0.01
<0.01
1.5
ZnO
20-200
20, 500 and 800
+27
41.3
78.5
1.1
TiO2
20-100
8 and 400
+6
0.1
0.1
1.3
NiO
2-70
20 and 500
+30
0.9
5.1
>10
CeO2
4-30
8 and 80
+4
<0.01
<0.01
1.3
Ag 10
10
6, 40 and 200
N/A
3.0
21.6
0.97
Ag 40
40
10-40 and 200
−41.5
0.2
10.6
0.93
aSize, estimated from TEM images, and zeta potential (in MilliQ water with 1 mM NaCl) were analyzed in previous studies (Karlsson et al., 2008 [17], Kain et al.,
2012 [18]), Ag NPs were received from Nanocomposix and were carefully characterized in Gliga et al., 2014 [31]. bAcellular ROS: Times increase compared to control in ROS kinetics (mean slope per min), 10 μg/mL particle dispersions. Table 1 Physico-chemical characterization of the metal and metal oxide nanoparticles aSize, estimated from TEM images, and zeta potential (in MilliQ water with 1 mM NaCl) were analyzed in previous studies (Karlsson
2012 [18]), Ag NPs were received from Nanocomposix and were carefully characterized in Gliga et al., 2014 [31]. bAcellular ROS: Times increase compared to control in ROS kinetics (mean slope per min), 10 μg/mL particle dispersions. viable cells. Stem cells are generally very sensitive to cellu-
lar damage and in response rapidly activate their cellular
signaling pathways and apoptotic programs. These proper-
ties make stem cells a highly sensitive system for detection
of the primary toxic properties of chemicals and nanoma-
terials. The induction was observed for doses at which at
least 40% of the cells were still viable and started from
10 μg/mL for the CuO and 25 μg/mL for the NiO NPs. The ZnO NPs showed induction of the oxidative stress re-
porter at concentrations from 20–30 μg/mL, but high
levels of cytotoxicity (>75%) at these concentrations make
the ToxTracker results inconclusive. Cellular uptake, but no evidence for nuclear localization
of metal oxide NPs None of the particles
induced the DNA replication stress reporter (Bscl2-GFP),
indicating that none of the tested NPs induced significant
levels of replication-blocking DNA lesions. In addition to
the oxidative stress reporter, the NiO NPs also induced
the p53-dependent Btg2-GFP reporter, suggesting that the
NiO NPs induced other types of biological damage to the
cells. Exposure of the ToxTracker cells to CeO2, Fe3O4,
TiO2 and Ag NPs did not result in any reporter activation
(Figure 2A) nor induced any significant cytotoxicity at in-
vestigated doses (Figure 2B). The cell viability results
based on flow cytometry were confirmed using the Ala-
mar Blue cell viability assay (Additional file 1: Figure S4). viable cells. Stem cells are generally very sensitive to cellu-
lar damage and in response rapidly activate their cellular
signaling pathways and apoptotic programs. These proper-
ties make stem cells a highly sensitive system for detection
of the primary toxic properties of chemicals and nanoma-
terials. The induction was observed for doses at which at
least 40% of the cells were still viable and started from
10 μg/mL for the CuO and 25 μg/mL for the NiO NPs. The ZnO NPs showed induction of the oxidative stress re-
porter at concentrations from 20–30 μg/mL, but high
levels of cytotoxicity (>75%) at these concentrations make
the ToxTracker results inconclusive. None of the particles
induced the DNA replication stress reporter (Bscl2-GFP),
indicating that none of the tested NPs induced significant
levels of replication-blocking DNA lesions. In addition to
the oxidative stress reporter, the NiO NPs also induced
the p53-dependent Btg2-GFP reporter, suggesting that the
NiO NPs induced other types of biological damage to the
cells. Exposure of the ToxTracker cells to CeO2, Fe3O4,
TiO2 and Ag NPs did not result in any reporter activation
(Figure 2A) nor induced any significant cytotoxicity at in-
vestigated doses (Figure 2B). The cell viability results
based on flow cytometry were confirmed using the Ala-
mar Blue cell viability assay (Additional file 1: Figure S4). nanoparticle toxicity testing (Figure 1A), we first investi-
gated the ability of the mES cells to internalize NPs by
means of TEM and by analyzing the side scatter shift by
using flow cytometry. Indeed, NPs may cause genotoxic
effects via indirect mechanisms [19], but without cellular
uptake the applicability of the assay for NP testing will
be limited as in the case for the Ames mutagenicity test
[20]. The ToxTracker assay identifies distinct toxicity responses
to NPs Next, we exposed the ToxTracker cell lines to the eight
different metal-based NPs for 24 h and recorded induc-
tion of the GFP reporters for DNA damage, oxidative
stress and global cellular stress together with cell viabi-
lity using a 96-well plate-based flow cytometer. Based on
previous validation of the ToxTracker assay, a 1.5-fold
induction of GFP expression was considered as cut-off
for a positive test score [16]. A clear induction of the
oxidative stress reporter (Srxn1-GFP) was observed for
the CuO and NiO NPs (Figure 2A). Importantly, GFP re-
porter induction is exclusively determined in the intact, In order to confirm the ToxTracker responses we tested
the NPs in conventional DNA damage assays. First, we
performed alkaline as well as FPG (Formamidopyrimi-
dine DNA glycosylase) comet assay following mES cell
exposure to the NPs at 20 μg/mL for 4 h (non-cytotoxic
conditions). The alkaline comet assay is able to detect a
wide range of DNA damage including strand breaks and
alkaline labile sites (ALSs) whereas the FPG comet assay
mainly detects oxidized purines [12]. Exposure to CuO
and NiO NPs, as well as TiO2 NPs, gave significant tail-
ing in the alkaline version of the comet assay (Figure 3A). Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 5 of 14 Page 5 of 14 0
2
4
6
0
5
10
15
20
CuO
GFP induction
Conc. (µg/mL)
0
2
4
6
0
10
20
30
ZnO
GFP induction
Conc. (µg/mL)
0
2
4
6
0
25
50
75
100
NiO
GFP induction
Conc. (µg/mL)
0
2
4
6
0
25
50
75
100
CeO2
GFP induction
Conc. (µg/mL)
0
25
50
75
100
Fe3O4
Conc. (µg/mL)
0
2
4
6
0
25
50
75
100
TiO2
GFP induction
Conc. (µg/mL)
0
2
4
6
0
10 20 30 40 50
Ag-10
GFP induction
Conc. (µg/mL)
0
2
4
6
0
10 20 30 40 50
Ag-40
GFP induction
Conc. The ToxTracker assay identifies distinct toxicity responses
to NPs (µg/mL)
0
0.2
0.4
0.6
0.8
1.0
1.2
CuO
Cell viability
NiO
Fe3O4
0
2
4
6
GFP induction
A
B
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
Ag-10
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
10
20
30
Conc. (µg/mL)
0
25
50
75
100
Conc. (µg/mL)
ZnO
TiO2
0
25
50
75
100
Conc. (µg/mL)
0
10 20 30 40 50
Conc. (µg/mL)
CeO2
Ag-40
0
25
50
75
100
Conc. (µg/mL)
0
10 20 30 40 50
Conc. (µg/mL)
0
5
10
15
20
Conc. (µg/mL)
0
25
50
75
100
Conc. (µg/mL)
Bscl2-GFP
Srxn1-GFP
Btg2-GFP
0
2
4
6
8
0
2
4
6
8
10
CisPt
GFP induction
Conc. (µM)
0
4
8
12
16
0 50 100 150 200 250
DEM
GFP induction
0
0.2
0.4
0.6
0.8
1.0
1.2
0
2
4
6
8
10
CisPt
GFP induction
Conc. (µM)
0
0.2
0.4
0.6
0.8
1.0
1.2
0 50 100 150 200 250
DEM
GFP induction
Conc. (µM)
Conc. (µM)
Figure 2 (See legend on next page.) 0
25
50
75
100
Fe3O4
Conc. (µg/mL)
0
2
4
6
GFP induction
Srxn1-GFP
Btg2-GFP Fe3O4
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
25
50
75
100
Conc. (µg/mL) 0
2
4
6
0
5
10
15
20
CuO
GFP induction
Conc. (µg/mL)
A
Bscl2-GFP
S 0
0.2
0.4
0.6
0.8
1.0
1.2
CuO
Cell viability
Fe3O4
B
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
5
10
15
20
Conc. (µg/mL)
0
25
50
75
100
Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
CuO
Cell viability
B
0
5
10
15
20
Conc. (µg/mL) B A Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
25
50
75
100
Conc. (µg/mL)
TiO2 0
2
4
6
0
25
50
75
100
TiO2
GFP induction
Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
10
20
30
Conc. (µg/mL)
ZnO 0
2
4
6
0
10
20
30
ZnO
GFP induction
Conc. (µg/mL) Conc. (µg/mL) Conc. (µg/mL) Conc. (µg/mL) Conc. (µg/mL) Conc. The ToxTracker assay identifies distinct toxicity responses
to NPs (B) Cytotoxicity of the tested NPs was determined by
measuring the fraction of intact cells after 24 h exposure using flow cytometry. ssDNA breaks in mES cells [22]. RAD51 is a protein re-
cruited to DSBs and plays an essential role in repair of
DSBs by homologous recombination [23]. Exposure of
mES cells to CuO, ZnO and NiO led to an increase
in H2AX phosphorylation, although the levels were low
compared with the control exposure to 10 Gy ionizing ra-
diation (IR) (Figure 3C). In contrast, no RAD51 foci for-
mation was observed after exposure to these NPs. Taken
together, these results indicate that CuO induce ssDNA
breaks and oxidative DNA lesions and thus confirms oxi-
dative stress as a main mechanism as identified by the
ToxTracker. NiO predominantly induce ssDNA breaks at
non-cytotoxic conditions and possibly act via additional
mechanisms that lead to p53 induction, as shown by Tox-
Tracker. Clearly, the particles were not potent in inducing The other NPs did not induce detectable DNA dam-
age at these non-cytotoxic conditions. Furthermore, only
CuO NPs caused an 5-fold induction of FPG sensitive
sites, suggesting that oxidative stress is a main mechanism
for CuO toxicity (Figure 3B). The ZnO also induced tail-
ing in the alkaline comet assay at higher concentrations
(Additional file 1: Figure S5). Additional analyses using
the neutral comet assay, which is more indicative for dou-
ble strand DNA breaks, showed no induction by CuO or
NiO and only slight induction by ZnO (Additional file 1:
Figure S5). The metal oxide NPs that showed a positive
response in the ToxTracker were further investigated for
their ability to induce γH2AX and RAD51 foci. The his-
tone variant H2AX is rapidly phosphorylated at dsDNA
breaks (DSB) but also at sites of chromatin relaxation and The other NPs did not induce detectable DNA dam-
age at these non-cytotoxic conditions. Furthermore, only
CuO NPs caused an 5-fold induction of FPG sensitive
sites, suggesting that oxidative stress is a main mechanism
for CuO toxicity (Figure 3B). The ZnO also induced tail-
ing in the alkaline comet assay at higher concentrations
(Additional file 1: Figure S5). Additional analyses using
the neutral comet assay, which is more indicative for dou-
ble strand DNA breaks, showed no induction by CuO or
NiO and only slight induction by ZnO (Additional file 1:
Figure S5). The ToxTracker assay identifies distinct toxicity responses
to NPs (µg/mL) 0
2
4
6
0
25
50
75
100
NiO
GFP induction
Conc. (µg/mL) NiO
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
Ag-10
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
25
50
75
100
Conc. (µg/mL)
0
10 20 30 40 50
Conc. (µg/mL) Ag-10
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
10 20 30 40 50
Conc. (µg/mL) NiO
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
25
50
75
100
Conc. (µg/mL) 0
2
4
6
0
10 20 30 40 50
Ag-10
GFP induction
Conc. (µg/mL) Conc. (µg/mL) Conc. (µg/mL) Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
CeO2
0
25
50
75
100
Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
CeO2
Ag-40
0
25
50
75
100
Conc. (µg/mL)
0
10 20 30 40 50
Conc. (µg/mL) 0
2
4
6
0
10 20 30 40 50
Ag-40
GFP induction
Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
Cell viability
Ag-40
0
10 20 30 40 50
Conc. (µg/mL) 0
2
4
6
0
25
50
75
100
CeO2
GFP induction
Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
0
2
4
6
8
10
CisPt
GFP induction
Conc. (µM)
0
0.2
0.4
0.6
0.8
1.0
1.2
0 50 100 150 200 250
DEM
GFP induction
Conc. (µM) 0
2
4
6
8
0
2
4
6
8
10
CisPt
GFP induction
Conc. (µM)
Figure 2 (See legend on next page.) 0
4
8
12
16
0 50 100 150 200 250
DEM
GFP induction
Conc. (µM) Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 6 of 14 (See figure on previous page.)
Figure 2 The ToxTracker assay can identify genotoxic properties of NPs. (A) The ToxTracker reporter cell lines Bscl2-GFP for DNA replication
stress, Srxn1-GFP for oxidative stress and Btg2-GFP for p53-associated cellular stress were used to provide mechanistic insight into the biological
damage that is induced by various metal-based NPs. Induction of the GFP reporters was determined by flow cytometry after 24 h exposure. The
data show the mean of four independent experiments ± standard deviation of the mean. The ToxTracker assay identifies distinct toxicity responses
to NPs The metal oxide NPs that showed a positive
response in the ToxTracker were further investigated for
their ability to induce γH2AX and RAD51 foci. The his-
tone variant H2AX is rapidly phosphorylated at dsDNA
breaks (DSB) but also at sites of chromatin relaxation and A
B
C
γH2AX
RAD51
Untreated
IR
CuO
ZnO
NiO
0
8
16
24
Control
CuO
NiO
TiO2
ZnO
CeO2
Fe3O4
Ag-10
Ag-40
H2O2
% DNA in tail
0
5
10
Control
CuO
NiO
TiO2
ZnO
CeO2
Fe3O4
Ag-10
Ag-40
% DNA in tail
*
*
*
*
Figure 3 Conventional DNA damage assays confirm the ToxTracker response. (A) Induction of DNA strand breaks by the metal oxide NPs
was determined by the comet assay under alkaline conditions. Wild type mES cells were exposed to NPs (20 μg/mL) for 4 h. H2O2 (10 μM for
10 min on ice) was used as positive control. DNA damage was quantified as percentage of DNA in the comet tail. Results are presented as
mean ± standerd deviation of 3 independent experiments. (B) Induction of oxidative DNA lesions was determined by FPG comet. Wild type mES cells
were exposed to NPs (20 μg/mL) for 4 h and results are expressed as net FPG sites. (C) Induction of γH2AX and RAD51 foci after 4 or 8 h exposure of
mES cells to CuO (20 μg/mL), ZnO (30 μg/mL) and NiO (100 μg/mL) NPs as determined by immunocytochemistry. DSBs induction after 10 Gy IR was
used as positive control. B
0
5
10
Control
CuO
NiO
TiO2
ZnO
CeO2
Fe3O4
Ag-10
Ag-40
% DNA in tail
* A
0
8
16
24
Control
CuO
NiO
TiO2
ZnO
CeO2
Fe3O4
Ag-10
Ag-40
H2O2
% DNA in tail
*
*
* B A C C
γH2AX
RAD51
Untreated
IR
CuO
ZnO
NiO Figure 3 Conventional DNA damage assays confirm the ToxTracker response. (A) Induction of DNA strand breaks by the metal oxide NPs
was determined by the comet assay under alkaline conditions. Wild type mES cells were exposed to NPs (20 μg/mL) for 4 h. H2O2 (10 μM for
10 min on ice) was used as positive control. DNA damage was quantified as percentage of DNA in the comet tail. Results are presented as
mean ± standerd deviation of 3 independent experiments. (B) Induction of oxidative DNA lesions was determined by FPG comet. The ToxTracker assay identifies distinct toxicity responses
to NPs Wild type mES cells
were exposed to NPs (20 μg/mL) for 4 h and results are expressed as net FPG sites. (C) Induction of γH2AX and RAD51 foci after 4 or 8 h exposure of
mES cells to CuO (20 μg/mL), ZnO (30 μg/mL) and NiO (100 μg/mL) NPs as determined by immunocytochemistry. DSBs induction after 10 Gy IR was
used as positive control. Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 7 of 14 Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 dsDNA breaks. ZnO only caused effects at doses that at
later time points were highly cytotoxic. therefore is difficult to mimic. Furthermore, complexation
between dissolved metal ions and cell medium compo-
nents changes the metal speciation, reducing the free
ion concentration with time. Dissolved Cu from CuSO4
(assuming 100% free Cu ions) induced the Srxn1-GFP
oxidative stress reporter at doses starting from 50 μM
(Figure 4A) without any effects on cell viability (Figure 4B). When compared with released concentrations of Cu
from the CuO NPs (20 μg/L) after 0 h (8 μM) and
24 h (117 μM) it is evident that the dissolved Cu
fraction from CuO NPs after 24 h was sufficient to induce
oxidative stress. In the case of dissolved Zn from ZnSO4 The toxicity of CuO is mediated via ions whereas that of
NiO is particle-mediated Since the NPs that showed induction of the ToxTracker
reporters (CuO, ZnO and NiO) dissolve to various ex-
tents in the cell culture medium, we investigated the
ToxTracker response induced by dissolved metal ions
from easily soluble metal salts at different concentra-
tions. It should be noted that the metal dissolution from
the oxide NPs occurs over time and that the cellular effect Bscl2-GFP
Srxn1-GFP
Btg2-GFP
0
1
2
3
4
0 250 500 750 1000
ZnSO4
GFP induction
Conc. (µM)
0
2
4
6
8
0 250 500 750 1000
NiCl2
GFP induction
Conc. (µM)
0
0.2
0.4
0.6
0.8
1.0
1.2
ZnSO4
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
NiCl2
Cell viability
0
3
6
9
12
0 100 200 300 400
CuSO4
GFP induction
Conc. (µM)
0
0.2
0.4
0.6
0.8
1.0
1.2
CuSO4
Cell viability
0 100 200 300 400
Conc. (µM)
0 250 500 750 1000
Conc. (µM)
0 250 500 750 1000
Conc. (µM)
A
B
Figure 4 Induction of the ToxTracker assay by metal ions. mES cells were exposed to CuSO4 (25 – 1000 μM), ZnSO4 (50 – 1000 μM), NiCl2
(50 – 1000 μM) for 24 h. GFP induction and cell viability were determined with flow cytometry. Results are presented as mean ± standard
deviation of three independent experiments. Btg2-GFP
0
0.2
0.4
0.6
0.8
1.0
1.2
ZnSO4
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
NiCl2
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
CuSO4
Cell viability
0 100 200 300 400
Conc. (µM)
0 250 500 750 1000
Conc. (µM)
B Btg2-GFP
0
0.2
0.4
0.6
0.8
1.0
1.2
ZnSO4
Cell viability
0
0.2
0.4
0.6
0.8
1.0
1.2
CuSO4
Cell viability
0 100 200 300 400
Conc. (µM)
0 250 500 750 1000
Conc. (µM)
B Bscl2-GFP
Srxn1-G
0
3
6
9
12
0 100 200 300 400
CuSO4
GFP induction
Conc. (µM)
A Btg2-GFP
0
0.2
0.4
0.6
0.8
1.0
1.2
CuSO4
Cell viability
0 100 200 300 400
Conc. (µM)
B B A 0
1
2
3
4
0 250 500 750 1000
ZnSO4
GFP induction
Conc. (µM) 0
0.2
0.4
0.6
0.8
1.0
1.2
ZnSO4
Cell viability
0 250 500 750 1000
Conc. (µM) 0
0.2
0.4
0.6
0.8
1.0
1.2
NiCl2
Cell viability
0 250 500 750 1000
Conc. Discussion In order to benchmark the observed response to a par-
ticle control material we exposed the ToxTracker repor-
ter cell lines to the carcinogenic quartz particles (DQ12). DQ12 is not a nanomaterial, but has often been used as
a positive reactive particle control in both in vitro and
in vivo studies on nanomaterials since it has high surface
reactivity, inflammatory effects and induce oxidative
DNA lesions at higher doses [24-26]. We also investi-
gated whether the ToxTracker reporters were induced
upon exposure to diesel particles (standard reference ma-
terial SRM1650b) and carbon nanotubes (MWCNTs). Ex-
posure to quartz particles clearly induced the Srxn1-GFP
reporter at non-cytotoxic doses, starting from 50 μg/mL
(Figure 5) supporting previous findings showing that ROS
generation and more specifically hydroxyl radicals, play a
major role for DQ12 induced genotoxicity [27]. On the
other hand, no acellular ROS production was detected
from the DQ12 particles (data not shown). In contrast,
the MWCNTs and diesel particles did not induce the Tox-
Tracker reporters. TEM images of mES cells exposed to
MWCNTs indicated some uptake and there was also an
increased side scatter shift analyzed by flow cytometry for
both MWCNTs and diesel particles (Additional file 1: The fast expansion of manufacturing and use of nano-
sized materials requires toxicity testing that should ideally
be rapid, reliable, possible to use in a high-throughput
manner and should provide information regarding the
mechanisms of toxicity. These requirements are challen-
ging to meet and will require development or implemen-
tation of novel tools. In this study we applied a novel
fluorescence-based reporter assay for mechanism-based
genotoxicity testing of various nanoparticles, in line with
the 21st century paradigm for nanomaterial testing which
emphasizes the need for mechanism-based screening as-
says [2,30]. The ToxTracker reporter assay has previously
been shown to discriminate between compounds that can
directly interact with DNA causing stalled replication
forks (that may ultimately lead to mutations and genome
instability) and compounds that act via oxidative stress
and subsequent oxidative DNA lesions. The mES cells
that are used in the ToxTracker assay are untransformed,
proficient in all major DNA damage and cellular stress re-
sponse pathways and shown in this study to efficiently en-
gulf nanoparticles. The toxicity of CuO is mediated via ions whereas that of
NiO is particle-mediated Treatment of mES cells with
diesel particles in the presence of S9 did not lead to any
reporter activation (Figure 5B). Thus, under the condi-
tions tested in the present study, no pronounced genotoxi-
city/oxidative stress was observed for either the MWCNT
or the diesel particles. (assuming 100% free Zn ions), the Srxn1-GFP reporter re-
sponse was mainly induced at concentrations exceeding
500 μM and at these concentrations high cytotoxicity was
observed making the ToxTracker results inconclusive (as
for ZnO NPs). The particle effect was, however, pro-
nounced for the NiO NPs compared to the response for
dissolved Ni from NiCl2 (assuming 100% free Ni ions). For NiCl2, the ToxTracker Srxn1-GFP reporter was in-
duced at concentrations ≥500 μM and the Btg2-GFP re-
porter at concentrations of 1000 μM. Measured released
concentrations from the NiO NPs after 24 h in cell
medium were only minor (17 μM) compared to these con-
centrations and could therefore not explain the observed
toxicity. A comparison between the concentrations used
for NPs (CuO, NiO, ZnO) and their corresponding ions as
well as the effect observed in ToxTracker is summarized
in Table 2. The toxicity of CuO is mediated via ions whereas that of
NiO is particle-mediated (µM) 0
2
4
6
8
0 250 500 750 1000
NiCl2
GFP induction
Conc. (µM) Conc. (µM) Figure 4 Induction of the ToxTracker assay by metal ions. mES cells were exposed to CuSO4 (25 – 1000 μM), ZnSO4 (50 – 1000 μM), NiCl2
(50 – 1000 μM) for 24 h. GFP induction and cell viability were determined with flow cytometry. Results are presented as mean ± standard
deviation of three independent experiments. Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 8 of 14 Figure S2 and Additional file 1: Figure S3). Thus, lack of
uptake is not a likely explanation for the lack of effect in
the ToxTracker reporters. Diesel exhaust particles consist
of a mixture of polycyclic aromatic hydrocarbons (PAH),
transition metals and quinones adsorbed on a carbon core
that can lead to genotoxicity mainly via PAH-DNA bulky
adduct formation and partly by oxidative DNA damage
[28,29]. Since PAHs require metabolic activation by cyto-
chrome P450 enzymes in the liver and the lung before
they become reactive, the effect of the diesel particles was
also investigated in the presence of S9 rat liver extract. As
a control for the activity of the S9 liver enzymes we
treated the ToxTracker cells with the genotoxic com-
pound aflatoxin B1 [16]. Treatment of mES cells with
diesel particles in the presence of S9 did not lead to any
reporter activation (Figure 5B). Thus, under the condi-
tions tested in the present study, no pronounced genotoxi-
city/oxidative stress was observed for either the MWCNT
or the diesel particles. Figure S2 and Additional file 1: Figure S3). Thus, lack of
uptake is not a likely explanation for the lack of effect in
the ToxTracker reporters. Diesel exhaust particles consist
of a mixture of polycyclic aromatic hydrocarbons (PAH),
transition metals and quinones adsorbed on a carbon core
that can lead to genotoxicity mainly via PAH-DNA bulky
adduct formation and partly by oxidative DNA damage
[28,29]. Since PAHs require metabolic activation by cyto-
chrome P450 enzymes in the liver and the lung before
they become reactive, the effect of the diesel particles was
also investigated in the presence of S9 rat liver extract. As
a control for the activity of the S9 liver enzymes we
treated the ToxTracker cells with the genotoxic com-
pound aflatoxin B1 [16]. Discussion Exposure of the ToxTracker cells to
CuO and NiO induced the Srxn-1 reporter but not the Table 2 The ability of NPs and their corresponding metal ions to induce the ToxTracker reporters
μg/mL
μM
μg metal/mL
ToxTracker induction
μg released metal/mL
in cell medium
Is the released metal in cell
medium likely to induce ToxTracker? CuO NPs
20
-
15,8
Yes
7,4
YES →ionic effect
CuSO4
-
50
3,2
Yes
-
NiO NPs
20
-
15,6
Yes
1
NO →particle effect
NiCl2
-
250
14,7
No
-
ZnO NPs
20
-
16,1
Yes (↑Tox)
15,7
Inconclusive due to high cytotoxicity
ZnSO4
-
500
32,7
Yes (↑Tox)
- Table 2 The ability of NPs and their corresponding metal ions to induce the ToxTracker reporters s and their corresponding metal ions to induce the ToxTracker reporters Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 9 of 14 Page 9 of 14 Bscl2-GFP
Srxn1-GFP
Btg2-GFP
0
1
2
3
4
0
25
50
75
100
Carbon nanotubes
GFP induction
Conc. (µg/mL)
0
1
2
3
4
0
25
50
75
100
Diesel
GFP induction
Conc. (µg/mL)
0
1
2
3
4
0
25
50
75
100
Quartz
GFP induction
Conc. (µg/mL)
0
0.2
0.4
0.6
0.8
1.0
1.2
0
25
50
75
100
Diesel
Cell survival
Conc. (µg/mL)
0
0.2
0.4
0.6
0.8
1.0
1.2
0
25
50
75
100
Quartz
Cell survival
Conc. (µg/mL)
A
B
0
0.2
0.4
0.6
0.8
1.0
1.2
0
25
50
75
100
Carbon nanotubes
Cell survival
Conc. (µg/mL)
0
1
2
3
4
0
25
50
75
100
Diesel + S9
GFP induction
Conc. (µg/mL)
0
2
4
6
Bscl2
Srxn1
Btg2
GFP induction
- S9
+ S9
Aflatoxin B1
Figure 5 ToxTracker induction by non-metal nanoparticles. (A) The ToxTracker mES cells were exposed to quartz, multi-walled carbon
nanotubes and diesel exhaust particles (6.25 – 100 μg/mL) for 24 h. GFP induction and cell viability were determined with flow cytometry. Results
are presented as mean ± standard deviation of three independent experiments. (B) The ToxTracker mES cells were exposed to diesel exhaust
particles in the presence of S9 rat liver extract for 3 h. GFP induction was determined after 24 h by flow cytometry. Results are presented as
mean ± standard deviation of three independent experiments. Aflatoxin B1 was used as a positive control. Discussion This might be explained by differences in sensi-
tivity or possibly also that photocatalytic TiO2 NPs create
additional strand breaks during the comet assay perform-
ance [12]. The Ag NPs included in the study showed no
genotoxicity (comet assay and reporter cells) as well as no
clear effect on the cell viability. Especially the lack of ef-
fects on cell viability after 24 h of the Ag-10 NPs was sur-
prising considering the high cytotoxicity observed in
BEAS-2B cells of these NPs [31]. salts, we revealed that the effect observed for the CuO
NPs could be explained by the released ions whereas the
reporter activation (oxidative stress and p53 dependent
stress) by NiO was related to the particles per se (Table 2). Indeed, our previous studies on lung epithelial cells sug-
gested a high DNA damage potential of CuO and NiO
NPs compared to other metal oxide NPs [17,18] and the
p53-dependent cellular stress response suggests additional
reactivity of the NiO NPs compared to CuO. The uptake
of the NiO was high as assessed by the TEM images. Likely, intracellular release of Ni ions could be important
for the reporter response, which would be in line with the
Ni-ion bioavailability theory describing that the carcino-
genicity of a Ni-compound depends on the cellular uptake
and subsequent availability of Ni in the cell nucleus [32]. Nickel ions from particles can be transported to the nu-
cleus [33] and have been suggested to interact with DNA
in a manner that causes silencing of tumor suppressor
genes, i.e. an epigenetic effect [34]. Thus, even though we
did not observe any NPs in the nucleus, metal ions from
the NPs may still be present in the nucleus and interact
with DNA, but in a manner that does not cause replica-
tion stress according to our results. Bscl2 reporter (Figure 2), indicating that the main mech-
anism of toxicity is oxidative stress rather than direct
DNA binding and subsequent interference with DNA rep-
lication. This is also in line with the positive response in
the alkaline comet assay (detecting ssDNA breaks and al-
kaline labile sites), the positive FPG comets for CuO, and
the limited response in the neutral version of the assay as
well as the lack of RAD51 foci formation (Figure 3). Discussion B
0
1
2
3
4
0
25
50
75
100
Diesel + S9
GFP induction
Conc. (µg/mL) Btg2-GFP
0
1
2
3
4
0
25
50
75
100
Diesel
GFP induction
Conc. (µg/mL) Bsc
0
1
2
3
4
0
25
50
75
100
Quartz
GFP induction
Conc. (µg/mL)
A A B cl2-GFP
Srxn1-GFP
0
1
2
3
4
0
25
50
75
100
Carbon nanotubes
GFP induction
Conc. (µg/mL) Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
0
25
50
75
100
Carbon nanotubes
Cell survival
Conc. (µg/mL) 0
2
4
6
Bscl2
Srxn1
Btg2
GFP induction
- S9
+ S9
Aflatoxin B1 0
0.2
0.4
0.6
0.8
1.0
1.2
0
25
50
75
100
Quartz
Cell survival
Conc. (µg/mL) 0
0.2
0.4
0.6
0.8
1.0
1.2
0
25
50
75
100
Diesel
Cell survival
Conc. (µg/mL) Cell survival Conc. (µg/mL) Conc. (µg/mL) Conc. (µg/mL) Figure 5 ToxTracker induction by non-metal nanoparticles. (A) The ToxTracker mES cells were exposed to quartz, multi-walled carbon
nanotubes and diesel exhaust particles (6.25 – 100 μg/mL) for 24 h. GFP induction and cell viability were determined with flow cytometry. Results
are presented as mean ± standard deviation of three independent experiments. (B) The ToxTracker mES cells were exposed to diesel exhaust
particles in the presence of S9 rat liver extract for 3 h. GFP induction was determined after 24 h by flow cytometry. Results are presented as
mean ± standard deviation of three independent experiments. Aflatoxin B1 was used as a positive control. Bscl2 reporter (Figure 2), indicating that the main mech-
anism of toxicity is oxidative stress rather than direct
DNA binding and subsequent interference with DNA rep-
lication. This is also in line with the positive response in
the alkaline comet assay (detecting ssDNA breaks and al-
kaline labile sites), the positive FPG comets for CuO, and
the limited response in the neutral version of the assay as
well as the lack of RAD51 foci formation (Figure 3). Iden-
tification of oxidative stress as primary mechanism of gen-
otoxicity strongly decreases the carcinogenic hazard of
these particles compared to agents that interact directly
with DNA and interfere with DNA replication. The TiO2
NPs were positive in the comet assay but not in the Tox-
Tracker. Conclusions In this study, we have shown that the ToxTracker mES
reporter cell assay can be applied as a rapid mechanism-
based tool for assessing genotoxic effects of NPs. The
assay is adapted to a 96-well plate format thus enabling
medium/high throughput screening. CuO and NiO NPs
caused a substantial reporter cell response that was re-
lated to oxidative stress rather than to direct interaction
with DNA and stalled replication forks. NiO also induced
the p53 dependent cellular stress reporter suggesting ad-
ditional reactivity compared to CuO. Furthermore, the
reporter cell induction appeared to be mediated by dis-
solved metal ions from CuO whereas the responses ob-
served from NiO were related to the particles per se. The
assay was validated for metal oxide NPs whereas the
applicability for carbon based nanomaterials needs to
be further investigated. Obtained results indicate that
the ToxTracker reporter system can be used as a rapid
mechanism-based tool for assessing genotoxicity of metal
oxide NPs. MWCNTs and diesel particles did not induce the Tox-
Tracker reporters suggesting lack of direct DNA inter-
action and comparatively low oxidative stress potential. Previous studies have also reported low ROS generation
of these materials, although a slight increase in DNA
damage has been observed for the MWCNTs [17] and
an increase in the mutation frequency following repeated
lung cell exposure to diesel particles [37]. The ToxTracker
reporter cell lines were thus not activated following expos-
ure to low-reactive carbon-based materials. ToxTracker
response following exposure to carbon particles with
higher reactivity, such as Printex 90 [37], requires further
testing. In this study, we show the applicability of the Tox-
Tracker assay for rapid screening of genotoxic properties
of NPs. Other approaches have also been suggested for
this purpose. Recently, Li and co-workers [38] proposed
that DNA-binding assays can be useful in this regard
and showed that NPs (size range 3–46 nm) with a high
affinity for DNA strongly inhibited DNA replication
(tested acellularly), whereas NPs with low affinity had no
or minimal effect. However, such experimental acellular
studies do not consider important factors such as the
ability of the NPs to enter the nucleus and the fact that
DNA is highly packed in mammalian cells. The likeli-
hood for nuclear localization and DNA interaction de-
pends on the NP size as well as its charge. Nabiev et al. Discussion Iden-
tification of oxidative stress as primary mechanism of gen-
otoxicity strongly decreases the carcinogenic hazard of
these particles compared to agents that interact directly
with DNA and interfere with DNA replication. The TiO2
NPs were positive in the comet assay but not in the Tox-
Tracker. This might be explained by differences in sensi-
tivity or possibly also that photocatalytic TiO2 NPs create
additional strand breaks during the comet assay perform-
ance [12]. The Ag NPs included in the study showed no
genotoxicity (comet assay and reporter cells) as well as no
clear effect on the cell viability. Especially the lack of ef-
fects on cell viability after 24 h of the Ag-10 NPs was sur-
prising considering the high cytotoxicity observed in
BEAS-2B cells of these NPs [31]. In the present study, we carefully evaluated time-
dependent dissolution/metal ion release from the metal
(oxide) nanoparticles in cell medium by means of ICP-
MS. In combination with experiments using soluble metal In order to benchmark the observed responses in the
reporter assay after metal NP exposure, we compared
responses to the carcinogenic quartz material called Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 Page 10 of 14 with the major grooves of DNA, which was suggested to
be the reason for the high toxicity of these NPs [40]. Interestingly, only marginally smaller or larger particles
showed significantly reduced toxicity [41]. Some studies
have reported direct interaction of NPs with DNA in
bacterial cells [42-44]. Possibly, DNA binding may be a
more relevant mechanism in bacterial cells where DNA
is more “naked” (histone-free) in contrast to mammalian
cells where DNA is packed in nucleosomes and chro-
mosomes. DNA may still be more exposed during spon-
taneously conformational fluctuations, in which DNA
transiently unwraps the histone core [45] or at transcrip-
tionally active sites. At present, the general importance
of such mechanisms for the genotoxicity of nanomater-
ials for mammalian cells remains unclear. DQ12 that have previously been shown to cause oxida-
tive stress and inflammation in various test systems
[24]. It is therefore often used as a positive insoluble
particle control in nanotoxicology studies [25]. In line
with earlier reporters, DQ12 showed a positive response
in the oxidative stress reporter cell line observed already
at sub-toxic doses (Figure 5). Conclusions demonstrated that green (2.1 nm) quantum dots (QDs)
but not red ones (3.4 nm) entered the nucleus of THP-1
cells via nuclear pore complexes [8] and Conroy et al. [39] furthermore showed that QDs preferentially bind to
the positively charged core histone proteins as opposed
to the DNA. Size-dependent effect has also been re-
ported for gold NPs as those with a very specific size of
1.4 nm have been shown to interact in a unique manner Discussion Furthermore, both CuO
and (especially) NiO showed intrinsic ROS generating
ability (as observed in the DCFH acellular ROS generating
assay), but this was not observed for DQ12. This supports
our previous work suggesting that cellular interaction/
lipid peroxidation is important for SiO2 NPs-induced tox-
icity [35]. ZnO did not produce acellular ROS and both
ZnO and ZnSO4 only induced the oxidative stress reporter
at highly cytotoxic doses (Figure 4B). This suggests that
the reporter induction could be a secondary effect of the
cytotoxicity in line with previous results that indicate that
oxidative stress is not the primary cause of toxicity for
ZnO [35,36]. Cellular uptake and localization by TEM For TEM analysis, wild type B4418 mES cells were ex-
posed to 20 μg/mL CuO, 30 μg/mL ZnO, 100 μg/mL NiO,
100 μg/mL CeO2 NPs, 10 μg/mL Ag-10 and 20 μg/mL
MWCNT for 24 h and subsequently fixed in 4% glutaral-
dehyde, rinsed in phosphate buffer (PB) and centrifuged. The pellets were then post fixed in 2% osmium tetroxide
in 0.1 M PB, pH 7.4 at 4°C for 2 h, dehydrated in ethanol
followed by acetone, and embedded in LX-112 (Ladd,
Burlington, Vermont, USA). Ultrathin sections (approx. 60–80 nm) were cut by a Leica ultracut UCT (Leica, Nanomaterials and soluble metal salts
l
f
(
) Nanoparticles of CuO (20–40 nm), ZnO (20–200 nm),
NiO (2–70 nm), CeO2 (4–30 nm), Fe3O4 (20–40 nm)
and TiO2 (20–100 nm, mix of rutile and anatase) as well as
multi-walled carbon nanotubes (MWCNTs, 100–200 nm
in diameter and 3–7 μm in length) were obtained from
Sigma Aldrich. TEM images of these particles have been
provided in previous studies [17,18]. Citrate coated Ag NPs
(10 and 40 nm, 1 mg/mL dispersion in aqueous 2 mM so-
dium citrate) were purchased from NanoComposix, Inc
(San Diego, CA), diesel exhaust particles (powder, SRM
1650b) were obtained from National Institute of Standards
and Technology (Gaithersburg, MD, USA) and quartz
particles (crystalline silica DQ12) were a kind gift from
Prof. Roel Schins, Leibniz Research Institute for Envi-
ronmental Medicine, Düsseldorf, Germany. Soluble metal Page 11 of 14 Page 11 of 14 Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
http://www.particleandfibretoxicology.com/content/11/1/41 salts of CuSO4, ZnSO4 and NiCl2 were purchased from
Sigma Aldrich. solution). This solution was then incubated with the NPs
in final concentrations of 10 and 50 μg/mL of NPs and
15 μM DCFH in 37°C for 30 min. Fluorescence was re-
corded every 5 min over 30 min (excitation 485 nm, emis-
sion 535 nm) using a plate reader (Tecan Infinite F200) at
37°C and ROS generation was calculated as mean slope
per min and normalized to the blank. ROS generation was
considered increased when the value was 1.5 or above. Hydrodynamic particle size in cell medium The size distribution in cell medium was investigated
using dynamic light scattering (DLS) on an instrument
employing photon cross correlation spectroscopy (PCCS)
(NanoPhox, Sympatec, Germany). 20 μg/mL dispersions
were prepared and analyzed directly after preparation. Sin-
gle samples were measured three times at 25°C. Data from
the unique measurements was integrated to produce a
single distribution with the PCCS software. Standard latex
samples (20 ± 2 nm) and blank samples were tested prior
to analysis to ensure the accuracy of the measurements. The cell medium components resulted in a background
contribution that was subtracted from the measured dis-
tribution for all nanomaterials investigated. Preparation of nanoparticle dispersions All powder particles were dispersed in cold mES culture
medium by two times 10 min sonication at maximum
power on ice (Bioruptor, Diagenode). The NP dispersions
were diluted in warm BRL-conditioned mES culture me-
dium, vortexed thoroughly and immediately added to the
ToxTracker reporter cells. The Ag NPs were diluted dir-
ectly in warm BRL-conditioned mES culture medium. NP
dispersions were freshly prepared prior to exposure. Metal release in cell medium and cellular uptake of CuO
The amount of released metals in mES cell culture me-
dium was determined by inductively coupled plasma op-
tical emission spectroscopy, ICP-OES (Thermo Scientific
iCAP 6500 duo). 20 μg/mL dispersions of CuO, ZnO,
NiO, TiO2, CeO2, Fe3O4 NPs and of Ag-10 and Ag-40
NPs were prepared in complete cell culture medium. Metal release investigations were performed immediately
after dispersion preparation (0 h) or kept at 37°C for 24 h
after particle separation using a two-fold centrifugation
procedure (30 min, 13000 rpm, 0°C). Successful particle
separation was ensured using PCCS measurements. Total
concentrations (limits of detection (LOD) in parenthesis)
of Cu (0.3 μg/L), Zn (0.1 μg/L), Ni (0.4 μg/L), Ce (5 μg/L),
Fe (0.3 μg/L), Ti (0.1 μg/L) and Ag (0.4 μg/L) in solution
were analyzed using standard operational procedures with
multiple standards for calibration (0, 100, 1000 μg/L) and
triplicate measurements of each sample. Parallel measure-
ments were performed on selected elements (Ag, Ti, Zn)
for quality control using atomic absorption spectroscopy-
graphite furnace, AAS-GF, (Perkin–Elmer Analyst 800). Mean metal concentrations of each element in solution
are based on three replicate readings of each sample, inde-
pendent of analytical method. Quality control samples
were analyzed continuously throughout all analysis. All re-
sults are expressed as the released metal mass fraction of
the total amount of exposed particles. The cellular uptake
of CuO NPs was analyzed using AAS-GF as previously
described [21]. mES cell culture and treatments Culture of the ToxTracker mES cells was performed as
described previously [16]. The mES cells were maintained
in the presence of irradiated mouse embryonic fibroblasts
as feeder cells in Knockout DMEM containing 10% fetal
bovine serum, 2 mM GlutaMAX, 1 mM sodium pyruvate,
100 μM β-mercaptoethanol, and leukemia inhibitory fac-
tor (LIF). For NP analysis, cells were seeded 24 h prior to
exposure on gelatin-coated plates using buffalo rat liver
cell (BRL)-conditioned mES cell medium in the absence
of feeder cells. Cells were continuously exposed for 24 h
before GFP reporter analysis. The tested NP concentra-
tions are based on cytotoxicity in mES cells after 24 h con-
tinuous exposure to a maximum test concentration at
50-75% cytotoxicity. In case of no observed cytotoxicity, a
top concentration of 50 or 100 μg/mL was used. For ana-
lysis of diesel exhaust particles and compounds that re-
quire metabolic activation, cells were exposed for 3 h in
the presence of 1% S9 rat liver extract in 3.2 mM KCl,
0.8 mM MgCl2, 0.5 mM glucose-6-phosphate and 0.4 mM
NADP. After 3 h, the cells were washed with PBS and cul-
tured for 24 h in BRL-conditioned medium without any
nanomaterials. The ToxTracker assay Bscl2-GFP, Srxn1-GFP and Btg2-GFP mES reporter cell
lines were seeded in gelatin-coated 96-wells plates and
exposed to various concentrations of NPs as described
above. Induction of the GFP reporters was measured af-
ter 24 h continuous exposure using a 96-well Guava flow
cytometer (Millipore) as described previously [16]. Sim-
ultaneously, cytotoxicity of the NPs was determined by
measuring the concentration of intact cells after expos-
ure using flow cytometry. All presented figures show the
average GFP induction of at least three independent ex-
periments. Error bars represent the standard error of the
mean. The ToxTracker assay is considered positive above
1.5 fold increase in GFP signals. This limit is statistically
based on 5 times the standard deviation of untreated con-
trols and has been extensively validated in previous studies
[16]. GFP induction levels at exposure concentration that
induce >75% cytotoxicity after 24 h exposure are dis-
carded for ToxTracker analysis. Competing interests The authors declare that they have no competing interests. Authors’ contribution
d
d
l HLK initiated and planned the study together with GH, performed some of
the experiments including alkaline and FPG comet assay, was involved in
data interpretation and wrote the final version of the manuscript. ARG
participated in the design of the study, carried out part of the cellular work,
prepared samples for the particle characterization, was involved in data
interpretation and drafted the manuscript. FC and CG carried out part of the
cellular work including alkaline and neutral comet assay. IOW supervised the
particle characterization, metal dissolution and ICP-OES analyses and their
interpretations and was involved in manuscript writing. BF and HV were
involved in data interpretation and in manuscript writing. GH initiated and
planned the study together with HLK, performed most of the cellular work
and wrote a first draft of the manuscript. All authors have given approval to
the final version of the manuscript. Abbreviations AAS-GF: Atomic absorption spectroscopy-graphite furnace; ALS: Alkaline
labile sites; ATR: Ataxia telangiectasia mutated and Rad3-related;
AgNPs: Silver nanoparticles; DCFH-DA: Dichlorodihydrofluorescein diacetate;
DLS: Dynamic light scattering; DSB: Double strand DNA breaks;
FPG: Formamidopyrimidine DNA glycosylase; GFP: Green fluorescent protein;
HBSS: Hank’s buffered salt solution; ICP-OES: Inductively coupled plasma
optical emission spectroscopy; IR: Ionizing radiation; mES: Mouse embryonic
stem cells; MWCNTs: Multi-walled carbon nanotubes; Nrf2: Nuclear factor
erythroid 2-related factor 2; PAH: Polycyclic aromatic hydrocarbons;
PCCS: Photon cross correlation spectroscopy; ROS: Reactive oxygen species;
TEM: Transmission electron microscopy; QDs: Quantum dots. Alkaline and FPG Comet Assay y
The mES cells were exposed to 20 μg/mL of nanoparti-
cles for 4 h. The alkaline and FPG version of the comet
assay was then performed as previously described [17]
with some modifications. In short, cells were embedded
in agarose and lysed for 1 h. For alkaline comet assay, al-
kaline unwinding and electrophoresis (29 V, 1.15 V/cm)
was then performed for 40 and 30 min, respectively. For
analysis of FPG-sensitive sites, following lysis cells were
immersed (3 × 5 min) in FPG enzyme buffer and 30 μL
of diluted (1:2500) FPG enzyme (kindly provided by Pro-
fessor A. R. Collins, Department of Nutrition, School of
Medicine, University of Oslo, Norway), or enzyme buffer,
was added to each gel. Parafilm was placed on the slides,
and incubation was performed in a humidity chamber at
37°C for 35 min. DNA unwinding and electrophoresis
(1.15 V/cm) were then carried out for 30 and 20 min, re-
spectively. The comets were examined on a fluorescence
microscope (Leica DMLB and Leica Axioplan2, Hous-
ton, TX) with Comet Assay IV (Perceptive Instruments)
and CometScore (TriTek). software. For each sample, 50
comets were evaluated in each experiment. The level of
FPG-sites was obtained by subtracting the value of % tail
obtained with no enzyme added from the value when
FPG-enzyme was present. Immunocytochemistry Immunocytochemistry Wien, Austria) and contrasted with uranyl acetate fol-
lowed by lead citrate and examined with in Tecnai 12
Spirit Bio TWIN transmission electron microscope (Fei
company, Eindhoven, The Netherlands) at 100 kV. Digital
images were captured by using a Veleta camera (Olympus
Soft Imaging Solutions, GmbH, Münster, Germany). For detection of the DNA damage response after expo-
sure to NPs, wild type B4418 mES cells were seeded on
fibronectin-coated glass cover slips in BRL-conditioned
mES cell medium. Cells were continuously exposed to
20 μg/mL CuO, 30 μg/mL ZnO, 100 μg/mL NiO NPs or
to 10 Gy ionizing radiation as positive control. After 4
or 8 h, cells were fixed in 3% paraformaldehyde and
stained for γH2AX or RAD51 as described previously
[46]. RAD51 antibodies were a kind gift from Prof. R. Kanaar, rabbit anti-γH2AX and alexa488-labeled sec-
ondary antibodies were purchased from Millipore and
Invitrogen, respectively. Images were taken with a Zeiss
Axioplan2 microscope equipped with a Zeiss Axiocam
MRm camera using either a Plan-NEOFLUAR 40x/1.30
or a 63x/1.25 objective. Acellular ROS generation To measure acelullar ROS production, the 2',7'-dichlo-
rofluorescein diacetate (DCFH-DA) assay was used. In
brief, sodium hydroxide (0.01 M) was added to DCFH-
DA to cleave the DA from the DCFH. The reaction was
stopped by the addition of HBSS (Hank’s buffered salt Karlsson et al. Particle and Fibre Toxicology 2014, 11:41
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Cite this article as: Karlsson et al.: Mechanism-based genotoxicity
screening of metal oxide nanoparticles using the ToxTracker panel of
reporter cell lines. Particle and Fibre Toxicology 2014 11:41. doi:10.1186/s12989-014-0041-9
Cite this article as: Karlsson et al.: Mechanism-based genotoxicity
screening of metal oxide nanoparticles using the ToxTracker panel of
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Dyslexia as a Neurodevelopmental Disorder and What Makes It Different from a Chess Disorder
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Keywords: dyslexia; disorder; neurodevelopmental Keywords: dyslexia; disorder; neurodevelopmental p
Dyslexia as a Neurodevelopmental Disorder and
What Makes It Different from a Chess Disorder Gorka Fraga González 1,2,3,*, Iliana I. Karipidis 3,4 and Jurgen Tijms 1,2,5 1
Department of Developmental Psychology, University of Amsterdam,
Postbus 15916 1001 NK Amsterdam, The Netherlands; jurgentijms@gmail.com
2
Rudolf Berlin Center, 1018 WS Amsterdam, The Netherlands
3
Department of Child and Adolescent Psychiatry and Psychotherapy, Psychiatric Hospital,
University of Zurich, 8032 Zurich, Switzerland; iliana.karipidis@kjpd.uzh.ch
4
Neuroscience Center Zurich, University of Zurich and ETH Zurich, 8057 Zurich, Switzerland
5
RID, 1018 WS Amsterdam, The Netherlands
*
Correspondence: gorka.fragagonzalez@uzh.ch; Tel.: +41-779-55-29-75 1
Department of Developmental Psychology, University of Amsterdam,
Postbus 15916 1001 NK Amsterdam, The Netherlands; jurgentijms@gmail.com
2
Rudolf Berlin Center, 1018 WS Amsterdam, The Netherlands
3
Department of Child and Adolescent Psychiatry and Psychotherapy, Psychiatric Hospital,
University of Zurich, 8032 Zurich, Switzerland; iliana.karipidis@kjpd.uzh.ch
4
Neuroscience Center Zurich, University of Zurich and ETH Zurich, 8057 Zurich, Switzerland
5
RID, 1018 WS Amsterdam, The Netherlands
*
Correspondence: gorka.fragagonzalez@uzh.ch; Tel.: +41-779-55-29-75 Brain Sci. 2018, 8, 189; doi:10.3390/brainsci8100189 brain
sciences brain
sciences Received: 21 September 2018; Accepted: 18 October 2018; Published: 19 October 2018 Abstract: The convenience of referring to dyslexia as a neurodevelopmental disorder has been
repeatedly brought into question. In this opinion article, we argue in favor of the current diagnosis
of dyslexia based on the criteria of harm and dysfunction. We discuss the favorable clinical and
educational outcomes of a neuroscience-informed approach of dyslexia as a disorder. Furthermore,
we discuss insights derived from neuroimaging studies and their importance to address problems
related to developmental dyslexia. 1. Introduction Individuals with dyslexia are characterized by specific and persistent reading problems. Prevalence rates vary widely, although usually it is estimated that 3–7% of the population suffers from
dyslexia [1,2]. Over the years, the convenience of labeling dyslexia as a (neurodevelopmental) disorder has been
repeatedly brought into question [3–5]. One of the main arguments against the term is that people
differ in their reading proficiency along a continuum, and dyslexia is simply the lower-end tail of
this continuum, cut-off from the rest of the distribution by some artificial, arbitrary criterion. To be
a disorder, the problem in question should be of a categorical nature [4,6,7]. A logical consequence
of this is that there is no need to categorize poor readers as a different group than good readers,
some children simply are better at learning how to read than others. In a similar line of reasoning,
the observed brain differences between dyslexics and non-dyslexics merely account for individual
differences in reading skills, which will necessarily have a brain basis as any other cognitive ability
has [4]. In sum, this view of dyslexia seems to emphasize individual differences in learning and
performance, in contrast to the notion of typical/atypical development that is implied in terms such as
‘neurodevelopmental disorder’. p
Additional arguments against the use of the term dyslexia center on the alleged causes of poor
reading. It has been claimed that what is called dyslexia is in fact the consequence of poor teaching
methods [5] and that good-quality teaching would prevent children from becoming (labelled as)
dyslexic. The argument, which in our opinion conflicts with the presence of dyslexia across very
diverse educational systems [8], also underscores the fact that there is no neurodevelopmental and
genetic predisposition as to how fast and easy fluent reading skills are acquired. A related point Brain Sci. 2018, 8, 189; doi:10.3390/brainsci8100189 www.mdpi.com/journal/brainsci 2 of 12 Brain Sci. 2018, 8, 189 is that reading is not an innate ability, but a skill to be learned related to a cultural artifact, that is,
the writing system. In this sense, learning to read is the same as learning to sing, dance or to play
chess [4]. Some children excel in chess, while others are horrible even after years of practice, just like
we see differences in children’s reading skills. So, what makes a reading disorder different from a chess
disorder? Terminology Before we can look into the evidence in favor or against dyslexia as a neurodevelopmental disorder,
it is vital to start by clarifying the terminology. First, what constitutes a disorder? According to the
Diagnostic and Statistical Manual of Mental Disorders (DSM-5), a mental disorder is a syndrome
characterized by a clinically significant disturbance in an individual’s cognition, emotion regulation,
or behavior that reflects a dysfunction in the psychological, biological, or developmental processes
underlying mental functioning. Mental disorders are usually associated with significant distress or
disability in social, occupational, or other important activities [9]. Thus, to be considered a disorder, the
symptoms have to represent a dysfunction in the underlying neuropsychological processes, and they
should cause harm to the individual due to a clinically significant disturbance in the individual’s
functioning in society. That is, a condition needs to meet the requirements of both harm and
dysfunction [10]. Furthermore, in DSM-5, neurodevelopmental disorders (which encompass specific
learning disorders) are considered a group of conditions with onset in the developmental period,
typically early in development, before the child enters school, and are characterized by developmental
deficits that produce impairments of personal, social, academic, or occupational functioning [9,11]. More specifically related to the term neurodevelopmental disorder, it is assumed that the deficits
represent a biologically-based deviation from the typical neurodevelopmental trajectory [12]. Finally, the term disorder is itself of dynamic nature. In particular, the concept of Research
Domain Criteria (RDoC), and the network modeling approaches for understanding psychopathology
are relevant vis-à-vis the interpretation of neurodevelopmental disorders. Instead of primarily focusing
on symptoms for disorder classification, RDoC aims to provide a way to classify disorders based
on multiple dimensions of observable behavioral and neurobiological measures [13,14]. Therefore,
we will also discuss dyslexia within these conceptual frameworks. 1. Introduction In the following, we aim to examine dyslexia according to the essential characteristics of
a disorder in general, and in specific, to those of a neurodevelopmental disorder. Here, we argue in favor of the notion of dyslexia as a neurodevelopmental disorder by examining
the harm and dysfunction associated with it. This view is not incompatible with the place of dyslexia
at the tail of the continuum of reading abilities. However, importantly, it provides a necessary
instrumental framework to plan informed interventions from which individuals with reading problems
are the ultimate beneficiaries. We emphasize the insights from fundamental neuroscientific research as
central to our theoretical understanding of complex abilities like reading, and, therefore, they should
have an impact upon our characterization of reading disorders. Furthermore, we discuss the practical
implications of this approach in the clinical and educational domains. Brain Sci. 2018, 8, 189 Brain Sci. 2018, 8, 189 3 of 12 The most persistent symptom of dyslexia is dysfluent reading. The failure to become
a fluent reader is not simply a reflection of a developmental delay, but it is persistent throughout
adulthood [21,22]. In fact, DSM-5 incorporates a reference to response to intervention (RTI) to include
this resistance as a fundamental symptom for diagnosing dyslexia as a specific learning disorder. Indeed, studies on the effects of remedial teaching show that dyslexics’ reading fluency problems are
highly resistant to specialized educational support and even after a prolonged period of school-based
remediation, children with dyslexia typically remain dysfluent readers [23–25]. y
yp
y
y
So it is obvious that the impairments in dyslexia limit one’s possibilities to fulfill his or her potential
in life, as it interferes with a fundamental skill to function in our society. It has a negative impact on
quality of life, and children with dyslexia are exposed to significant stressors and are at severe risk of
negative psychosocial consequences, including symptoms of depression and anxiety [26,27]. Therefore,
it is of primary interest to investigate to what extent dyslexia impacts quality of life, which ultimately
defines its clinical relevance. A standard method to quantify the loss in quality of life due to a disease,
handicap or disorder is the Quality-Adjusted Life Year (QALY) measure [28]. Using this measure,
a study into quality of life of individuals with severe dyslexia (prevalence 4% of the population) in the
Netherlands [29] revealed that they experience a substantial loss in quality of life, comparable to the
loss of individuals diagnosed with epilepsy and with attention deficit hyperactivity disorder (ADHD). That study also highlights the importance of good identification and early intervention in terms of
economic and societal costs. 3. Dysfunction: Atypical Neurodevelopment of Reading Networks Now that we have provided evidence for the harm related to dyslexia, we turn our attention
to a more challenging element of disorder: dysfunction. One of the main arguments raised against
dyslexia as a neurodevelopmental disorder refers to the fact that the core symptom, reading, is of
a continuous nature, thus obstructing the ability to differentiate disordered from non-disordered
presentations. This continuity problem is, however, not limited to dyslexia. Virtually every psychiatric
symptom characterizing a mental disorder can occur in a certain gradation or form in a normally
functioning person [30,31]. Moreover, in contrast to many medical conditions, in most cases the
symptoms of mental disorders are much harder to be validated by measuring specific biomarkers
(such as an objectively identifiable tumor) [32,33]. Consequently, establishing the presence of an internal
dysfunction in mental disorders is much more challenging than that of harm. To do so, a disorder
should be captured in a set of symptoms constructed in such a way that they cannot reasonably be
considered normal, but lend support to an inference to underlying dysfunction or pathology [30]. To qualify symptoms in this context, some researchers use the term ‘pathosuggestive’ [30,31]. In this
section, we address the state of research on neurobiological aspects related to atypical reading to
provide a window on the element of dysfunction in dyslexia. 2. Harm: Dyslexia and Its Impact on the Individual’s Quality of Life Reading is an essential skill for functioning in our modern societies dominated by the written word. Reading is the basis for an individual’s ability to learn in an academic context, and is fundamental
for academic success [15]. The workplace has become more and more literacy-dependent over the
last decades, and even low-paid, insecure jobs require adequate literacy skills nowadays [16,17]. In addition to literacy’s role as an essential kernel to learning and employment opportunities, good
literacy skills are needed to manage one’s health, stay socially active, make informed decisions and
engage politically [18]. Literacy is, therefore, considered an essential life skill [19], and as such declared
a fundamental human right by the United Nations Convention on the Rights of the Child [18,20]. Fluent reading is thus considered of primordial importance to obtain socio-economic success in our
knowledge society. 3.2. Dysfunctional Brain Networks in Dyslexia A growing number of studies has targeted the neurobiological basis of dyslexia and provided
evidence in support for atypical development of brain networks in dyslexic readers. Most studies
focused on specific brain regions previously associated with reading. With regard to neuroanatomy,
several studies have suggested that dyslexics may present impaired structural connectivity across
the main white matter tracts that constitute the anatomical basis of the reading network (see review
in [43]), as well as asymmetries in key areas for auditory processing [44]. A larger corpus of studies
has focused on functional neuroimaging to examine brain activity in a variety of reading-related
tasks. Evidence from functional magnetic resonance (fMRI) studies shows differences in brain
activations of specific brain systems [45,46]. Dyslexics seem to exhibit anomalous specialization
of visual areas for word recognition [47], phonological processing in the auditory cortex [48,49],
and integration of grapheme and phonemes in multisensory regions [50,51]. Several studies revealed
that the very elemental neural process of recognizing whether a letter and a sound are matching
or not, may remain deviant in adult dyslexics even after years of reading exposure, and thus being
highly overlearned [52], further supporting the argument for atypical development. In addition,
electroencephalography (EEG) and magnetoencephalography (MEG) studies compliment the fMRI
findings with insights about their temporal specificity [53,54] and suggest additional deficits involving
general brain oscillatory mechanisms [55–58]. More recently, the focus of part of neuroimaging dyslexia
research has shifted towards connectivity, emphasizing the dynamic interplay between the systems in
the reading network [45,59–61]. We should acknowledge that our understanding of neural dysfunction
underlying dyslexia is currently still limited. A recent meta-analysis with focus on neuroanatomy
highlighted inconsistencies across studies and the need for more large-scale research [62]. Notably,
some of the inconsistencies may be explained by differences in selection criteria as many studies
equate dyslexia to poor reading [63]. In any case, besides the incompleteness, not uncommon to
neuroimaging research in general, qualitative and quantitative reviews strongly support a convergent
pattern of abnormal brain activations underlying dyslexia across languages [45,64–66]. The terms
dysfunctional and disrupted are often used in the literature when describing neural activations or
networks in dyslexics vs. typical readers. This has been criticized by some authors on the basis that the
findings merely indicate deviations in brain activity between two groups and no ‘core’ brain function
is affected in dyslexia [4]. 3.1. Brain Systems for Reading Reading is a highly complex cognitive skill which is multimodal in nature. The act of reading
must involve coordinated activity of both lower and higher-order processing brain areas, and can
hardly be described by a single set of brain regions working independently. With this caveat in mind,
neuroimaging research has been successful in delineating a set of brain systems that specialize for
various sub-skills involved in reading, conforming a ‘reading network’. A failure in the specialization
of one or several of those systems or in bringing them to act in concert is presumed to severely hinder
fluent reading acquisition in dyslexics. Since the very early neuropsychological evidence [34], the advent of modern neuroimaging has
considerably advanced our knowledge on the specific brain systems involved in reading. Studies on
illiterate and late literate individuals have allowed us to directly investigate how learning to read
transforms core brain function (see review in [35]). Such transformation has been hypothesized
as a sort of ‘cultural recycling’ of brain areas which evolved for diverse basic functions and can 4 of 12 Brain Sci. 2018, 8, 189 be specialized to fulfil a culturally developed skill such as reading [36]. In addition, longitudinal
research has provided insights into the developmental trajectory of specific neural activations with
reading expertise (see reviews in [37,38]). Furthermore, the causal role in literacy of some of these
brain systems has been supported by lesion studies examining patients with brain damage resulting
in impaired reading (e.g., [39–41]). The neural basis for reading seems to combine fairly universal
elements, such as specialization of visual areas to recognize a particular set of symbols, while other
elements like the way symbols, sounds and meanings are integrated, seem to vary more from
alphabetic to logographic languages [42]. So far, a primary contribution of the research reviewed
above has been to neurocognitively decompose fluent reading into different sub-processes and the
corresponding brain systems. Importantly, the various neuroimaging techniques allow for temporal
and differential characterization of these components that may otherwise be elusive to behavioral tests. Thus, they reveal unique cognitive elements that are essential for a more thorough understanding of
reading (dis)abilities. 3.3. Atypical Brain Development as Most Probable Cause The notion that dyslexia is a neurodevelopmental disorder with a neurobiological component
underscores that it is not caused by extraneous factors like socioeconomic status, poor instruction or
lack of practice. The broad category of neurodevelopmental disorders include a heterogeneous group
of conditions with a varying degree of genetic causes, which are in many cases poorly understood. The evaluation of genetic and environmental influences is important as it has consequences to clinical
description, risk assessment and expected outcomes. In relation to this, as highlighted in a recent
article, some of the assumed causal processes of dyslexia could in fact be also consequences of
poor reading experience [68]. Dyslexics are likely to read less outside school settings and to have
qualitatively poorer reading experiences and this fact complicates the distinction between cause and
effect making it difficult to define underlying dysfunction [68,69]. In view of this, the explanatory
value of the proposed neurobiological indicators of dyslexia may be compromised if they merely
reflect the consequence of long-term poor reading instead of informing about an underlying cause. Importantly, however, several sources of evidence may be able to provide actual causal insights. First,
some of the neural deviances in dyslexia are presumed to be present at birth or, at least, to precede
reading onset [70–77]. Indeed, neural activity in pre-reading stages has been correlated with the
probability of developing reading problems in later stages [72,74,78]. For example, Leppänen and
colleagues revealed that infants neural responses to speech stimuli had a strong predictive power for
reading fluency levels 14 years later [79]. In addition, differences in brain responses before reading
onset have also been related to the risk dyslexia [71,80,81]. Moreover, there is evidence supporting
the role of a genetic predisposition to the development of reading difficulties [82,83] and associated
brain structural abnormalities [84,85]. Notably, a large behavioral–genetic twin study [86] showed
that individual differences in reading ability are strongly heritable in contrast to those in reading
exposure, and, importantly, that reading ability predicts reading exposure rather than vice versa. Altogether, these studies support the idea that dyslexia is unlikely to occur due to environmental
factors. These findings suggest that in the context of dyslexia, less reading experience is not so much
a cause, but is better seen as a mediating factor exaggerating symptoms in an atypically developing
(reading) network. 3.2. Dysfunctional Brain Networks in Dyslexia It has also been argued that a neurobiological origin does not necessarily
imply brain dysfunction and a neurodevelopmental disorder, such as dyslexia, but may instead be
considered as a natural product of ‘neurodiversity’ [67]. Although this argument seems reasoned, it is
difficult to reconcile it with the need for dyslexia treatment in literate societies (harm). Thus, we argue
that the denomination of dyslexia as a disorder and of the associated brain differences as dysfunctional
are relevant. The brain findings in dyslexics reflect dysfunctionality as they demonstrate persistent
deficits in enabling a skill with a high impact upon quality of life (see Section 2), and atypicality, as they 5 of 12 Brain Sci. 2018, 8, 189 show deviation from a reading network that is efficiently developed in the majority of individuals. Importantly, this does not necessarily imply a discontinuous distribution and categorical distinction
between groups, similar to brain differences associated with other mental disorders such as autism,
anxiety and depression. 3.3. Atypical Brain Development as Most Probable Cause Interesting complements to this evidence are the findings from paradigms using
artificial script learning in a controlled setting. Those studies show that neural sensitivity to newly
learned orthographies [78] and performance in learning new script [87] in pre-readers can predict
acquisition of reading skills in later years. Similarly, learning artificial letter-speech sounds predicted
individual differences in reading and treatment outcomes in older children with dyslexia after a few
years of reading instruction [88,89]. These results strengthen the causal role of atypical development
rather than deficient teaching or poor reading experience. To sum up, there is compelling evidence suggesting that impairments in dyslexia may indeed be
caused by atypical specialization of brain networks and begin early in development. This makes the
case for dyslexia as a neurodevelopmental disorder according to the terminology of the DSM-5 and
emphasizes the need for adequate identification and early specialized intervention. 4. Clinical and Educational Implications of a Neurodevelopmental Disorder Perspective We believe that a neuroscience-informed view of dyslexia as neurodevelopmental disorder is
useful and has positive clinical and educational outcomes that outweigh the alleged shortcomings
(e.g., stigmatization, undermining the need for improvement in educational practices). A first benefit relates to the clinical specification of dyslexia. As we discussed in the previous
section, symptoms differ in the extent to which they lend support to an inference to dysfunction, 6 of 12 Brain Sci. 2018, 8, 189 which is sometimes referred to as their pathosuggestiveness. One can argue that poor reading,
being related to a learned skill, is in itself not particularly pathosuggestive, as it is a function of both
quantity and quality of instruction, as well as potentially dependent on other external influences. However, the discussed evidence from neuroscientific studies, has yielded progress in characterizing
dysfunction underlying reading deficits in dyslexia. A good example is the case of deficits in
auditory-phonological processing and crossmodal letter-speech sound mapping, that typically co-occur
with specific, persistent reading abilities, that are clearly more pathosuggestive. As discussed above,
deficits in these fundamental neural processes precede and predict later reading failure, and are still
deviant on the most basic level in adult dyslexics. Demonstrating co-occurrence of these deficits
makes a strong case for an atypical or dysfunctional development of the neural reading network in
children with dyslexia, thus anchoring it as a neurodevelopmental disorder. A behavioral validation of
these biological markers comes from a study [90] showing that when dyslexia was defined on reading
symptoms only, the percentages of affected pupils varied widely between schools. However, when the
criteria for dyslexia included both reading and phonological–orthographic deficits, the percentages of
positive cases hardly varied any more between schools [90]. This illustrates the utility of neuroscience
to specify cognitive sub-skills that are important to reading fluency and improve our operational
definition of dyslexia. In addition, studies with children at risk have shown improved prediction
of reading difficulties when including neuroimaging measures over using behavioral tests only [91]. While the results still do not have a direct clinical application, neurobiological measures can help in
disambiguating diagnostic criteria and refining current theories of reading disabilities [92]. For these
purposes, an important success factor will be to find adequate markers of specific dysfunctions
underlying dyslexia. Furthermore, neuroimaging research can aid our definition and prediction of responsiveness to
treatment. 4. Clinical and Educational Implications of a Neurodevelopmental Disorder Perspective Currently, symptom persistency and resistance to remediation constitute an important factor
in the diagnosis of dyslexia. The RTI framework encourages evidence-based instruction at different
tiers of the school system and aims at better distinguishing children with poor reading due to external
factors, like inadequate instruction, and those with a reading disability [93]. Nonetheless, there is no
ample agreement on the inclusion of RTI in the definition of dyslexia [94,95], as this approach presents
difficulties. It requires a definition of responsiveness that can be ambiguous and vary depending
on the RTI model applied [96]. Perhaps more importantly, the operationalization of persistence in
this approach can hinder early diagnosis of dyslexia, thereby delaying the application of specialized
treatment [97], and prolonging the period of affliction in dyslexic readers. In addition, it conflicts with
research showing that early interventions have a more favorable outcome than those provided at a later
age [21,25,98]. Thus, we believe it is important to aim to include indexes of atypical neural development
in diagnostic assessments in order to be able to detect dyslexia at earlier stages. To do so, one promising
approach is to complement the RTI framework by a dynamic assessment approach, which focuses on
factors moderating responsiveness and on the child’s learning potential [89,99]. As described above,
there are promising efforts from neuroscience addressing differences between responders and poor
responders [56,100,101] and simulating reading acquisition with artificial scripts [78,87] that could
significantly contribute to our understanding of individual differences in treatment outcomes. General Conclusions In sum, unlike other learned abilities of similar complexity and specificity, e.g., playing chess,
a persistent difficulty in learning how to read has a negative impact in an individual’s wellbeing,
and thus is clearly harmful. Hence, we need to define a reading disorder but not a chess disorder. We believe there is compelling evidence suggesting that impairments in dyslexia may indeed be
caused by atypical, specialization of brain networks, have a start early in development, and are
persistent over time. This qualifies neurobiological mechanisms that impede a typical acquisition of
reading skills as dysfunctional and makes the case for dyslexia as a neurodevelopmental disorder
Additionally, we would like to emphasize that the view of dyslexia as a mental disorder is an 7 of 12 Brain Sci. 2018, 8, 189 essential tool to facilitate adequate support to individuals facing academic as well as psychological
and/or socio-emotional problems. Although we acknowledge that the current model of disorders has
conceptual limitations, we believe its utility is a matter of practical convenience for elaborating policies
that can be effectively implemented in our healthcare systems. It is clear that not all poor readers
have the same requirements when it comes to improving their reading. The more we understand
about dysfunction in dyslexia, the more opportunities will arise to characterize individual differences
and, thus, to develop tailored support. Eventually, this knowledge should facilitate a shift from
the current, somewhat categorical, model of disorders to neuroscience-informed multidimensional
models of individual differences. In this regard, the view of dyslexia as a neurodevelopmental
disorder should also benefit from a more modern view of psychopathology, such as that derived from
the network theory of mental disorders [102,103]. Whereas classification models as DSM-5 and the
International Classification of Diseases (ICD-11) have a strong reliance on a classical, medical approach
of disorder, in which an empirically identifiable condition unidirectionally causes a set of symptoms,
these network theories aim to be more in line with the fact that in mental disorders complex, reciprocal
influences between an atypical initial state (of the network), mediating factors and symptoms are
present [102]. Basically, this theory conceptualizes disorders as a network of complex interactions
between symptoms with direct causal connections between the symptoms that may be grounded on
different levels (e.g., biological, psychological, societal, etc.). General Conclusions In the same vein, the RDoC approach of
the National Institute of Mental Health (NIMH) promotes the conceptualization of mental disorders as
dimensional rather than categorical, and the inclusion of different levels of analysis (e.g., neural circuit,
neurocognitive, and behavior) that instantiate the constructs associated with mental disorders [104]. Multidimensional approaches also offer a framework to understand and investigate common factors
among often co-morbid conditions, such as in the case of dyslexia and ADHD. These instrumental
views, rather than that of a common underlying cause, offer an appealing organizational framework in
which to accommodate the heterogeneity of behavioral and neuroscientific findings in the dyslexia
literature and to advance the clinical valorization of insights. Funding: This research received no external funding. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. References De Beer, J.; Engels, J.; Heerkens, Y.; Van Der Klink, J. Factors Influencing Work Participation of Adults with
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Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state
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Claudia AntoniID1☯, Dennis QuentinID1☯, Alexander E. Lang2, Klaus AktoriesID2,
Christos Gatsogiannis1, Stefan RaunserID1* Claudia AntoniID1☯, Dennis QuentinID1☯, Alexander E. Lang2, Klaus AktoriesID2,
Christos Gatsogiannis1, Stefan RaunserID1* 1 Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany,
2 Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of
Freiburg, Freiburg, Germany a1111111111
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a1111111111 ☯These authors contributed equally to this work. * stefan.raunser@mpi-dortmund.mpg.de OPEN ACCESS Citation: Antoni C, Quentin D, Lang AE, Aktories K,
Gatsogiannis C, Raunser S (2020) Cryo-EM
structure of the fully-loaded asymmetric anthrax
lethal toxin in its heptameric pre-pore state. PLoS
Pathog 16(8): e1008530. https://doi.org/10.1371/
journal.ppat.1008530 Editor: Karla J.F. Satchell, Northwestern University,
Feinberg School of Medicine, UNITED STATES Received: April 5, 2020
Accepted: June 27, 2020
Published: August 18, 2020 Published: August 18, 2020 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.ppat.1008530 Abstract Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mor-
tality in humans and other mammals. It consists of a membrane translocase, known as pro-
tective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF)
and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to
the heptameric PA pre-pore and subsequent translocation, however, are not well under-
stood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax
lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of
LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding
change their conformation to form a continuous chain of head-to-tail interactions. As a result
of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact
with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a
higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a
model for toxin assembly, in which the relative position of the N-terminal α-helices in the
three LFs determines which factor is translocated first. PLOS PATHOGENS PLOS PATHOGENS Author summary Copyright: © 2020 Antoni 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. Anthrax is a life-threatening infectious disease that affects primarily livestock and wild
animals, but can also cause high mortality in humans. Due to its suitability as a bioweapon
and the search for an antidote, it is important to understand the molecular mechanism of
infection of the anthrax pathogen and in particular of the anthrax toxin. Although the pro-
cess of poisoning by anthrax toxin has been extensively investigated, many details are still
missing that are required to understand the mechanism of action in molecular detail. Here, we used single-particle electron cryo microscopy to determine structures of the Data Availability Statement: The cryo-EM density
maps of the PA7LF3-masked, PA7LF(2+1B),
PA7LF(2+1A) and PA7LF(2+1A)’, complexes are
deposited into the Electron Microscopy Data Bank 1 / 19 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state with the accession codes EMD-11522, EMD-
11523, EMD-11524 and EMD-11525, respectively. Corresponding coordinates for PA7LF3-masked,
PA7LF2+1B and PA7LF2+1A have been deposited
in the Protein Data Bank under accession number
6ZXJ, 6ZXK and 6ZXL. All relevant data is in the
manuscript or has been deposited to the EMDB
and PDB and publicly available. with the accession codes EMD-11522, EMD-
11523, EMD-11524 and EMD-11525, respectively. Corresponding coordinates for PA7LF3-masked,
PA7LF2+1B and PA7LF2+1A have been deposited
in the Protein Data Bank under accession number
6ZXJ, 6ZXK and 6ZXL. All relevant data is in the
manuscript or has been deposited to the EMDB
and PDB and publicly available. fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state. The struc-
tures reveal that three lethal factors interact with the heptameric protective antigen and
upon binding change their conformation to form a continuous chain of head-to-tail inter-
actions. Based on our data, we propose a model for toxin assembly, in which the relative
position of the N-terminal region in the three lethal factors determines which factor is
translocated first. Our studies provide novel insights into the organization of the anthrax
lethal toxin and advance our understanding of toxin assembly and translocation. Introduction Anthrax is a life-threatening infectious disease that affects primarily livestock and wild ani-
mals, but can also cause high mortality in humans [1]. During the early and late steps of infec-
tion with the Gram-positive bacterium B. anthracis, the tripartite anthrax toxin is secreted as
major virulence factor in order to kill host immune cells such as macrophages or neutrophils
[2,3]. Like other AB-type toxins, it is composed of a surface binding/translocation moiety, the
protective antigen (PA, 83 kDa), and two cytotoxic subunits, lethal factor (LF, 90 kDa) and
edema factor (EF, 93 kDa) [4,5]. Competing interests: The authors have declared
that no competing interests exist. To execute their toxicity, both the zinc-dependent metalloproteinase LF and/or the adenyl-
ate cyclase EF need to enter the host cytoplasm [6,7]. For that purpose, PA monomers first
attach to the cell surface through binding to one of the two known membrane receptors, capil-
lary morphogenesis gene 2 (CMG-2) and tumor endothelial marker 8 (TEM8) [8,9]. After
cleavage by furin-like proteases, the truncated 63 kDa-sized PA monomer oligomerizes either
into homo-heptamers (PA7) or homo-octamers (PA8) [10–12]. These ring-shaped oligomers,
enriched in lipid raft regions, are in a pre-pore conformation as they do not penetrate the host
membrane [13]. Due to the enhanced stability of PA8 under diverse physiological conditions,
it is proposed that the octameric form could circulate in the blood to reach and exert toxicity
even in distant tissues [14]. This suggests that both oligomeric forms play an important role in
intoxication, endowing B. anthracis with greater versatility against its host. In the next step, the holotoxin is assembled by recruiting LFs/EFs. While PA8 can bind up
to four factors, only three of them can simultaneously bind to PA7. Both enzymatic substrates
bind to the upper rim of the PA oligomer via their N-terminal domains in a competitive man-
ner [15]. Loaded complexes are then endocytosed [16,17], followed by a conformational
change from the pre-pore to pore state which is triggered by the low pH in the endosome [18]. The central feature of the pore state is an 18 nm long 14-stranded β-barrel that spans the endo-
somal membrane with its narrowest point in the channel lumen being ~6 Å in width [19]. To
pass through this hydrophobic restriction, called F-clamp, the substrate needs to be unfolded
prior to translocation [20]. Author summary Funding: This work was supported by the Max
Planck Society (to S.R.) and the European Council
under the European Union’s Seventh Framework
Programme (FP7/ 2007–2013) (grant no. 615984)
(to S.R.). D.Q. is a fellow of Fonds der Chemischen
Industrie. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Introduction Structural and functional studies on the pre-pore PA octamer bound to four LFs revealed
that an amphipathic cleft between two adjacent PA protomers, termed α-clamp by Krantz and
coworkers, assists in the unfolding process [21]. More specifically, the first α-helix and β-
strand (α1-β1) of LF almost completely unfold and change their position respective to the rest
of the protein when interacting with the α-clamp of the PA oligomer [21]. After transition into
the pore state, the unidirectional translocation of LF is driven by a proton-motive force, com-
prising the proton gradient between the two compartments and the membrane potential. It is
thought that the acidic pH present in the endosome destabilizes the LF and thus promotes
unfolding of its N-terminus [22]. Ultimately, it is believed that the translocation follows a
‘charge-dependent Brownian ratchet’ mechanism [23]. The required unfolding and refolding PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 2 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state of translocated enzymes is facilitated in vivo by chaperones, but can occur in vitro without the
need of accessory proteins [24,25]. Crystallographic studies provided us with structural insights pertinent to the molecular
action of the anthrax toxin. This includes structures of the individual complex subunits such
as LF, EF and the PA pre-pore in both, its heptameric and octameric form [12,26–29]. The PA
monomer was also co-crystallized with its receptor CMG-2, delineating the surface attachment
to the host cell in molecular detail. More recently, the elusive pore state of PA7 was determined
by electron cryo-microscopy (cryo-EM) in which Jiang et al. made use of an elegant on-grid
pore induction approach [30]. In contrast, high resolution information on holotoxin complexes is rather scarce. The only
obtained crystallographic structure is the aforementioned PA8 pre-pore in complex with four
LFs [21]. In this structure, however, the C-terminal domain of LF is absent. Unlike PA8, loaded
PA7 was mainly studied by cryo-EM [31–35], presumably because its asymmetry impeded
crystallization efforts. Earlier this year, the PA7 pore state decorated with a single LF molecule
and with up to two EF molecules was determined, in which it was shown that EF undergoes a
large conformational rearrangement as opposed to LF [36]. However, cryo-EM studies of the
loaded heptameric pre-pore were so far limited to a resolution of ~16 Å [31,32,34]. Introduction In addition,
the number of LFs bound to PA7 varied between one and three in these structures. Here, we present three cryo-EM structures of the fully loaded anthrax lethal toxin in the
heptameric pre-pore state (PA7LF3), in which three LF molecules are bound to the rim of the
PA7 ring, forming a continuous chain of head-to-tail interactions. The position and conforma-
tion of the LFs, however, varies between the structures. Unexpectedly, only one of three LFs
interacts with the α-clamp of PA, adopting the “open” conformation as reported in the
PA8LF4 structure [21]. Since we could neither observe a similar interaction for the other two
LFs, nor them being in the “closed” conformation, we propose that they adopt an “intermedi-
ate” state between holotoxin assembly and translocation. Our findings allow us to propose a
model for anthrax lethal toxin assembly, in which the LF translocation sequence is dictated by
the position of the N-terminal α-helix of the LFs. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Structure of the fully-loaded anthrax lethal toxin in the heptameric pre-
pore state To ensure that our purified and reconstituted PA7 complexes (Materials and Methods) are
indeed intact, we tested their membrane insertion capacity by reconstituting them in lipo-
somes or nanodiscs (S1 Fig). We then evaluated different molar ratios of LF:PA7 and only
obtained fully-loaded anthrax lethal toxin (PA7LF3) when using a 10:1 molar ratio as judged by
size exclusion chromatography (S2A and S2B Fig). We then determined the structure of the PA7LF3 pre-pore complex by single particle cryo-
EM to an average resolution of 3.5 Å. However, in this initial 3D reconstruction the densities
corresponding to LF represented a mixture of assemblies and were partly unassignable (S3
Fig). This can be either due to the symmetry mismatch that emerges when three lethal factors
bind simultaneously to PA7 or to possible different conformations of the individual LFs bound
to PA7. To address these points, we established an image processing workflow that includes
sequential 3-D classifications and rotation of classes (S3 Fig). This resulted in three reconstruc-
tions with resolutions of 3.8 Å, 4.2 Å and 4.3 Å that differed in the position of the third LF
bound to PA7 (1A and Fig 1B, S2, S3 and S4 Figs) and the conformation of LF (Fig 1C, S2, S3
and S4 Figs). The densities corresponding to the lethal factors in the 4.3 Å structure were not
resolved well enough to allow the fitting of an atomic model (S2F, S2I and S4B Figs). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 3 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Fig 1. Cryo-EM structures of the PA7LF3 complexes. (A) Top view and side view of the color-coded segmented cryo-EM density map of PA7LF2+1A, with PA7 in blue,
1LF in pink, 2LF in gold and 3LF in cyan. The naming of the three LFs is based on their respective local resolution with 1LF being best-resolved, while 3LF showed the
weakest density in all reconstructions. Three lethal factors bind to the PA7 ring and form a continuous chain of head-to-tail interactions. Schematic representation is Fig 1. Cryo-EM structures of the PA7LF3 complexes. (A) Top view and side view of the color-coded segmented cryo-EM density map of PA7LF2+1A, with PA7 in blue,
1LF in pink, 2LF in gold and 3LF in cyan. Structure of the anthrax heptameric pre-pore state shown on the left, corresponding atomic model on the right. (B) Same as in (A) for the PA7LF2+1B complex. (C) Same as in (A) for the PA7LF(2+1A)’ complex. Notably, two
LFs interact in their peripheral region (C-terminal domain) with each other close to the central axis. Segmented maps are shown at different thresholds for visualization. (D) Schematic representation of the last step in PA7LF3 toxin assembly, in which the third lethal factor can bind to one of two empty PA sites, resulting in two different
complexes, PA7LF2+1A and PA7LF2+1B. Top and side views are shown, with the same color code as in (A), except that PA protomers alternate in light and dark blue. https://doi.org/10.1371/journal.ppat.1008530.g001 https://doi.org/10.1371/journal.ppat.1008530.g001 Therefore, we proceeded with the remaining two structures, combined the two particle stacks
and masked out the density of the third LF to improve the resolution of the rest of the complex
to 3.5 Å (S2H, S3 and S4D Figs). Using a combination of the maps, we then build atomic mod-
els for the 3.8 Å and 4.2 Å reconstructions (Fig 1A and 1B, S1 Table). The structures reveal that PA7 forms a seven-fold symmetric ring structure with a ~25 Å
wide central opening. With the exception of a few poorly resolved loop regions in the periph-
ery of PA7, our structures almost perfectly superimpose with the crystal structure of the PA7
pre-pore (PDB:1TZO; RMSD of 0.92 Å) [26] (S5A Fig), indicating that the binding of LF does
not induce conformational changes in PA7. This is in contrast to Ren et al. who suggested that
LF binding results in a distortion of the symmetric PA7 ring, thereby facilitating the passage of
cargo through the enlarged lumen [31,37]. Noteworthy, the 2β2-2β3 loop region (residues
300–323) which is implicated in pore formation was not resolved in our map. This indicates a
high flexibility of this loop, which is in line with previous MD simulations [38]. In all PA7LF3 structures, the three LFs sit on top of PA7. The densities corresponding to the
LFs show a resolution gradient from the central N-terminal domain which is resolved best to
the peripheral C-terminal domain (S4A–S4D Fig). This indicates that this region is quite flexi-
ble compared to the rest of the toxin complex. We named the three LFs based on their respec-
tive resolution with 1LF being best resolved and 3LF having the weakest density. Structure of the anthrax heptameric pre-pore state The LFs do not only interact with PA7 but also form a continuous chain of head-to-tail
interactions with each other. Binding of LF to a single PA protomer is mediated via the N-ter-
minal domain of LF, orienting its bulky C-terminal domain such that the adjacent PA proto-
mer is not accessible for binding. In this way a single lethal factor de facto occupies two of the
seven binding sites of PA7. In the chain of LFs, the C-terminal domain of the anterior LF binds
to the N-terminal domain of the following one, creating a directionality in the complex (Fig
1D). Consequently, if two LFs are bound, three free PA binding sites are available, of which
only two can potentially be occupied due to steric clashes (Fig 1D). This results in the two
complexes PA7LF2+1A and PA7LF2+1B, that differ in the binding position of the third LF (Fig
1). Since each LF occupies two potential binding sites in these structures, this leads to a sym-
metry mismatch and leaves one PA unoccupied. PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Structure of the fully-loaded anthrax lethal toxin in the heptameric pre-
pore state The naming of the three LFs is based on their respective local resolution with 1LF being best-resolved, while 3LF showed the
weakest density in all reconstructions. Three lethal factors bind to the PA7 ring and form a continuous chain of head-to-tail interactions. Schematic representation is PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 4 / 19 PLOS PATHOGENS PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Crucial interactions in the PA7LF3 complex Interfaces between lethal factor and protective antigen. (A) The N-terminal domain of LF mediates binding to two adjacent PA molecules,
forming a large planar interface. The positions of 2LF (gold), PA and PA’(blue) are shown relative to the overall shape of the complex that is represented as
transparent, low-pass filtered volume. A black square indicates the interaction interface between all three molecules. The inset shows a close-up of the
interaction regions, with contributing residues labeled. They form a central hydrophobic core, that is surrounded by electrostatic interactions. (B) The
second LF-PA interface, indicated by a dashed square in (A), is formed by the N-terminal α-helix of LF, interacting with the α-clamp region, located
between two adjacent PA molecules. The four panels depict a close-up of this region for the three different LFs (3LF can adopt two different positions, i.e. the PA7LF2+1A or PA7LF2+1B complex) with half-transparent densities shown for PA (white), PA’ (light blue) and the LF (1LF—pink; 2LF—gold; 3LF–cyan). Notably, only 2LF interacts with the α-clamp. Fig 2. Interfaces between lethal factor and protective antigen. (A) The N-terminal domain of LF mediates binding to two adjacent PA molecules,
forming a large planar interface. The positions of 2LF (gold), PA and PA’(blue) are shown relative to the overall shape of the complex that is represented as
transparent, low-pass filtered volume. A black square indicates the interaction interface between all three molecules. The inset shows a close-up of the
interaction regions, with contributing residues labeled. They form a central hydrophobic core, that is surrounded by electrostatic interactions. (B) The
second LF-PA interface, indicated by a dashed square in (A), is formed by the N-terminal α-helix of LF, interacting with the α-clamp region, located
between two adjacent PA molecules. The four panels depict a close-up of this region for the three different LFs (3LF can adopt two different positions, i.e. the PA7LF2+1A or PA7LF2+1B complex) with half-transparent densities shown for PA (white), PA’ (light blue) and the LF (1LF—pink; 2LF—gold; 3LF–cyan). Notably, only 2LF interacts with the α-clamp. https //doi org/10 1371/jo rnal ppat 1008530 g002 Fig 2. Interfaces between lethal factor and protective antigen. (A) The N-terminal domain of LF mediates binding to two adjacent PA molecules,
forming a large planar interface. The positions of 2LF (gold), PA and PA’(blue) are shown relative to the overall shape of the complex that is represented as
transparent, low-pass filtered volume. Crucial interactions in the PA7LF3 complex LF and PA interact mainly via a large planar interface at which domain I of LF interacts with
the LF/EF binding sites of two adjacent PAs (Fig 2, S5B Fig). The interaction with LF is medi-
ated primarily by one PA, whereas the contribution of the second PA is only minor (Fig 2A). The LF-PA interface is well resolved for all LFs and almost identical in the different structures
(Fig 2A, S5A and S5C Fig). The interaction is primarily mediated by an extensive hydrophobic
core that is further surrounded by electrostatic interactions (Fig 2A). The interface in our
structure is very similar to the one previously described for PA8LF4 [21]. There, the second
LF-PA interface is formed by the N-terminal α-helix of LF that interacts with the α-clamp
located at the interface of two PAs. This “open” conformation differs from the “closed” confor-
mation of this region as observed in the structure of the unbound LF [27]. When comparing
the LFs in our structure with that of the unbound LF [27], we observed that the C-terminal
domain of the LFs in PA7LF3 is rotated in relation to the N-terminal domain, bringing them PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 5 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Fig 2. Interfaces between lethal factor and protective antigen. (A) The N-terminal domain of LF mediates binding to two adjacent PA molecules,
forming a large planar interface. The positions of 2LF (gold), PA and PA’(blue) are shown relative to the overall shape of the complex that is represented as
transparent, low-pass filtered volume. A black square indicates the interaction interface between all three molecules. The inset shows a close-up of the
interaction regions, with contributing residues labeled. They form a central hydrophobic core, that is surrounded by electrostatic interactions. (B) The
second LF-PA interface, indicated by a dashed square in (A), is formed by the N-terminal α-helix of LF, interacting with the α-clamp region, located
between two adjacent PA molecules. The four panels depict a close-up of this region for the three different LFs (3LF can adopt two different positions, i.e. the PA7LF2+1A or PA7LF2+1B complex) with half-transparent densities shown for PA (white), PA’ (light blue) and the LF (1LF—pink; 2LF—gold; 3LF–cyan). Notably, only 2LF interacts with the α-clamp. htt
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PATHOGENS
Structure of the anthrax heptameric pre-pore state Fig 2. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Crucial interactions in the PA7LF3 complex A black square indicates the interaction interface between all three molecules. The inset shows a close-up of the
interaction regions, with contributing residues labeled. They form a central hydrophobic core, that is surrounded by electrostatic interactions. (B) The
second LF-PA interface, indicated by a dashed square in (A), is formed by the N-terminal α-helix of LF, interacting with the α-clamp region, located
between two adjacent PA molecules. The four panels depict a close-up of this region for the three different LFs (3LF can adopt two different positions, i.e. the PA7LF2+1A or PA7LF2+1B complex) with half-transparent densities shown for PA (white), PA’ (light blue) and the LF (1LF—pink; 2LF—gold; 3LF–cyan). Notably, only 2LF interacts with the α-clamp. https://doi.org/10.1371/journal.ppat.1008530.g002 https://doi.org/10.1371/journal.ppat.1008530.g002 closer together (Fig 3, S1 Movie). However, we only found that the N-terminal region of one
LF (2LF) resides in the α-clamp, adopting the “open” conformation as described for PA8LF4
[21]. In the other LFs (1LF, 3LF), this region is flexible and not interacting with the α-clamp
(Fig 2B). A steric clash between the loop region (residues 576–579) of 1LF and α1-β1 of 2LF
(Fig 4A) prevents the N-terminal α-helix of 2LF from remaining in the “closed” conformation. Since 1LF and 3LF neither take the “open”, nor the “closed” conformation, we propose that
they reside in an “intermediate” conformational state between toxin assembly and transloca-
tion. We further hypothesize that 2LF is the first of the three lethal factors that is unfolded by
PA7 and is also the first one to be translocated. As described above, the LFs interact via their N- and C-terminal regions. In two of our
structures, PA7LF2+1A and PA7LF2+1B, two LFs only interact at one position which is located
next to the major LF-PA interface. In the third structure, which we designate as PA7LF(2+1A)’, PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 6 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Fig 3. Conformational change of LF upon PA binding. Superposition of 1LF (green, “intermediate” conformation), 2LF (yellow,
“open” conformation) and unbound LF (green, “closed” conformation, PDB: 1J7N), aligned via their N-terminal domains. Red and
green arrows indicate similar positions in 1LF and unbound LF, respectively. When compared with the crystal structure of the
unbound lethal factor (PDB: 1J7N), the three LFs undergo a conformational change upon interaction with PA7. Crucial interactions in the PA7LF3 complex The C-terminal
domain rotates with respect to the N-terminal domain such that the LFs come closer to form a continuous chain of head-to-tail
interactions. A schematic representation illustrates the rotation of the C-terminal domain that occurs between unbound (green)
and bound (red) LF conformation. See also S1 Movie. htt
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t 1008530 003 Fig 3. Conformational change of LF upon PA binding. Superposition of 1LF (green, “intermediate” conformation), 2LF (yellow,
“open” conformation) and unbound LF (green, “closed” conformation, PDB: 1J7N), aligned via their N-terminal domains. Red and
green arrows indicate similar positions in 1LF and unbound LF, respectively. When compared with the crystal structure of the
unbound lethal factor (PDB: 1J7N), the three LFs undergo a conformational change upon interaction with PA7. The C-terminal
domain rotates with respect to the N-terminal domain such that the LFs come closer to form a continuous chain of head-to-tail
interactions. A schematic representation illustrates the rotation of the C-terminal domain that occurs between unbound (green)
and bound (red) LF conformation. See also S1 Movie. Fig 3. Conformational change of LF upon PA binding. Superposition of 1LF (green, “intermediate” conformation), 2LF (yellow,
“open” conformation) and unbound LF (green, “closed” conformation, PDB: 1J7N), aligned via their N-terminal domains. Red and
green arrows indicate similar positions in 1LF and unbound LF, respectively. When compared with the crystal structure of the
unbound lethal factor (PDB: 1J7N), the three LFs undergo a conformational change upon interaction with PA7. The C-terminal
domain rotates with respect to the N-terminal domain such that the LFs come closer to form a continuous chain of head-to-tail
interactions. A schematic representation illustrates the rotation of the C-terminal domain that occurs between unbound (green)
and bound (red) LF conformation. See also S1 Movie. https://doi.org/10.1371/journal.ppat.1008530.g003 two LFs likely interact with each other also via their C-terminal region close to the central axis
of the complex (Fig 5, S2 Movie). However, the position of this additional interface that we
observe in our PA7LF(2+1A)’ structure differs from the putative interface, that has been pro-
posed by Fabre et al, located further away from the central axis [34]. The main 2LF-1LF inter-
face, that is present in all three structures, consists of the helix-loop region (residues 572–579)
of the first lethal factor (1LF) which forms a relatively small interaction surface with the helix-
helix-β-sheet motif (residues 52–84) of the adjacent lethal factor (2LF) (Fig 4B). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Crucial interactions in the PA7LF3 complex (A) Potential steric clash between the C-terminal domain of LF (red) and the N-terminal domain of
an adjacent LF when it adopts the “closed” conformation (blue). The clash is highlighted as fading red spot in the background. In contrast, the “open”
conformation (gold), i.e. the N-terminal α-helix interacts with the α-clamp region of PA, does not result in a steric clash. The N-terminal region of the red
LF, which adopts an “intermediate” conformation, is not shown in this view. (B) A relatively small interaction interface mediates binding of the C-terminal
domain of LF to the N-terminal domain of an adjacent LF. The positions of 1LF (pink) and 2LF (gold) are shown relative to the overall shape of the complex
that is represented as transparent, low-pass filtered volume. A black square indicates the interaction interface between the two LFs. Insets show close-ups of
the interacting regions in different orientations, with contributing residues labeled. Fig 4. Molecular interface between two lethal factors. (A) Potential steric clash between the C-terminal domain of LF (red) and the N-terminal domain of
an adjacent LF when it adopts the “closed” conformation (blue). The clash is highlighted as fading red spot in the background. In contrast, the “open”
conformation (gold), i.e. the N-terminal α-helix interacts with the α-clamp region of PA, does not result in a steric clash. The N-terminal region of the red
LF, which adopts an “intermediate” conformation, is not shown in this view. (B) A relatively small interaction interface mediates binding of the C-terminal
domain of LF to the N-terminal domain of an adjacent LF. The positions of 1LF (pink) and 2LF (gold) are shown relative to the overall shape of the complex
that is represented as transparent, low-pass filtered volume. A black square indicates the interaction interface between the two LFs. Insets show close-ups of
the interacting regions in different orientations, with contributing residues labeled. https://doi.org/10.1371/journal.ppat.1008530.g004 https://doi.org/10.1371/journal.ppat.1008530.g004 backbone level (RMSD of 0.84 Å and 0.96 Å) (S5F Fig), we expect them to exhibit a similar net-
work of interactions. Both interfaces, LF-PA and LF-LF that we describe here limit the free-
dom of movement mainly in the N-terminal region of LF, but still allow a certain level of
flexibility in the rest of the protein. In all structures, the LFs show a gradient in flexibility (Fig 1A–1C, S4A–S4D Fig). This was
previously not observed at lower resolution [34]. Crucial interactions in the PA7LF3 complex Residues L63,
L71 and I81 of 2LF form a central hydrophobic cavity that interacts with Y579 of 1LF. In the β-
sheet region of 2LF, we identified a potential backbone-backbone hydrogen bond between
K578 and I81 of 1LF. In addition, P577 forms a hydrophobic interaction with Y82, which is
further stabilized by H91. K572, being located on the α-helix next to the loop region in 1LF,
could potentially form a salt bridge interaction with E52 or D85 of 2LF. Together these interac-
tions mediate the binding between two LFs. Although the local resolution at the 2LF-3ALF and
3BLF-1LF interfaces does not allow the fitting of side chains (S4A–S4D Fig), we could flexibly
fit in the structures of 1LF and 2LF at this position. Since all structures are almost identical at two LFs likely interact with each other also via their C-terminal region close to the central axis
of the complex (Fig 5, S2 Movie). However, the position of this additional interface that we
observe in our PA7LF(2+1A)’ structure differs from the putative interface, that has been pro-
posed by Fabre et al, located further away from the central axis [34]. The main 2LF-1LF inter-
face, that is present in all three structures, consists of the helix-loop region (residues 572–579)
of the first lethal factor (1LF) which forms a relatively small interaction surface with the helix-
helix-β-sheet motif (residues 52–84) of the adjacent lethal factor (2LF) (Fig 4B). Residues L63,
L71 and I81 of 2LF form a central hydrophobic cavity that interacts with Y579 of 1LF. In the β-
sheet region of 2LF, we identified a potential backbone-backbone hydrogen bond between
K578 and I81 of 1LF. In addition, P577 forms a hydrophobic interaction with Y82, which is
further stabilized by H91. K572, being located on the α-helix next to the loop region in 1LF,
could potentially form a salt bridge interaction with E52 or D85 of 2LF. Together these interac-
tions mediate the binding between two LFs. Although the local resolution at the 2LF-3ALF and
3BLF-1LF interfaces does not allow the fitting of side chains (S4A–S4D Fig), we could flexibly
fit in the structures of 1LF and 2LF at this position. Since all structures are almost identical at PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 7 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Fig 4. Molecular interface between two lethal factors. Crucial interactions in the PA7LF3 complex 1LF is resolved best in all structures, followed
by 2LF and 3LF has the weakest density in all reconstructions. Since the N-terminal domain is
well resolved in all LFs, this cannot be due to a varying occupancy of the binding sites, but
must stem from a flexibility of the C-terminal domain. As expected, all free C-terminal
domains, i.e. those that are not stabilized by an adjacent LF are more flexible than those with a
binding partner. However, there is one exception, namely 2LF. In this case, the C-terminal
domain is always flexible, independent of a stabilizing binding partner. Interestingly, 2LF is
also the only lethal factor where the N-terminal α-helix of LF is ordered and resides in the α-
clamp, suggesting that this interaction results in a destabilization of the C-terminal domain of
the molecule. This is in line with a recently reported structure of the PA7LF1 pore state where
the C-terminal domain of the single LF bound was not resolved while the N-terminal α-helix
is also bound to the α-clamp [36]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Discussion We determined three structures of the fully-loaded heptameric anthrax lethal toxin complex,
which differ in the position and conformations of the bound LFs. Due to a symmetry mis-
match, three LFs occupy six binding sites of the heptameric PA7 complex, leaving one PA site PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 8 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Fig 5. LFs can interact via their C-terminal domain. Top and side views of the low-passed filtered maps of the three PA7LF3 complexes,
with PA7LF2+1B in orange, PA7LF2+1A in yellow and PA7LF(2+1A)’ in light blue. Volumes are shown at the same threshold. While the three
LFs interact in all structures via their N- and C-terminal domains in a head-to-tail manner, an additional interface was identified in the
PA7LF(2+1A)’ reconstruction. Here, the C-terminal domains of 1LF and 2LF, interact with each other close to the central axis of the PA7LF3
complex. This region is highlighted by dashed red circles. See also S2 Movie. https://doi.org/10.1371/journal.ppat.1008530.g005 Fig 5. LFs can interact via their C-terminal domain. Top and side views of the low-passed filtered maps of the three PA7LF3 complexes,
with PA7LF2+1B in orange, PA7LF2+1A in yellow and PA7LF(2+1A)’ in light blue. Volumes are shown at the same threshold. While the three
LFs interact in all structures via their N- and C-terminal domains in a head-to-tail manner, an additional interface was identified in the
PA7LF(2+1A)’ reconstruction. Here, the C-terminal domains of 1LF and 2LF, interact with each other close to the central axis of the PA7LF3
complex. This region is highlighted by dashed red circles. See also S2 Movie. https://doi org/10 1371/journal ppat 1008530 g005 Fig 5. LFs can interact via their C-terminal domain. Top and side views of the low-passed filtered maps of the three PA7LF3 complexes,
with PA7LF2+1B in orange, PA7LF2+1A in yellow and PA7LF(2+1A)’ in light blue. Volumes are shown at the same threshold. While the three
LFs interact in all structures via their N- and C-terminal domains in a head-to-tail manner, an additional interface was identified in the
PA7LF(2+1A)’ reconstruction. Here, the C-terminal domains of 1LF and 2LF, interact with each other close to the central axis of the PA7LF3
complex. This region is highlighted by dashed red circles. See also S2 Movie. https://doi.org/10.1371/journal.ppat.1008530.g005 empty. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Discussion Compared to the “closed” state as observed in the crystal structure of LF [27], the C-ter-
minal domain of the LFs in PA7LF3 is rotated respective to the N-terminal domain. However,
only 2LF adopts the “open” conformation which was reported for the structure of PA8LF4 [21],
i.e. the N-terminal α-helix interacts with the α-clamp of PA. 1LF and 3LF do not show this
interaction, but can also not be in the “closed” conformation because of a steric clash with an
adjacent LF. We therefore propose that they are in an “intermediate” state between toxin
assembly and translocation. Loading of PA7 with one or more substrates is crucial for the anthrax toxin to mediate tox-
icity during infection. The high binding affinity of LF and EF to PA7 enables the formation of
a stable toxin complex, which is required to withstand the rather hostile environment present
within the host during the various steps of the infection process. A dissociation constant (KD)
in the range of 1–2 nM has been determined by both, surface plasmon resonance (SPR) and
binding studies using radiolabeled substrate with receptor-bound PA on the surface of L6 cells
[39]. Head-to-tail binding of LFs as observed in our fully loaded PA7LF3 structures further PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 9 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state contributes to the overall stability of the complex. This increased stability might be important
during the internalization step and subsequent endosomal trafficking, before finally reaching
its cytosolic target. It might reduce the susceptibility towards proteases and the probability to
prematurely transition into the pore-state. Studies addressing the saturation state of PA7 (and PA8) with LF/EF ligands in vivo are
rather limited. While one study of cutaneous infection in immune-competent mice has
focused on the level of both substrates and their respective ratio over the course of infection,
the number of bound substrates to PA7 or PA8 has not been determined [40]. As has been sug-
gested before, it is likely that this number can vary depending on the stage of the infection pro-
cess, as well as the location within the host. Interestingly, mass spectrometry data of toxin
assembly in vitro did not identify PA7 or PA8 ring intermediates decorated with less than three
or four LFs, respectively [12]. This observation, however, could be a consequence of the two
different assembly routes that will be discussed later. Discussion Furthermore, it has been shown by
Zhang et al. that the number of ligands bound to PA7 did not affect the translocation efficiency
[41]. By varying either the ligand concentration or the available ligand binding sites in PA7,
they excluded both positive and negative cooperativity in the translocation process of radiola-
beled LF. These findings suggest that LF and EF ligands are translocated independently of the
number of bound ligands. In our structures, we observed that two LFs adopt a so called “intermediate” state, in which
their N-terminal α-helix neither interacts with the α-clamp of PA, nor adopts the “closed” con-
formation [27]. Why has this state not been observed in the crystal structure of PA8LF4? It
could have been missed due to averaging of the asymmetric unit of the PA8LF4 crystals, which
is composed of two PAs and one LF. Another possibility is that compared to PA7, the PA8 pre-
pore provides more space for the N-termini of the LFs to arrange in the “open” conformation
in comparison to the PA7 pre-pore. However, if all LFs were indeed in a “ready-to-be-translo-
cated position” which LF would then be translocated first through the narrow PA pore that
only allows the passage of a single unfolded LF at a time? The process could in principle be sto-
chastic, but our PA7LF3 structures offer an alternative explanation. Already based on the low-resolution structure of the PA7LF3 pre-pore [34], it has been sug-
gested that the order of translocation is non-stochastic and that the first LF, whose N-terminal
domain is not interacting with an adjacent LF, is translocated first. After the translocation of
this factor, the second LF would be released from the inhibitory bond of the first LF and then
be translocated and so on [34]. However, our data indicate that such an order in which the
translocation of one LF is dependent on the translocation of an adjacent factor is rather
unlikely. Although we can as well only speculate about the exact order of translocation, based on our
cryo-EM structures, two alternative scenarios are conceivable: In the first one, the factor in the
“open” state, 2LF, is translocated first, followed by 1LF or 3LF which are in the “intermediate”
state (Fig 6). The second possibility would be that 1LF and 3LF are translocated before 2LF (Fig
6). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Discussion Due to the different arrangements in the complexes, both alternatives exclude a depen-
dency on a neighboring factor for LF translocation. While we cannot exclude the second scenario, we think that the first one is more likely. Being in the “open” conformation, the N-terminal α-helix of 2LF interacts with the α-clamp of
PA. The α-clamp is known to unfold polypeptides in a sequence-independent manner. The
current theory is that this crucial region formed by two adjacent PA molecules first stabilizes
unfolding intermediates, and introduces mechanical strain before the unfolded structure is fed
further down the central pore [21]. In this way the α-clamp would facilitate the rapid unfolding
of the entire 2LF molecule upon transition into the pore state. We therefore believe that 2LF is
translocated before 1LF and 3LF. The higher flexibility in the C-terminal domain of 2LF in the PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 10 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Fig 6. Model for PA7LF3 assembly and translocation. After anthrax-receptor-mediated PA7 formation on the surface of the host cell, three LFs bind to PA7 to assemble
into the fully loaded anthrax lethal toxin. As a consequence of the symmetry mismatch between PA7 and three LFs, the third LF can attach to two different PA sites,
resulting in two distinct PA7LF3 complexes. In both arrangements, the three LFs form a continuous chain of head-to-tail interactions. One LF, namely 2LF, undergoes a
conformational change from the “closed” state as observed in the unbound LF structure to an “open” state, which is characterized by an interaction of its N-terminal α-
helix with the α-clamp region of PA. The other two LFs, 1LF and 3LF, adopt an “intermediate” state, i.e. being neither in an “open” nor “closed” conformation. Two
different scenarios for the translocation of the 1st LF after transitioning into the pore state are conceivable: In scenario 1, 2LF, being in the “open” state is translocated first,
whereas either 1LF or 3LF are translocated first in scenario 2. https://doi.org/10.1371/journal.ppat.1008530.g006 Fig 6. Model for PA7LF3 assembly and translocation. After anthrax-receptor-mediated PA7 formation on the surface of the host cell, three LFs bind to PA7 to assemble
into the fully loaded anthrax lethal toxin. As a consequence of the symmetry mismatch between PA7 and three LFs, the third LF can attach to two different PA sites,
resulting in two distinct PA7LF3 complexes. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Discussion In both arrangements, the three LFs form a continuous chain of head-to-tail interactions. One LF, namely 2LF, undergoes a
conformational change from the “closed” state as observed in the unbound LF structure to an “open” state, which is characterized by an interaction of its N-terminal α-
helix with the α-clamp region of PA. The other two LFs, 1LF and 3LF, adopt an “intermediate” state, i.e. being neither in an “open” nor “closed” conformation. Two
different scenarios for the translocation of the 1st LF after transitioning into the pore state are conceivable: In scenario 1, 2LF, being in the “open” state is translocated first,
whereas either 1LF or 3LF are translocated first in scenario 2. htt
//d i
/10 1371/j
l
t 1008530 006 https://doi.org/10.1371/journal.ppat.1008530.g006 presence of potentially stabilizing neighboring LFs suggests that the interaction of the N-termi-
nal domain with the α-clamp results in a destabilization of the molecule. This in turn lowers
the energy barrier for the unfolding of the entire LF molecule and further supports the
assumption that 2LF is translocated first. Once 2LF is translocated, either 1LF or 3LF can follow. As these two LFs both adopt an “intermediate” conformation in our structures, we cannot pre-
dict which LF is translocated next. Currently, two possible routes for toxin assembly have become established within the
anthrax field: (I) Anthrax-receptor (ATR)-mediated assembly on the cell surface and (II)
ATR-independent assembly in solution, i.e. the bloodstream. It is important to note that both
routes are not mutually exclusive. In route I, ATR first recruits full-length PA to form PA7 and
PA8, where PA is cleaved into its mature form, followed by LF/EF substrate recruitment. In
vitro mass spectrometry data indicated that the PA oligomerization process occurs via even-
numbered PA2 and PA4 intermediates, being consistent with a proposed dimerization of ATR
[42]. In route II, binding of LF/EF to two PA molecules facilitates the assembly of both, the
heptameric and octameric PA ring. Similar to route I, assembly route II is populated with
dimeric and tetrameric PA intermediates, bound to one and two LFs, respectively [12]. As we PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 11 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state have pre-assembled the PA7 ring before LF was added, our assembly approach resembles
rather route I. Protein expression and purification Protective antigen (PA) from Bacillus anthracis was cloned into a pET19b vector (Novagen),
resulting in a N-terminal His10-tag fusion construct. E. coli BL21(DE3) were transformed with
the pET19b::His10-PA plasmid and expression was induced immediately after transformation
by the addition of 75 μM IPTG. Following incubation at 28˚C for 24 h in LB medium, cells
were pelleted, resuspended in lysis-buffer (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 500 μM
EDTA, 5 μg/ml DNAse, 1 mg/ml Lysozym plus Protease inhibitor cOmplete tablets from
Sigma Aldrich) and lysed by sonication. Soluble proteins were separated from cell fragments
by ultracentrifugation (15,000 rpm, 45 min, 4˚ C) and loaded onto Ni-IDA beads (Cube Bio-
tech). After several washing steps, the protein was eluted with elution buffer (500 mM imidaz-
ole, 20 mM Tris-HCl pH 8.5, 500 mM NaCl, 1 mM EDTA). Protein-containing fractions were
pooled and dialyzed against buffer containing 50 mM Tris-HCl pH 8.5, 150 mM NaCl, 1 mM
EDTA. Subsequently, the sample was further purified using anion-exchange Mono Q (GE
Healthcare) with a no-salt buffer (20 mM Tris-HCl pH 8.5) and high-salt buffer (20 mM Tris-
HCl pH 8.5, 1M NaCl), applying a gradient from 0 to 40%. Next, oligomerization of PA was
induced by addition of trypsin (1 μg enzyme for each mg of PA), followed by incubation on ice
for 30 min. Upon addition of double molar excess of trypsin inhibitor (Sigma Aldrich), PA7
was further purified by size exclusion chromatography using a Superdex 200 column (GE
Healthcare). Lyophilized LF (List Biological Lab. Inc., Lot#1692A1B) were resuspended in
water according to the manufacturer’s manual and mixed with PA7 in a molar ration of 10:1. Ultimately, loaded complexes were further purified in a final size exclusion chromatography
step (20 mM Tris-HCl pH 8.5, 150 mM NaCl) using a Superdex 200i column (GE Healthcare),
before being used in down-stream applications. Discussion We believe that 2LF is not only the first LF being translocated, but might also be
the first one that binds to PA7 during toxin assembly (Fig 6). Upon binding to PA, 2LF under-
goes a conformational change from the “closed” to the “open” state. The two subsequent LFs
adopt an “intermediate” state instead of transitioning into the “open” conformation. Since the
third LF can attach to two different PA sites, this results in two different complexes (Fig 6). In
this way, the assembled toxin has three LFs bound to PA7 with two in an “intermediate” and
one in the “open” conformation (Fig 6). In summary, our high-resolution cryo-EM structures provide us with novel insights into
the organization of the fully-loaded heptameric anthrax lethal toxin and thus advance our
understanding of toxin assembly and translocation. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Cryo-EM data acquisition Cryo-EM data sets of PA7LF3 were collected on a Titan Krios transmission electron micro-
scope (FEI) equipped with a high-brightened field-emission gun (XFEG), operated at an accel-
eration voltage of 300 kV. Micrographs were recorded on a K2 direct electron detector (Gatan)
at 130,000 x magnification in counting mode, corresponding to a pixel size of 1.07 Å. 40 frames
taken at intervals of 375 ms (1.86 e-/Å2) were collected during each exposure, resulting in a
total exposure time of 15 s and total electron dose of 74.4 e-/Å2. Using the automated data col-
lection software EPU (FEI), a total of 5238 micrographs with a defocus range between -1.2 and
-2.6 μm was automatically collected. Negative-stain electron microscopy Complex purity and integrity were assessed by negative stain electron microscopy prior to
cryo-EM grid preparation and image acquisition. For negative stain, 4 μl of purified PA7LF3
complex at a concentration of ~0.04 mg/ml was applied onto a freshly glow discharged car-
bon-coated copper grid (Agar Scientific; G400C) and incubated for 45 s. Subsequently, excess
liquid was blotted away with Whatman no. 4 filter papers. The sample was stained with 0.8%
(w/v) uranyl acetate (Sigma Aldrich). Micrographs were recorded manually using a JEOL
JEM-1400 TEM, operated at an acceleration voltage of 120 kV, equipped with a 4,000 × 4,000
CMOS detector F416 (TVIPS) and a pixel size of 1.84 Å/px. Reconstitution of PA7 in lipid-mimetic systems For nanodisc insertion, Ni-NTA column material was first washed with ddH20 and subse-
quently equilibrated with buffer A (50 mM NaCl, 20 mM Tris-HCl–pH 8.5, 0.05% Octyl β-D-
glucopyranoside (w/v)). In the next step, 500 μL of 0.2 μM PA7 in the pre-pore state was added
and incubated for 25 min at room temperature. An additional washing step with buffer A was
performed to remove unbound PA7 pre-pore, followed by a 5 min incubation step with 1 M
urea at 37˚C and another wash with buffer A. MSP1D1:POPC:sodium cholate ratio and prepa-
ration was done according to Akkaladevi et al [33]. After dialysis (MWCO of 12-14k) for 24 to
72 h against buffer B (50 mM NaCl, 20 mM Tris-HCl pH 7.5), excess of nanodiscs was col-
lected from five washing steps with 500 μL of buffer B. To elute PA7 pores inserted into nano-
discs, column material was incubated for 10 min on ice in buffer C (500 mM NaCl, 50 mM PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 12 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Tris-HCl pH 7.5, 50 mM imidazole). The eluted sample was concentrated and subsequently
used for negative staining EM. For the preparation of pre-formed liposomes, POPC was initially solubilized in 5% Octyl β-
D-glucopyranoside. Solubilized lipids were dialyzed (MWCO: 12–14 k) for 8–12 h at 4˚ C
against buffer A and subsequently PA7 pre-pores were added to the lipids in a 1: 10 molar
ratio. Following 24–72 h dialysis (MWCO:12–14 k) against buffer D (50 mM NaCl 50 mM
NaOAc, pH 5.0), samples were used for negative staining EM. Sample vitrification For Cryo-EM sample preparation, 4 μl of purified PA7LF3 at a concentration of ~0.06 mg/ml
was applied onto freshly glow discharged grids (Quantifoil R 1.2/1.3 holey carbon with a 2 nm
additional carbon support) and incubated for 45 s. Subsequently, grids were blotted automati-
cally (3 s blotting time) and plunged into liquid ethane using a CryoPlunge3 (Gatan) at a
humidity of ~ 95%. Grid quality was screened before data collection using a JEOL JEM-1400
TEM electron microscope (same settings as for negative-stain electron microscopy) or with an
Arctica microscope (FEI), operated at 200 kV. Grids were kept in liquid nitrogen for long-
term storage. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Image processing and 3-D reconstruction The final densities were filtered according to local resolution or the local de-noising
filter LAFTER was applied to recover features with more signal than noise (based on half
maps) [47]. Image processing and 3-D reconstruction Micrographs of the dataset were inspected visually and ones with extensive ice contamination
or high drift were discarded. Next, frames were aligned and summed using MotionCor2 (in 3
x 3 patch mode) [43]. By doing so, dose-weighted and un-weighted full-dose images were gen-
erated. Image and data processing were performed with the SPHIRE/EMAN2 software pack-
age [44]. Un-weighted full-dose images were used for defocus and astigmatism estimation by
CTER [45]. With the help of the drift assessment tool in SPHIRE, drift-corrected micrographs
were further sorted to discard high defocus as well as high drift images that could not be com-
pensated for by frame alignment. 13 / 19 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state For the PA7LF3 dataset, particles were automatically selected based on a trained model
using the crYOLO software, implemented in SPHIRE [46]. In total, 382 k particles were
extracted from the dose-weighted full dose images with a final window size of 336 x 336 pixel. Two-dimensional classification was performed using the iterative and stable alignment and
clustering (ISAC) algorithm implemented in SPHIRE. Several rounds of 2-D classification
yielded a total number of 213 k ‘clean’ dose-weighted and drift-corrected particles. During the
manual inspection of the 2-D class averages, top views of the particles were excluded. A generated composite crystal structure consisting of PA7 (PDB:1TZO) decorated with
three full-length LF (PDB:1J7N), docked with their N-terminal domain to PA as observed in
the PA8LF4 structure (PDB: 3KWV), was converted into electron density (sp_pdb2em func-
tionality in SPHIRE). After filtering to 30 Å, this map served as reference in the initial 3-D
refinement. The 3-D refinement without imposed symmetry (sxmeridien in SPHIRE, C1)
yielded an initial 3.5 Å electron density map of the PA7LF3 complex. Several rounds of 3-D
classification and rotation of certain classes were necessary to separate particles belonging to
PA7LF2+1A, PA7LF2+1B and PA7LF(2+1A)’ complexes. The respective classes obtained by 3D
sorting or from previous 3D refinements served as reference for all subsequent 3D refine-
ments. The flowchart of the image processing strategy including the obtained 3-D classes as
well as the number of particles that they contained is described in detail in S3 Fig (S3 Fig). Global resolutions of the final maps were calculated between two independently refined
half maps at the 0.143 FSC criterion, local resolution was calculated using sp_locres in
SPHIRE. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Model building, refinement and validation To build the PA7 model, a single monomer of the PA8 crystal structure (PDB: 3HVD) was
placed seven times into the density corresponding to the heptameric PA ring of the PA7LF3--
masked map using rigid body fitting in Chimera [48]. Next, two copies of the monomeric lethal
factor of the unbound crystal structure (PDB:1J7N) were fitted similarly into the correspond-
ing LF density located atop of the PA7 ring. The resulting model was then flexibly fitted using
iMODFIT [49] and subsequently further refined using a combination of manual model build-
ing in COOT [50] and real-space refinement in PHENIX [51]. Unresolved loop regions were
deleted and side chain information was removed for less well-resolved regions. The resulting models for 1LF, 2LF and PA7 from the PA7LF3-masked model served again as
starting point for the PA7LF2+1B structure and were placed into the corresponding density
using rigid-body fit in Chimera. Chain H (1LF) was duplicated and placed similarly into the
density corresponding to the third LF. For the entire model a restrained refinement in PHE-
NIX was performed. The resulting model was further refined using a combination of COOT
and real-space refinement in PHENIX. Like for the PA7LF3-masked model, unresolved loop
regions were deleted and side chain information was removed for less well-resolved regions. The PA7LF2+1B model served as starting model for PA7LF2+1A and was initially fitted into
the density map using rigid-body fitting. Chain J (3LF) was placed into the density correspond-
ing to the third LF. Similar to PA7LF2+1B, a restrained refinement in PHENIX was performed
with the entire model, followed by an additional refinement using a combination of COOT
and real-space refinement in PHENIX. Unresolved loop regions were deleted and side chain
information was removed for less well-resolved regions. Geometries of the final refined models
were obtained either from PHENIX or calculated with the Molprobity server (http://
molprobity.biochem.duke.edu). Data statistics are summarized in S1 Table (S1 Table). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 14 / 19 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state Structure analysis and visualization UCSF Chimera was used for structure analysis, visualization and figure preparation. The angu-
lar distribution plots as well as beautified 2-D class averages were calculated using SPHIRE. Supporting information S1 Fig. Reconstitution of PA7 into lipid mimetic systems after pore transition. (A) Repre-
sentative negatively stained electron micrograph areas of PA7 reconstituted into POPC lipo-
somes (top panels), with individual inserted particles highlighted by white arrowheads. Selection of inserted particles in smaller lipid vesicles (bottom panel). Scale bar: 15 nm. Parti-
cles are clearly accumulated at lipid membranes. (B) Representative negatively stained electron
micrograph area of PA7 reconstituted in lipid nanodiscs (MSP1D1), with individual inserted
particles highlighted by black arrowheads. Scale bar: 20 nm (C) Model of PA7 complexes
inserted into lipid nanodiscs with additional examples of individual particles after reconstitu-
tion (same nanodiscs as in B). Scale bar: 20 nm. S2 Fig. Purification and cryo-EM of PA7LF3. (A) Coomassie-stained SDS-PAGE of purified
PA7LF3 complex. (B) Size exclusion chromatography profile of the PA7LF3 complex using a
Superdex 200 column. Sample fraction used for cryo-EM studies is indicated by black arrow. (C) Representative digital micrograph area of vitrified PA7LF3 complex. Scale bar: 20 nm. (D)
Representative 2-D class averages corresponding to C. Scale bar: 10 nm. (E-H) Rotated views
of the 3-D reconstruction of PA7LF2+1A (E), PA7LF(2+1A)’ (F), PA7LF2+1B (G), and PA7LF3--
masked (H), respectively. (I) FSC curves between two independently refined half-maps of
PA7LF2+1A (green), PA7LF(2+1A)’ (red), PA7LF2+1B (blue) and PA7LF3-masked (purple). (TIF) S3 Fig. Flowchart of image processing strategy in SPHIRE. The single particle processing
workflow is shown that included multiple 3-D classification steps as well as rotation of individ-
ual classes (indicated by rotation symbol). Number of particles in each class is provided as
orange box below the respective structure and the obtained resolution of the map after 3-D
refinement is indicated. For each structure a top and side view is shown (in top views PA7 den-
sity is partially clipped to focus on the bound LFs). Mask for masking out third LF is provided
in dashed box. Final electron density maps are highlighted by green boxes. Abbreviations:
cla3D – 3-D classification, cla2D – 2-D classification, ref-3D – 3-D refinement. (TIF) S3 Fig. Flowchart of image processing strategy in SPHIRE. The single particle processing
workflow is shown that included multiple 3-D classification steps as well as rotation of individ-
ual classes (indicated by rotation symbol). Number of particles in each class is provided as
orange box below the respective structure and the obtained resolution of the map after 3-D
refinement is indicated. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 Supporting information For each structure a top and side view is shown (in top views PA7 den-
sity is partially clipped to focus on the bound LFs). Mask for masking out third LF is provided
in dashed box. Final electron density maps are highlighted by green boxes. Abbreviations:
cla3D – 3-D classification, cla2D – 2-D classification, ref-3D – 3-D refinement. (TIF) S4 Fig. Local resolution and 3-D orientation plots. (A-D) Rotated views of the reconstruc-
tions, PA7LF2+1A (A), PA7LF(2+1A)’ (B), PA7LF2+1B (C), and PA7LF3-masked (D), respectively,
colored by local resolution. Corresponding color key of local resolution is provided on the
right. The position of the N-terminal domain of 1LF is indicated by a dashed orange ellipse for
orientation. (E) Selected examples of side chain densities corresponding to PA and LF with
atomic models fitted. (F) Rotated views of the 3-D angular distribution plot for the PA7LF2+1A
reconstruction, in which the relative height of bars represents the number of containing parti-
cles. Corresponding 2-D histogram is shown on the right. (G-I) Same as in F for PA7LF(2+1A)’
(G), PA7LF2+1B (H), and PA7LF3-masked (I). (TIF) S5 Fig. Structure comparison of PAs and LFs. (A) Superposition of the seven PA protomers
in PA7LF3, which are colored in different blue hues (left panel), and a single PA subunit (blue)
with the known crystal structure (PDB: 1TZO, green, right panel). Loop region 2β2-2β3 15 / 19 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
August 18, 2020 PLOS PATHOGENS Structure of the anthrax heptameric pre-pore state (residues 300–323), resolved only in the crystal structure, is highlighted by a black arrowhead. (B) Domain organization of LF with individual domains highlighted by different colors. (C)
Superposition of individual LFs in the PA7LF3 structures with 1LF in pink, 2LF in gold, 3BLFN
in cyan and 3ALFN in dark green. (D) Superposition of 1LF (pink), 2LF (gold) and unbound LF
(PDB: 1J7N, green), aligned via their C-terminal domain. Green and red arrows indicate simi-
lar positions in 1LF and unbound LF (PDB:1J7N), respectively. Comparison reveals that the C-
terminal domain is rotated respective to the N-terminal domain in the PA7LF3 structures. (E)
Superposition of the N-terminal domain of the three LFs in PA7LF3 (green), of LF in the
“open” conformation in PA8LF4 (PDB: 3KWV, dark yellow) and of unbound LF in the
“closed” conformation (PDB: 1J7N, purple). (F) Superposition of the three LF-LF interfaces
with 1LF-2LF in pink, 2LF-3ALF in green and 3BLF-1LF in blue. Supporting information (TIF) Acknowledgments We thank O. Hofnagel and D. Prumbaum for assistance with data collection. We thank O. Hofnagel and D. Prumbaum for assistance with data collection. S1 Table. Data collection, refinement and model building statistics.
(TIF) S1 Movie. Conformational change of LF upon PA binding. The C-terminal domain of the
three LF molecules rotates respective to the N-terminal domain upon binding to PA7 when
compared with the unbound LF structure (PDB:1J7N), to form a continuous chain of head-to-
tail interactions. Top view of the morph between both conformations is shown, with LFs in
blue and PA7 in transparent grey. (MP4) S2 Movie. LFs can interact via their C-terminal domains. In our PA7LF(2+1A)’ reconstruc-
tion, two LF molecules interact with each other via their C-terminal domain close to the cen-
tral axis of the complex, thus forming an additional LF-LF interface. Top view of the morph
between this conformation (light blue) and the one observed in the PA7LF2+1A (yellow) is
shown. Volumes are low-pass filtered and shown at the same threshold. (MP4) S2 Movie. LFs can interact via their C-terminal domains. In our PA7LF(2+1A)’ reconstruc-
tion, two LF molecules interact with each other via their C-terminal domain close to the cen-
tral axis of the complex, thus forming an additional LF-LF interface. Top view of the morph
between this conformation (light blue) and the one observed in the PA7LF2+1A (yellow) is
shown. Volumes are low-pass filtered and shown at the same threshold. (MP4) Author Contributions Conceptualization: Stefan Raunser. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008530
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S2059798319011471 PMID: 31588918 19 / 19
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https://zenodo.org/records/6445482/files/9_Eskiyeni_ISSN%202636-8536_Issue_46_March_2022_%20Article_7_pages_145-168_by_Mustafa_Bilal_%C3%96zt%C3%BCrk.pdf
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Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi
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Eskiyeni (Online)/Eskiyeni
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cc-by
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Yayın | Published: 20 Mart/March 2022
Kabul | Accepted: 21 Şubat/Fabruary 2022
Geliş | Received: 01 Ocak/January 2022
Değerlendirme | Peer Review: Dış Bağımsız | Externally Peer-reviewed.
Hakem Raporları | Peer Review Reports: https://dergipark.org.tr/tr/pub/eskiyeni/page/13397
Atıf | Citation: Mustafa Bilal Öztürk, “Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi =
Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The Definition of Knowledge”, Eskiyeni 46
(Mart/March 2022), 145-168. https://doi.org/10.37697/eskiyeni.1052186
© Mustafa Bilal Öztürk | CC BY-NC 4.0 Attribution-NonCommercial 4.0 International Araştırma Makalesi | Research Article Eskiyeni 46 (Mart/March 2022), 145-168
Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin
Hatibzâde Tenkidi
Mustafa Bilal Öztürk | Sorumlu Yazar/Corresponding Author
Dr. Araş. Gör., Dokuz Eylül Üniversitesi (ROR ID: 00dbd8b73)
İlahiyat Fakültesi, Kelâm Anabilim Dalı
Dr. Research Assistant, Dokuz Eylül University, Faculty of Theology, Department of Kalām,
İzmir/Turkey
gocmenbey@gmail.com ORCID: 0000-0001-6879-2620 Araştırma Makalesi | Research Article Mustafa Bilal Öztürk | Sorumlu Yazar/Corresponding Author
Dr. Araş. Gör., Dokuz Eylül Üniversitesi (ROR ID: 00dbd8b73)
İlahiyat Fakültesi, Kelâm Anabilim Dalı
Dr. Research Assistant, Dokuz Eylül University, Faculty of Theology, Department of Kalām,
İzmir/Turkey
gocmenbey@gmail.com ORCID: 0000-0001-6879-2620 Abstract Since the early period of Islamic thought, studies on the subject of existence, knowledge,
and value have been made by theologians. While some theology-philosophy books started
with the subject of existence, some of them started with the subject of knowledge. It is
known that knowledge is closely related to faith. Due to this close contact, theologians have
built their philosophy of knowledge around titles such as the definition, limits, sources and
possibilities of knowledge. Rather than discussing faith, which has a predominant subjec-
tive aspect, the field of knowledge, which is a more objective field, has been highlighted. Thanks to the transfer of faith to the knowledge field, people were able to conduct their
discussions on rational grounds. It is foreseen that the issue will be inconclusive when faith
is discussed alone. Every person is free to believe anything, regardless of right or wrong, but
the same freedom does not exist in the field of knowledge. Knowledge has an objective and
controllable structure. Therefore anyone who is trying to convey his or his/her belief to
others should definitely explain his/her belief on a rational basis. Belief should be brought
closer to knowledge in order to spread the sharing of faith. Belief cannot be examined, but
knowledge cannot be ruled out. The close relationship of knowledge with belief should not
be overlooked. On account of this for theologians faith and belief consist of affirmation. So
what is knowledge, what does it mean to know? Is knowing the same as believing? Is the
person who says he knows something different from the person who says he believes some-
thing? All these questions seem to necessitate the definition of knowledge as well as belief. The definition of knowledge changes according to schools and scholars in Islamic thought. Some scholars claim that absolute knowledge cannot be defined by counting its self-evident
(badīhī). However, according to the great majority, knowledge is theoretical and therefore Eskiyeni 46 (Mart/March 2022) 146 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi its definition can be made. In the history of kalām, many definitions of knowledge have
been made and criticized. In the article, the definition of knowledge as “an adjective that
obliges to differentiate” will be investigated. According to this definition, another impor-
tant feature of knowledge is that differentiation is not likely to contradict. Abstract In the final anal-
ysis, the category of knowledge is an adjective. Through the agency of this adjective, the
subject knows. Knowledge is a separate activity. For something known to be knowledge, it
must be certain and not otherwise likely. An imprecise comprehension is not considered
knowledge according to theologians. This kind of knowledge definition, which was pre-
sented perfectly in the theological books of the late period, was criticized by Khaṭībzāde (d. 901/1496) from ten aspects. Ḥusām Çelebi (d. 926/1520), on the other hand, found all ten
criticisms of Khaṭībzāde weak in different aspects. Çelebi found all ten criticisms of
Khaṭībzāde weak in different aspects. In the article, first Khaṭībzāde’s criticisms will be ex-
plained in order, then Çelebi’s counter-criticism will be examined and strengths and weak-
nesses in the statements of both will be pointed out. Keywords Kalām, Ḥusām Çelebi, Khaṭībzāde, Risāla fī taʿrīf al-ʿIlm, Knowledge www.dergipark.org.tr/tr/pub/eskiyeni 1 Babanzâde Ahmet Naim (öl. 1934) tarafından dile getirilen ilim, marifet ve irfan kelimelerinin farkları
ile İslam düşüncesinde üretilen ilim tarifleri için bk. İsmail Kara, Bir Felsefe Dili Kurmak: Modern Felsefe ve
Bilim Terimlerinin Türkiye’ye Girişi (İstanbul: Dergâh Yayınları, 2012), 203.
2 Adnan Bülent Baloğlu, “Doğru Bilgi Tanımına Ulaşma Çabası: Ebu’l-Mu‘în en-Nesefi Örneği”, Dokuz Eylül
Üniversitesi İlahiyat Fakültesi Dergisi 18 (2003), 8. Ayrıca bk. Mustafa Bozkurt, “Kelâmcılarda Bilginin
Tanımı Problemi”, Cumhuriyet Üniversitesi İlahiyat Fakültesi Dergisi 12/1 (2008), 259.
3 Mehmet Sait Özervarlı, “Alâeddin el-Üsmendî’nin Kelâmcılığı ve Bilgi Teorisi: Mâverâünnehir Kelâm
Düşüncesine Bir Katkı”, İslâm Araştırmaları Dergisi 10 [Doç. Dr. Ahmet Gürtaş Hatıra Sayısı] (2003), 49.
4 Mütekaddim dönemin bilgi anlayışının geniş bir biçimde araştırılması için bk. Hilmi Demir, Delil ve
İstidlâlin Mantıkî Yapısı: -İlk Dönem Sünnî Kelâm Örneği- (İstanbul: İslam Araştırmaları Merkezi Yayınları,
2012), 50.
5 Mantık ilmi bağlamında tasavvur-tasdik ayrımının arka planı için bk. Mehmet Özturan, “Seyyid Şerîf
Cürcânî’nin Risâle fi Taksîmi’l-İlm Adlı Eserinin Tahlil, Tahkik ve Tercümesi”, Nazariyat: İslâm Felsefe ve
Bilim Tarihi Araştırmaları Dergisi 1/2 (2015), 104. Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde
Tenkidi İslam düşüncesinin erken döneminden itibaren kelâmcılar tarafından varlık, bilgi ve değer
konusu hakkında çalışmalar yapılmıştır. Kimi kelâm-felsefe kitapları varlık konusu ile baş-
larken, kimileri de bilgi konusu ile başlatılmıştır. Bilginin iman ile yakın ilgisi bilinmektedir. Bu yakın irtibattan dolayı kelâmcılar; bilginin tanımı, sınırları, kaynakları, imkânı gibi baş-
lıklar etrafında kendilerine özgü bilgi felsefesi inşa etmiştir. Öznel yönü ağır basan imanı
tartışma konusu yapmak yerine daha nesnel bir alan olan bilgi alanı öne çıkarılmıştır. İma-
nın bilgiye aktarımı sayesinde insanlar rasyonel zeminde tartışmalarını yürütebilmiştir. İman tek başına tartışıldığında meselenin sonuçsuz kalacağı öngörülmüştür. Her insan doğ-
ru veya yanlış gözetmeksizin her şeye inanmakta özgürdür. Fakat aynı özgürlük bilgi ala-
nında yoktur. Bilginin nesnel ve denetlenebilir bir yapısı vardır. İnancını başkalarına ak-
tarmak gayretinde olan birinin mutlaka inancını rasyonel zeminde açıklaması gerekmekte-
dir. İnancın paylaşımını yaygınlaştırmak için imanın bilgiye yaklaştırılması gerekmektedir. İnancı denetlemek olası değildir, ama bilgi denetimden çıkarılamaz. Bilginin inançla olan
yakın ilgisi gözden kaçırılmamalıdır. Kelâmcılara göre inanç veya iman tasdikten ibarettir. Öyleyse bilgi nedir, bilmek ne demektir? Bilmek ile inanmak aynı mıdır? Bir şeyi bildiğini
söyleyen kişi, bir şeye inandığını söyleyen kişiden farklı mıdır? Tüm bu sorular inanç kadar
bilginin de tanımını yapmayı zorunlu kılmış gözükmektedir. İslam düşüncesinde ekollere ve
düşünürlere göre bilginin tanımı değişmektedir. Mutlak bilgiyi bedihi sayarak tanımlanma-
yacağı iddiasında olan bir kesim vardır. Ancak kahir ekseriyete göre bilgi nazarîdir ve bu
nedenle tanımı yapılabilir. Kelâm tarihinde farklı bilgi tanımları yapılmış ve eleştirilmiştir. Makalede bilginin “temyizi zorunlu kılan bir sıfat” şeklinde yapılan tanımı araştırılacaktır. Bu tanıma göre bilginin diğer önemli özelliği, temyizin çelişiğe ihtimali olmamasıdır. Nihai
tahlilde bilginin kategorisi bir sıfattır. Bu sıfat sayesinde özne bilmektedir. Bilgi bir ayrıştır-
ma faaliyetidir. Bilinen bir şey, bilgi sayılması için kesinlik taşımalı aksi ihtimal taşımamalı- Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 147 147 dır. Kesin olmayan bir idrak, kelâmcılara göre bilgi sayılmamaktadır. Müteahhir dönem ke-
lâm kitaplarında kusursuz olarak takdim edilen bu bilgi tanımını Hatibzâde (öl. 901/1496)
on açıdan eleştirmiştir. Buna karşı Hüsâm Çelebi (öl. 926/1520), Hatibzâde’nin on eleştirisi-
nin tamamını farklı açılardan zayıf bulmuştur. Makalede ilk önce Hatibzâde’nin tenkitleri
sırası ile izah edilecek, ardından Çelebi’nin karşı eleştirileri incelenecek ve tarafların açık-
lamalarındaki güçlü ve zayıf yönlere işaret edilecektir. Anahtar Kelimeler 1 Babanzâde Ahmet Naim (öl. 1934) tarafından dile getirilen ilim, marifet ve irfan kelimelerinin farkları
ile İslam düşüncesinde üretilen ilim tarifleri için bk. İsmail Kara, Bir Felsefe Dili Kurmak: Modern Felsefe ve
Bilim Terimlerinin Türkiye’ye Girişi (İstanbul: Dergâh Yayınları, 2012), 203. 5 Mantık ilmi bağlamında tasavvur-tasdik ayrımının arka planı için bk. Mehmet Özturan, “Seyyid Şerîf
Cürcânî’nin Risâle fi Taksîmi’l-İlm Adlı Eserinin Tahlil, Tahkik ve Tercümesi”, Nazariyat: İslâm Felsefe ve
Bilim Tarihi Araştırmaları Dergisi 1/2 (2015), 104. Giriş Varlık, bilgi ve değer alanlarını araştırma konusu yapan kelâm ilmi, bir bakıma
kavramlar ve yargılar üzerine kuruludur. Kuruluşundan itibaren kelâmcılar kav-
ram haritasına ve tanımlarına, özellikle de bilgi meselesine önem vermişlerdir.1
Kelâm ilminin erken döneminden başlayarak geç dönemine kadar ilerleyen süreç-
te bilgi tanımı sürekli güncellenmiştir. Farklı düşünce ekolleri tarafından yapılan
bilgi tanımları sürekli eleştirilmiştir.2 Müteahhir dönemde kaleme alınan kelâm ki-
taplarının kahir ekseriyeti konu bakımından epistemoloji ile başlatılmıştır.3 Burada
bilginin tarifi, kapsamı, imkânı araştırılmış, ilaveten bilginin taksimi ile bunun
üzerine bina edilen nazar, delil türleri, delil-medlül irtibatı vb. kavramların tarifi
üzerine hassasiyet gösterilmiştir. İlahiyat, nübüvvet ve ahiret konularının bir yö-
nüyle ontoloji, diğer yönüyle epistemoloji ve değerler ile irtibatları kurulmuştur. l
kl
l
l
k
l
k
l
d b l l Kavramlara yüklenen anlamlar, kurulacak yargıların yönünü de belirlemiştir. İslam düşüncesi başından itibaren kavramlara ve yargılara titizlik göstermiş-
tir. Konuyu belirginleştirmek için mevzu, konudaki sorunu araştırmak için mesele
ve araştırmanın ilkelerini göstermek için mebâdî olmak üzere yöntemde üç kritik
eşik benimsenmiştir. Mütekaddim-müteahhir dönem farkı gözetilmeksizin sözü
edilen yöntemin izlerini, bilgi hakkında yapılan incelemelerin kendisinde takip
etmek mümkündür.4 Mantık biliminde bilgiyi tasavvur ve tasdik diye taksim etmek ne kadar
önemli ise kelâmda da bilginin zarûrî ve kesbî ayrımı o kadar önemlidir.5 Kelâm 5 Mantık ilmi bağlamında tasavvur-tasdik ayrımının arka planı için bk. Mehmet Özturan, “Seyyid Şerîf
Cürcânî’nin Risâle fi Taksîmi’l-İlm Adlı Eserinin Tahlil, Tahkik ve Tercümesi”, Nazariyat: İslâm Felsefe ve
Bilim Tarihi Araştırmaları Dergisi 1/2 (2015), 104. Eskiyeni 46 (Mart/March 2022) 148 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi kitaplarına varlık veya bilgi ile başlamak mütekaddim ile başlayan ve müteahhir
dönem kelâmcılarıyla devam ettirilen bir gelenek olmuştur. Bilginin hakikati,
tanımı, zarûrî-kesbî ayrımı, bu iki bilgi türünün birbirine dönüşüp dönüşemeye-
ceği, bilginin mertebeleri, bir bilginin iki bilinene ilişmesi, bir şeyin yönüyle bili-
nen diğer yönüyle bilinmeyen olabilmesi,6 bilinensiz bilginin imkânı temel tar-
tışma başlıklarıdır.7 Mütekaddim ve müteahhir İslam kelâmcıları ve felsefecileri, kendilerini bir
önceki dönemden bağımsız görmemişlerdir. Düşüncelerinde kendinden önceki-
lerin ilmi katkılarını kesinlikle göz ardı etmemişlerdir. Elbette bu, gelenek tara-
fından aktarılan birikimin tümüyle katıksız benimsendiği anlamına gelmemek-
tedir. Kendinden önce üretilen bilimsel çabalara bigâne kalmak ile onları düşün-
cenin konusu yapmak ve eleştiri süzgecinden geçirmek birbirinden farklıdır. Bu
hassas ayrım, belirli bir bilinç düzeyini ve farkındalığı göstermektedir. Klasik ke-
lâm-felsefe kitaplarının herhangi bir meselesinde bu bilinç düzeyini görmemek
imkânsızdır. 8 Seyyid Şerif Cürcânî, Şerhu’l-mevâkıf: Mevâkıf Şerhi, çev. Ömer Türker (İstanbul: Türkiye Yazma Eserler
Kurumu Başkanlığı, 2015), 1/280. Ayrıca bk. Sa‘düddîn Teftâzânî, Şerhu’l-makāsıd, thk. İbrahim
Şemseddin (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2011), 1/51. 6 Bir şeyi bilmek ile o şey hakkındaki bilgiyi bilmeyi ayıran Molla Lufti’nin (öl. 900/1495) tartışması için bk.
Ömer Mahir Alper - Yasin Apaydin, Molla Lutfi ve Fütûhât’i (Ankara: Türkiye Bilimler Akademisi, 2021), 133.
7 Fahreddin er-Râzî’nin katkılarına temas edilerek, İbn Sînâ özelinde yazılan bir araştırma için bk. Ömer
Türker, İbn Sînâ Felsefesinde Metafizik Bilginin İmkânı Sorunu (İstanbul: İslam Araştırmaları Merkezi
Yayınları, 2010), 173. 9 Teftâzânî bağlamında müteahhir dönemin bilgi serüvenine ışık tutan araştırmalar için bk. Süleyman
Akkuş - Ziya Erdinç, “Sa’düddîn Teftâzânî’de Bilgi Teorisi”, Usûl: İslam Araştırmaları 21 (2014), 13; Ziya
Erdinç, “Teftâzânî’nin Bilginin Tanımlanması Problemine Yaklaşımı”, Harran Üniversitesi İlahiyat
Fakültesi Dergisi 24/42 (2019), 263. Ömer Mahir Alper - Yasin Apaydin, Molla Lutfi ve Fütûhât i (Ankara: Türkiye Bilimler Akademisi, 2021), 133.
7 Fahreddin er-Râzî’nin katkılarına temas edilerek, İbn Sînâ özelinde yazılan bir araştırma için bk. Ömer
Türker, İbn Sînâ Felsefesinde Metafizik Bilginin İmkânı Sorunu (İstanbul: İslam Araştırmaları Merkezi
Yayınları, 2010), 173. 6 Bir şeyi bilmek ile o şey hakkındaki bilgiyi bilmeyi ayıran Molla Lufti’nin (öl. 900/1495) tartışması için bk.
Ömer Mahir Alper - Yasin Apaydin, Molla Lutfi ve Fütûhât’i (Ankara: Türkiye Bilimler Akademisi, 2021), 133.
7 Fahreddin er-Râzî’nin katkılarına temas edilerek, İbn Sînâ özelinde yazılan bir araştırma için bk. Ömer
Türker, İbn Sînâ Felsefesinde Metafizik Bilginin İmkânı Sorunu (İstanbul: İslam Araştırmaları Merkezi
Yayınları, 2010), 173.
8 Seyyid Şerif Cürcânî, Şerhu’l-mevâkıf: Mevâkıf Şerhi, çev. Ömer Türker (İstanbul: Türkiye Yazma Eserler
Kurumu Başkanlığı, 2015), 1/280. Ayrıca bk. Sa‘düddîn Teftâzânî, Şerhu’l-makāsıd, thk. İbrahim
Şemseddin (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2011), 1/51.
9 Teftâzânî bağlamında müteahhir dönemin bilgi serüvenine ışık tutan araştırmalar için bk. Süleyman
Akkuş - Ziya Erdinç, “Sa’düddîn Teftâzânî’de Bilgi Teorisi”, Usûl: İslam Araştırmaları 21 (2014), 13; Ziya
Erdinç, “Teftâzânî’nin Bilginin Tanımlanması Problemine Yaklaşımı”, Harran Üniversitesi İlahiyat
Fakültesi Dergisi 24/42 (2019), 263. 6 Bir şeyi bilmek ile o şey hakkındaki bilgiyi bilmeyi ayıran Molla Lufti’nin (öl. 900/1495) tartışması için bk.
Ömer Mahir Alper - Yasin Apaydin, Molla Lutfi ve Fütûhât’i (Ankara: Türkiye Bilimler Akademisi, 2021), 133. 6 Bir şeyi bilmek ile o şey hakkındaki bilgiyi bilmeyi ayıran Molla Lufti’nin (öl. 900/1495) tartışması için bk.
Ömer Mahir Alper - Yasin Apaydin, Molla Lutfi ve Fütûhât’i (Ankara: Türkiye Bilimler Akademisi, 2021), 133.
7 Fahreddin er-Râzî’nin katkılarına temas edilerek, İbn Sînâ özelinde yazılan bir araştırma için bk. Ömer
Tü k
İb
Sî â F l f i d
M
fi ik Bil i i
İ kâ
S
(İ t
b l İ l
A
t
l
M
k
i 10 Ziya Erdinç, “Teftâzânî’nin Düşünce Sisteminde İnsanî Nefsin Varlığı ve Hakikati”, Nazariyat: İslâm
Felsefe ve Bilim Tarihi Araştırmaları Dergisi 7/2 (2021), 182.
11 Adudüddin Îcî, Şerḥi muḫtaṣari’l-müntehâ (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2004), 1/184.
12 Cürcânî, Şerhu’l-mevâkıf, 1/171.
13 Bilginin sıfat veya izafet sayılmasını sorunlu gören Bahâeddinzâde’nin (öl. 952/1545) a’yân-ı sâbite
nazariyesi çerçevesindeki önerisine değinen bir çalışma için bk. Yasin Apaydın - Musakhanov Orkhan,
Kelâm ile Tasavvuf Arasında: Bahâeddinzâde ve Kader Anlayışı (İstanbul: Endülüs Yayınları, 2021), 39. Giriş Kitapların özellikle hacimli, derinlikli ve uzun soluklu (mutavvel)
olanlarında bir tür düşünce tarihi okuması yapıldığı göze çarpmaktadır. Bu bağlamda bilginin tanımı, kaynakları, imkânı, doğruluk ölçütleri, düşün-
cenin mahiyeti, doğru düşünmenin şartları, doğru düşünmenin doğru bilgi ile
ilişkisi, bilginin zihinle, hariçle ve nefsü’l-emr ile mutabakatı kelâm kitaplarında
tartışılanlar arasındadır.8 İslam düşüncesinin müteahhir döneminde öne çıkarılan ve mantık kuralları
bakımından doğruya en yakın kabul edilen bilgi tanımına nasıl ulaşıldığı önemli-
dir. İslam düşünce tarihi boyunca bilgi tanımına yönelik verilen uğraşıları ve
çabaları görmeksizin, ulaşılan son noktayı anlamak zordur. Son noktayı anlamak,
ilk noktadan itibaren gelişim ve değişim seyrini doğru anlamaya bağlıdır. Üreti-
len çeşitli bilgi tanımlarına yöneltilen eleştiriler, aslında ulaşılmak istenen bilgi
tanımını göstermektedir.9 Bu eleştirileri değerlendirmeye geçmeden önce mut-
lak bilginin mahiyeti bir kelâmcı için neden önemlidir? Sorusu çözümlenmelidir. Kelâmcılar, inanç ve bilgi arası ayrımın farkındadırlar. İnançlar inanç olmak
bakımından tartışmaya açık değildir. Akıl, düşünce ve bilgi zeminine taşımaksı- www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 149 149 zın inançları tartışmaya çalışmak, havanda su dövmeye benzemektedir. İnançları
düşünce ve bilgi alanına taşımak, sadece onlardan kurtulmak amacıyla yapılmaz. İnançtan bilgiye doğru geçiş, bilgiden inanca doğru geçişi denetlemek için yapı-
lır. Bilgiyi öne almak, inançları ötelemek, kötülemek, küçümsemek demek değil-
dir. Olsa olsa inançları, bilgi üzerine inşa etmektir. İnançları düşünce-bilgi zemi-
nine çekebilmenin ilk adımı onları bilginin konusu kılmaktan geçmektedir. İlk
adım öznel alanı, nesnel alana taşımak olarak da görülebilir. İnanç ile bilgi ay-
rımları olmak ile birlikte ortak yönleri de vardır. İnanç ve bilgi her ikisi de insan-
la ilişkilidir.10 İnsanın gönül-akıl, iman-düşün yetilerine ait fonksiyonlardır. Kimi
düzeylerde inanç-bilgi ayrımını gözetebilmek oldukça zordur. Fakat herhâlükâr-
da inançlar, öznel ve nedensiz olabilmektedir. Ancak bilgi nesnel ve nedenli ol-
mak zorundadır. İnanç inanç olması bakımından doğru ve yanlışın konusu olmaz
iken, bilgi doğru ve yanlışın konusudur. İnançları bilginin konusu yapabilmek
için ise bilginin ne olduğunu bilmek, bu yönde çaba sarf etmek ve önceden sarf
edilen çabaları değerlendirmek, eleştirmek önem arz etmektedir. 1. Eleştirisi Yapılan Bilgi Tanımı Müteahhir dönem kelâm edebiyatına damgasını vuran, kelâm-felsefenin dü-
şünce tarihinde yapılan bilgi tanımlarına yöneltilen eleştirilerin neticesinde
yanlışsız ve kusursuz bir bilgi tanımına ulaşıldığı İbnü’l-Hâcib (öl. 646/1249) ve
Adudüddîn Îcî (öl. 756/1355) tarafından açıkça ileri sürülmüştür. Bu ikisine göre
bilgi: “Çelişiğe ihtimali olmayan ayrıştırmayı zorunlu kılan bir sıfattır”. Bu tanım man-
tık kuralları çerçevesinde doğruya en yakın olması bakımından kimi yerde
esahhut-ta‘rîf,11 kimi yerde ta‘rîfü’l-muhtâr payesine layık görülmüştür.12 Bu ikisine
göre bilginin cinsi ve kategorisi sıfattır. Bilgiyi sıfatla açıklamak bilginin bilen
özne ile bağlantısı kurmak için kaçınılmazdır. Her sıfatın bir işlevi olduğu üzere
bilgi sıfatının da özneye katkısı ona temyiz yeteneği sağlamasıdır. Bilgi sıfatı öz-
nesine ayrıştırma özelliği kazandırmaktadır. Bilgi ile bilen arası bağlantının dü-
zeyi öznenin kimliğine göre değişkenlik arz etmektedir. Bilen Tanrı olduğunda
bilgi sıfatının içeriği zorunlulukla doldurulur.13 Bilen, Tanrı dışındakiler oldu-
ğunda ise bilgi-bilinen bağlantısında vesileci-ilişkisel nedene dayalı (âdet) irtibat
biçimi öngörülmektedir. Tarife getirilen “temyiz” kelimesiyle idrak dışında kalan tüm nefsânî olan ve
olmayan sıfatlar dışarıda bırakılmıştır. Nitekim bilgi dışındaki nefsânî sıfatlar- Eskiyeni 46 (Mart/March 2022) 150 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi dan, mesela cesaret veya nefsânî olmayan zencilik gibi hiçbirinin hem kendini
hem karşısındaki bizzat temyiz/ayrıştırma faaliyeti yoktur.14 Terim anlamı itiba-
riyle temyiz; öznenin idrakindeki ayırma/ayrıştırma faaliyetidir. Öyleyse bilgi,
anlamlar arasını ayrıştırma işlemidir. Özne ile ilişki olan temyiz kelimesini te-
meyyüz şeklinde okumak yanlıştır. Zira temeyyüz ayrılmak anlamıyla nesnede
gerçekleşir, öznede değil. Sözgelimi herhangi bir nesne siyah sıfatı ile beyaz sıfa-
tından ayrılmaktadır.15 Tanımda geçen sıfat, zorunluluk, temyiz kelimeleri bilgiyi; zan, kuşku, cehl-i
mürekkep ve taklit gibi diğer türlerinden ayrıştırmamıştır. Kelâm terminoloji-
sinde bilgi kesin olmakla kayıtlanmaktadır. Çünkü kelâm sisteminin idealine gö-
re kesin bilgiye ulaşılmalıdır. Yalnızca kesin bilgiyi bilgi saymak, kelâmcılara öz-
gü bir yaklaşımdır. Onlara göre bir şeyi bilmek; sanmaktan, kuşkulanmaktan,
bilmediğini bilmemekten ve taklit etmekten farklıdır. Bu farkı göstermek için
bilgi tanımına “çelişiğine ihtimali olmama” ilavesi eklenmiştir.16 Sözgelimi “Ah-
met canlıdır” yargısını bilen birinin bilgisi, Ahmet canlı kaldığı sürece çelişiği
olan “Ahmet ölüdür” yargısına izin vermez, vermemelidir. Bilindiği üzere zan ve
kuşku an itibariyle çelişiğine ihtimal taşırken, cehl-i mürekkep ve taklit gelecek
bakımından çelişiğine ihtimal taşımaktadır.17 Diğer tabirle zan ve şüphe edenin
bilgisi her an değişime açık iken, bilmediğini bilmeyen veya taklit ile bilen biri-
nin bilgisini değiştirmek zaman alır. 14 Cesaret ve zencilik örneklerini Cürcânî, bilgi sıfatının işlev alanını belirlemek ve diğer sıfatlarla olan
farklılığını göstermek adına özellikle kullanmaktadır. Cürcânî, Şerhu’l-Mevâkıf, 1/171.
15 Ebû Abdurrahman Abdulaziz b. Ahmed b. Hamîd Ferhârî, en-Nibrâs: Şerhu şerhi’l-akāid (İstanbul: Âsitâne
Kitabevi, 2009), 92.
16 Cürcânî, Şerhu’l-mevâkıf, 1/171.
17 Teftâzânî, Şerhu’l-makāsıd, 1/57.
18 Teftâzânî, Şerhu’l-makāsıd, 1/59.
19 Cürcânî, Şerhu’l-mevâkıf, 1/177.
20 Ebü’l-Muîn en-Nesefî bu tanımı İmâm Mâtürîdî’ye nispet etmiştir. Nesefî’nin aktarımına göre
Mâtürîdî’nin kitaplarında sözü edilen düzen ve sıra içinde bu tanım yoktur, ancak tanıma işaret eden
ifadeler vardır. Detaylı bilgi için bk. Ebü’l-Muîn Nesefî, Tebṣıratü’l-edille fî uṣûli’d-dîn, thk. Hüseyin Atay -
Şaban Ali Düzgün (Ankara: Diyanet İşleri Başkanlığı Yayınları, 2004), 2/19. www.dergipark.org.tr/tr/pub/eskiyeni 14 Cesaret ve zencilik örneklerini Cürcânî, bilgi sıfatının işlev alanını belirlemek ve diğer sıfatlarla olan
farklılığını göstermek adına özellikle kullanmaktadır. Cürcânî, Şerhu’l-Mevâkıf, 1/171.
15 Ebû Abdurrahman Abdulaziz b. Ahmed b. Hamîd Ferhârî, en-Nibrâs: Şerhu şerhi’l-akāid (İstanbul: Âsitâne
Kitabevi, 2009), 92. 20 Ebü’l-Muîn en-Nesefî bu tanımı İmâm Mâtürîdî’ye nispet etmiştir. Nesefî’nin aktarımına göre
Mâtürîdî’nin kitaplarında sözü edilen düzen ve sıra içinde bu tanım yoktur, ancak tanıma işaret eden
ifadeler vardır. Detaylı bilgi için bk. Ebü’l-Muîn Nesefî, Tebṣıratü’l-edille fî uṣûli’d-dîn, thk. Hüseyin Atay -
Şaban Ali Düzgün (Ankara: Diyanet İşleri Başkanlığı Yayınları, 2004), 2/19. 1. Eleştirisi Yapılan Bilgi Tanımı Murat
Polat, “Amasya Müftüsü Hüsâm Çelebi’nin (öl. 926/1520) ‘Risâle fî Raksi’l-Mutasavvıfa’ adlı Eserinde Mûsikî
ve Semaya Fıkhî Açıdan Bakış”, Geçmişten Günümüze Uluslararası Dinî Mûsikî Sempozyumu, ed. Şuayip
Özdemir- Ayşegül Gün (Ankara: Kıbrıs Balkanlar Avrasya Türk Edebiyatları Kurumu Başkent Klişe ve
Matbaacılık, 2017), 601. Mevâkıf hâşiyesi veya kelâm ilminin tanımına yönelik bu eserin mütemmim
parçası, bilgi tanımı ile ilgili risâle olmalıdır. Kelâm ilminin tanımı meselesi, Hüsâm Çelebi’yi bilginin
tanımını araştırmaya sevk etmiş gözükmektedir. 25 Risâlenin [Veliyüddin, 3244, 49a-50a] olmak üzere tek el yazma nüshası tespit edilmiştir. Risâlenin tam
adı nüshanın zahriyesinden alınmış, eserin Hüsâm Çelebi’ye ait olduğu yine bu nüshanın [41b]’e
kaydedilen not sayesinde belirlenmiştir. Hüsâmeddin Hüseyin b. Abdurrahman Hüsâm Çelebi, Hâşiye fî
mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i ‘itirâzât-ı Hatîbzâde (İstanbul: Beyazıt Devlet Kütüphanesi, Veliyüddin,
3244), 41b. Araştırma sırasında Hüsâm Çelebi’nin risâlesine oldukça benzer başka bir yazma risâle daha
vardır. Bu risâle de aynı şekilde Hatibzâde’nin eleştirilerini Çelebi gibi on açıdan eleştirmektedir. Ancak
Hüsâm Çelebi’nin risâlesinden farklılıklar barındırmaktadır. Dolayısıyla müellifi ayrı bir kişi olmalıdır. Anılan bilgi tanımı hakkındaki risâlenin mecmuanın zahriyesinde Celâleddîn ed-Devvânî’ye (?) nispeti
kuşku ile karşılanmalıdır. Her iki risâle üzerine yapılacak tahkikli neşir çalışması risâlelerin aynı ve ayrı
olduğu konusunda belirleyici olacaktır. Risâle fî ta‘rîfi’l-‘ilm (İstanbul: Beyazıt Devlet Kütüphanesi,
Veliyüddin, 3270), 118a. 25 Risâlenin [Veliyüddin, 3244, 49a-50a] olmak üzere tek el yazma nüshası tespit edilmiştir. Risâlenin tam
adı nüshanın zahriyesinden alınmış, eserin Hüsâm Çelebi’ye ait olduğu yine bu nüshanın [41b]’e
kaydedilen not sayesinde belirlenmiştir. Hüsâmeddin Hüseyin b. Abdurrahman Hüsâm Çelebi, Hâşiye fî
mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i ‘itirâzât-ı Hatîbzâde (İstanbul: Beyazıt Devlet Kütüphanesi, Veliyüddin,
3244), 41b. Araştırma sırasında Hüsâm Çelebi’nin risâlesine oldukça benzer başka bir yazma risâle daha
vardır. Bu risâle de aynı şekilde Hatibzâde’nin eleştirilerini Çelebi gibi on açıdan eleştirmektedir. Ancak
Hüsâm Çelebi’nin risâlesinden farklılıklar barındırmaktadır. Dolayısıyla müellifi ayrı bir kişi olmalıdır. Anılan bilgi tanımı hakkındaki risâlenin mecmuanın zahriyesinde Celâleddîn ed-Devvânî’ye (?) nispeti
kuşku ile karşılanmalıdır. Her iki risâle üzerine yapılacak tahkikli neşir çalışması risâlelerin aynı ve ayrı
olduğu konusunda belirleyici olacaktır. Risâle fî ta‘rîfi’l-‘ilm (İstanbul: Beyazıt Devlet Kütüphanesi,
Veliyüddin, 3270), 118a. 26 Hüsâm Çelebi’nin Hâşiye ala şerhi’t-Tecrîd’i bağlamında varlık felsefesine (umûr-ı âmme) temas eden bir
araştırma için bk. Yasin Apaydın, Metafiziğin Meselesini Temellendirmek: Tecrîd Geleneği Bağlamında Umûr-ı
Âmme Sorunu (İstanbul: Endülüs Yayınları, 2019), 276. 26 Hüsâm Çelebi’nin Hâşiye ala şerhi’t-Tecrîd’i bağlamında varlık felsefesine (umûr-ı âmme) temas eden bir
araştırma için bk. Yasin Apaydın, Metafiziğin Meselesini Temellendirmek: Tecrîd Geleneği Bağlamında Umûr-ı
Âmme Sorunu (İstanbul: Endülüs Yayınları, 2019), 276. 22 Ahlâkî değerlerin kaynağı ve insan fiillerinde Hatibzâde’nin Eş‘arî düşüncede olduğunu gösteren
çalışması ve hakkında yapılan bir değerlendirme yazısı için bk. Mustafa Borsbuğa - Coşkun Borsbuğa,
“Hatibzâde Muhyiddin Efendi’nin Hâşiye ʿale’l-Mukaddimâti’l-Erbaʿa Adlı Hâşiyesinin Tahkik ve
Tahlili”, Tahkik İslami İlimler Araştırma ve Neşir Dergisi 4/2 (31 Aralık 2021), 226. 21 Cürcânî, Şerhu’l-mevâkıf, 1/177. 1. Eleştirisi Yapılan Bilgi Tanımı Sonuç itibariyle sayılan dört çeşit bilgi türü
çelişiklerine ihtimal vermektedirler.18 Seyyid Şerif Cürcânî, Îcî’nin tercih ettiği bilgi tanımına açıktan bir eleştiri yö-
neltmese de imalı yeni bir teklifte bulunmuştur. İşte bu teklif, örtük bir eleştiri
olarak okunabilir. Cürcânî bilginin mahiyetini açığa çıkaran doğru tanımın; “her
kimde bulunursa bulunsun mezkûru tecelli ettiren bir sıfat” olarak yapılmasında bul-
muştur.19 Muhtemelen Cürcânî bu tanımı, Ebü’l-Muîn en-Nesefî’nin (öl. 508/1115)
Tebsıratü’l-edille’sinden almıştır.20 Tanımdaki “mezkûr” dile getirilebilmeyi ifade
etmekte; mevcut, madum, mümkün, müstahil olmak üzere ontolojik, ayrıca müf-
ret, mürekkep, tümel, tikel olmak üzere her türlü epistemik alanı kapsamaktadır. Benzer biçimde bulanık, karışık her türlü bilmek türünü dışarıda bırakan “tecelli”
tam inkişaf sağlamaktadır. Dolayısıyla tam inkişaf sayesinde tanımdan zan, ceha- Ş
f
20 Ebü’l-Muîn en-Nesefî bu tanımı İmâm Mâtürîdî’ye nispet etmiştir. Nesefî’nin aktarımına göre
Mâtürîdî’nin kitaplarında sözü edilen düzen ve sıra içinde bu tanım yoktur, ancak tanıma işaret eden
ifadeler vardır. Detaylı bilgi için bk. Ebü’l-Muîn Nesefî, Tebṣıratü’l-edille fî uṣûli’d-dîn, thk. Hüseyin Atay -
Şaban Ali Düzgün (Ankara: Diyanet İşleri Başkanlığı Yayınları, 2004), 2/19. Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 151 151 let, bilmediğini bilmemek (cehl-i mürekkep) ve ayrıca doğruyu taklit edenin itikadı
dışarı atılmıştır. Taklit ister doğruya ister yanlışa olsun bir bağlılık türüdür. Ancak
bu bağlılıkta dış açılım (inkişaf) ve iç açılım (inşirah) bulunmaz.21 Tüm bu bilgilerin ardından Hatibzâde’nin (öl. 901/1496)22 Risâle fî ta‘rîfi’l-‘ilm
adlı eseri çerçevesinde bilgi tanımına ilişkin eleştirileri özetlenecek,23 bilahare
Hüsâm Çelebi’nin (öl. 926/1520)24 Hâşiye fî mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i
‘itirâzât-ı Hatîbzâde25 isimli çalışması bağlamında karşı eleştirileri araştırılacak-
tır.26 Hüsâm Çelebi, Hatibzâde’nin Risâle fî ta‘rîfi’l-‘ilm çalışmasını kendi eserinde
birebir verir, ardından “ben derim ki” diyerek kendi görüşlerini açıklar.27 Yapı- 24 Taşköprizâde, Hüsâm Çelebi’nin vefat tarihini vermiş; Hâşiye: Şerhu’t-Tecrîd, Hâşiye: Şerhu’l-Vikāye, Risâle fî
istihlâfi Hatîb, Risâle fî cevâzi’z-zikri’l-cehrî adlı eserlerinden bahsetmiş ancak burada Hâşiye ala şerhi’l-mevâkıf fî
ta‘rîfi ilmi’l-kelâm ve nüshalarda bunun ardına gelen bilgi tanımı ile ilgili çalışmasına değinmemiştir. Ahmed
Efendi Taşköprizâde, Eş-şakā’iku’n-Nu’mâniyye fî ulemâi’d-devleti’l-Osmâniyye: Osmanlı âlimleri: (çeviri, eleştirmeli
metin), ed. Derya Örs (İstanbul: Türkiye Yazma Eserler Kurumu Başkanlığı, 2019), 610. Ayrıca akademik bir
araştırma, [Veliyüddin, 3244, 41b-48b] nüshasının [41b] varağına düşülen önemli nota binaen eseri Hüsâm
Çelebi’ye nispet etmiştir. Levent Öztürk, “Hüsâm Çelebi (öl. 926/1520)’nin Hayatı ve Eserleri”, Sakarya
Üniversitesi İlahiyat Fakültesi Dergisi 3 (2001), 465. Başka bir çalışma ise Adana İl Halk Kütüphanesi [01, Hk
115/2] koleksiyonuna dayanarak Risâle fî ta‘rîfi ilmi’l-kelâm eserini Hüsâm Çelebi’ye izafe etmiştir. 23 Hatibzâde’nin bilgi tanımına yönelik eleştirilerini birden ona kadar sıralı bir şekilde analiz eden bir çalışma
yapılmıştır. Ayrıca ilgili yazma risâlenin nüshaları da belirtilmiştir. Mebrure Hanife Özbakir,
“Hatîbzâde’nin el-Mevâkıf’ta Tercih Edilen Bilgi Tanımına Yönelik Eleştirileri”, Kilitbahir 18 (Mart 2021), 89. 1. Eleştirisi Yapılan Bilgi Tanımı 27 Hüsâm Çelebi’nin sıkı bir Hatibzâde eleştirmeni olduğuna dair güçlü işaretler vardır. Onun Hâşiye ala
şerhi’t-Tecrîd’inin derkenâr notlarına bakılırsa pek çok yerde Hatibzâde’yi hedef aldığı görülecektir. Hüsâmeddin Hüseyin b. Abdurrahman Hüsâm Çelebi, Hâşiye ala şerhi’t-Tecrîd (İstanbul: Süleymaniye 27 Hüsâm Çelebi’nin sıkı bir Hatibzâde eleştirmeni olduğuna dair güçlü işaretler vardır. Onun Hâşiye ala
şerhi’t-Tecrîd’inin derkenâr notlarına bakılırsa pek çok yerde Hatibzâde’yi hedef aldığı görülecektir. Hüsâmeddin Hüseyin b. Abdurrahman Hüsâm Çelebi, Hâşiye ala şerhi’t-Tecrîd (İstanbul: Süleymaniye Eskiyeni 46 (Mart/March 2022) 152 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi lan karşılaştırmalar neticesinde Çelebi’nin, Hatibzâde’nin risâlesini uzatma-
kısaltma yoluna gitmeden olduğu gibi verdiği tespit edilmiştir. Bu nedenle Çele-
bi’nin bulunan tek nüshasına dayalı bir biçimde araştırma yürütülecektir. Risâlede birden başlayarak ona kadar giden eleştiriler sıra ile verilmiştir. Bu ne-
denle takip kolaylığı sağlamak adına makalede bu sıra numaraları aynen korun-
muştur.28 28 Hüsâm Çelebi’nin iki risâlesi üzerine tahkik çalışması yapılmıştır. Kilisenin hukuki düzenlemesi
hakkında yapılan bir çalışma için bk. Levent Öztürk, “Hüsâm Çelebi’nin (öl. 926/1520) Risâle Ma’mûle li-
Beyâni Ahvâli’l-Kenâisi Şer‘an Adlı Eseri”, İslâm Araştırmaları Dergisi 5 (2001), 142. Sufilerin sosyal
yaşamdaki sürecini ilgilendiren başka bir çalışma için bk. Murat Polat, “Tahkîku risâle fî Raksi’l-
Mutasavvıfe li-Hüsâm Çelebî”, İslâm Araştırmaları Dergisi 41 (2019), 147. Kütüphanesi, Laleli, 2214), 1b. Ayrıca bk. Hüsâmeddin Hüseyin b. Abdurrahman Hüsâm Çelebi, Hâşiye ala
şerhi’t-Tecrîd (İstanbul: Süleymaniye Kütüphanesi, Şehit Ali Paşa, 2844), 125b. 29 Muhyiddin Mehmed Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (İstanbul: Süleymaniye Kütüphanesi, Ragıp Paşa,
1459), 188b. Beyâni Ahvâli’l-Kenâisi Şer‘an Adlı Eseri”, İslâm Araştırmaları Dergisi 5 (2001), 142. Sufilerin sosyal
yaşamdaki sürecini ilgilendiren başka bir çalışma için bk. Murat Polat, “Tahkîku risâle fî Raksi’l-
Mutasavvıfe li-Hüsâm Çelebî”, İslâm Araştırmaları Dergisi 41 (2019), 147.
29 Muhyiddin Mehmed Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (İstanbul: Süleymaniye Kütüphanesi, Ragıp Paşa,
1459), 188b. Kütüphanesi, Laleli, 2214), 1b. Ayrıca bk. Hüsâmeddin Hüseyin b. Abdurrahman Hüsâm Çelebi, Hâşiye ala
şerhi’t-Tecrîd (İstanbul: Süleymaniye Kütüphanesi, Şehit Ali Paşa, 2844), 125b.
28 Hüsâm Çelebi’nin iki risâlesi üzerine tahkik çalışması yapılmıştır. Kilisenin hukuki düzenlemesi
hakkında yapılan bir çalışma için bk. Levent Öztürk, “Hüsâm Çelebi’nin (öl. 926/1520) Risâle Ma’mûle li-
Beyâni Ahvâli’l-Kenâisi Şer‘an Adlı Eseri”, İslâm Araştırmaları Dergisi 5 (2001), 142. Sufilerin sosyal
yaşamdaki sürecini ilgilendiren başka bir çalışma için bk. Murat Polat, “Tahkîku risâle fî Raksi’l-
Mutasavvıfe li-Hüsâm Çelebî”, İslâm Araştırmaları Dergisi 41 (2019), 147.
29 Muhyiddin Mehmed Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (İstanbul: Süleymaniye Kütüphanesi, Ragıp Paşa,
1459), 188b. 30 Fahreddin er-Râzî, nazar faaliyetinin bilgiyi içerdiğini ve ikisini birbirinden tamamen ayrılmadığı
bildirmiştir. Genel kabule göre ilişkisel (âdet) nedenden ötürü düşüncenin ardından bilgi elde
edilmektedir. Ancak bunu ifade edenler aslında doğru düşüncenin kendi içinde bilgiyi tazammun
ettiğini açıklamıştır. “Tazammun”un anlamı; nazarî bilginin nazara bağlanması, nazarın da zarûrî
bilgiler dolayımında dolaşım olmasıdır. Fahreddin er-Râzî, Nihâyetü’l-‘ukûl fî dirâyeti’l-usûl, thk. Said
Fûde (Beyrut: Dârü’z-Zehâir, 2015), 1/188. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi Bir: Tanımın kapsam sorunudur. Şayet bilgi; anlamlar arasını, çelişiğine ihti-
mali bulunmayacak biçimde ayırt etmeyi zorunlu kılan bir sıfat ise burada duyu-
lara (mahsûsât) ilişkin tasavvurlar dışarıda bırakılmış, demektir. Oysa dil, din, ta-
rih ve gelenek gibi her bakımdan duyuya/nesneye ilişkin tasavvura bilgi denil-
mektedir. Mesela “Zeyd’i bildim” denir. Zaten ihsası bilgi kabul etmeyenler bile
duyulanlara ilişkin bilinen tasavvurları bilgi saymaktadırlar. Onlar için ihsas bilgi
değildir, ama mahsus bilgidir. Öyleyse ister gerçekliğin ve realitenin kendisine
(nefsü’l-emr) göre, isterse duyumsananlara ilişkin tasavvuru bilgi sayanlara göre
olsun sözü edilen tanım kapsaması gerekeni kapsamamıştır.29 İki: Hatibzâde’nin ikinci itirazı tanımın içlem sorunudur. Zira yapılan bilgi ta-
nımı, muhtevasına girmemesi gerekenleri içine almıştır. Hatibzâde’ye göre Îcî ve
Cürcânî’nin kelâm-felsefe ve mantık gibi farklı kitaplarına bakıldığında bilgi tanı-
mında kullanılan temyiz; “kendisi ile inkişaf olunan” anlamına gelmektedir. Kendisi
ile inkişaf olunan bilgi; tasdik bakımından ayıklama (ika) ve soyutlama (intizâ‘) faa-
liyetidir, tasavvur bakımından ise mutabık surettir. Şu hâlde bilgi tanımı içine al-
maması gereken unsurları içine almış gözükmektedir. Hatibzâde burada hem zihin
hem duyum açısından örnek vermektedir. Duyulardan başlanılacak olursa; kişi göz
organını, görülebilen bir nesneye doğru yönelttiğinde, bunun dışında başka bir şey
kullanmasına gerek kalmaksızın görünenin suretini/görüntüsünü o kişi zorunlu
olarak elde etmektedir. Sözü edilen suretin elde edilmesi göz kullanımının zorunlu
kılmasından başka bir aracıya bağlı olmaksızın gerçekleşmektedir. Aynı şeyler baş-
ka duyu organları için de geçerlilik arz etmektedir. Duyumdan teorik zihin düzeyine gelinirse kesin olarak işleyen bir süreç var-
dır. Doğru düşünmenin veya sahih nazarın kendisi bilgiyi zorunlu kılmaktadır. Mesela “evren sonludur” önermesi anlaşıldığı takdirde zorunlu bilginin ortaya www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 153 153 çıkması için başka bir düşünme safhasına gerek yoktur. Benzer bir durum “bir
ikinin yarısıdır”, önermesine yönelen bir zihin için de geçerlidir. Görüldüğü üze-
re bilginin sağladığı temyizi, doğru düşünme de sağlamaktadır. Yani bilgi, doğru
düşünceden ayrıştırılmamaktadır.30 Ancak bilginin, doğru düşünmekten başka
olduğu söylenmektedir. Hatibzâde’ye göre bilgi tanımı, farklı oldukları halde
kapsamaması gerekenleri içine almıştır. Bunlar tasdik bakımından doğru dü-
şünmek, tasavvur bakımından ise duyu algılarının suretleridir. Tanım bu ikisini
kapsadığı için sorunludur. Çünkü bilgi aracısız temyiz sağlamakta, doğru düşün-
ce veya duyu organını kullanmak da tek başına aracısız temyizi sağlamaktadır. 31 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 188b. 32 Seyyid Şerif Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, thk. Muhammed Hasan İsmail (Beyrut: Dârü’l-
Kütübi’l-‘İlmiyye, 2004), 1/187. 33 Cürcânî, Şerhu’l-mevâkıf, 1/543. 34 Hatibzâde burada İbn Mübârekşâh (öl. 784/1382) ile aynı düşünmektedir. Bkz. Murat Kaş, “Zihnî
Varlık Tartışmalarının Klasik Sonrası Dönemde Alımlanışı: Bilginin Mahiyeti ve Kategorisi
Bağlamında Bir İnceleme”, Nazariyat: İslâm Felsefe ve Bilim Tarihi Araştırmaları Dergisi 4/3 (2018), 55.
Ayrıca Ömer Türker’in özellikle 75. dipnotta vurguladığı üzere Fahreddin er-Râzî’nin husul görüşü
ile zihnî varlık görüşüne yönelttiği eleştirilerin benzer olması, Hatibzâde’nin husul ile zihnî varlığın
ayrılmayacağı yönündeki tespitini doğrular niteliktedir. Türker, İbn Sînâ Felsefesinde Metafizik Bilginin
İmkânı Sorunu, 66. 31 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 188b. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi Her temyiz bir bilgi olduğuna göre diğerleri de bilgi tanımında yer almaması ge-
rektiği halde almış demektir.31 Üç: Hatibzâde, Cürcânî’nin Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ’sındaki ifade-
sinden güç alarak bilgi tanımında istihdam edilen temyizin tasavvur bakımından
mutabık suret olduğunu belirtmiştir.32 Fakat temyize suret/form anlamını ver-
mek, Hatibzâde’ye göre bilgi tanımını sorunlu hale getirmektedir. Tanım sahip-
lerinin ekolüne göre zihnî varlık teorisi yadsınmaktadır.33 Zihnî varlık teorisi
bağlamında bir anlamı olan suretin zihinde hâsıl olmasının, kendi başına bir an-
lamı kalmayacaktır. Hatibzâde’ye göre zihnî varlığı kabul etmeyip yalnızca sure-
tin husulünü kabul etmek yanlıştır.34 Çünkü teorinin tamamını dışlayıp içindeki
tek bir öğeyi cımbızlayarak çekip çıkarmak, bağlam içinde anlamı olan unsuru
anlamsız bırakır.35 Şu hâlde Hatibzâde için tanımın ilâhî ilmi kapsamama sorunu
çıkmaktadır. Şayet bilgi, suretin temyizi ise temyizin artmasıyla kendinden sıralı
sonsuz suretlerin hâsıl olması gerekmektedir. Fakat bu, yanlışlığı üzerinde uzla- 34 Hatibzâde burada İbn Mübârekşâh (öl. 784/1382) ile aynı düşünmektedir. Bkz. Murat Kaş, “Zihnî
Varlık Tartışmalarının Klasik Sonrası Dönemde Alımlanışı: Bilginin Mahiyeti ve Kategorisi
Bağlamında Bir İnceleme”, Nazariyat: İslâm Felsefe ve Bilim Tarihi Araştırmaları Dergisi 4/3 (2018), 55. Ayrıca Ömer Türker’in özellikle 75. dipnotta vurguladığı üzere Fahreddin er-Râzî’nin husul görüşü
ile zihnî varlık görüşüne yönelttiği eleştirilerin benzer olması, Hatibzâde’nin husul ile zihnî varlığın
ayrılmayacağı yönündeki tespitini doğrular niteliktedir. Türker, İbn Sînâ Felsefesinde Metafizik Bilginin
İmkânı Sorunu, 66. 34 Hatibzâde burada İbn Mübârekşâh (öl. 784/1382) ile aynı düşünmektedir. Bkz. Murat Kaş, “Zihnî
Varlık Tartışmalarının Klasik Sonrası Dönemde Alımlanışı: Bilginin Mahiyeti ve Kategorisi
Bağlamında Bir İnceleme”, Nazariyat: İslâm Felsefe ve Bilim Tarihi Araştırmaları Dergisi 4/3 (2018), 55. Ayrıca Ömer Türker’in özellikle 75. dipnotta vurguladığı üzere Fahreddin er-Râzî’nin husul görüşü
ile zihnî varlık görüşüne yönelttiği eleştirilerin benzer olması, Hatibzâde’nin husul ile zihnî varlığın
ayrılmayacağı yönündeki tespitini doğrular niteliktedir. Türker, İbn Sînâ Felsefesinde Metafizik Bilginin
İmkânı Sorunu, 66. 35 Kelâmî sistemi temsil eden Fahreddin er-Râzî kimi kitaplarında zihni varlığı eleştirmiş, kimilerinde
ise kabul etmiştir. Şaban Haklı, “Fahreddin er-Râzî’nin Bilgi Teorisi”, İslâm Düşüncesinin Dönüşüm
Çağında Fahreddin er-Râzî, ed. Ömer Türker - Osman Demir (İstanbul: İslam Araştırmaları Merkezi
Yayınları, 2013), 439. Buradan Râzî’nin tadil edilmiş bir zihni varlık teorisine ulaşmaya çalıştığı
çıkarılabilir. Eskiyeni 46 (Mart/March 2022) 154 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi şılmış teselsüle yol açacaktır. Hakikaten ilâhî ilimde temyiz edilmiş suret var ise
sonsuz ilim, sonsuz suretlerin varlığını gerektirecektir.36 Dört: Hatibzâde tanımda kullanılan kelimelerin hakiki anlamının dışına çıka-
rılmasına karşı çıkmaktadır. Temyiz kelimesi buna bir örnektir. 36 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 188b.
37 Bu eleştiriyi dile getirerek eleştiren bir çalışma için bk. Muhammed Ebu’l-Fadl er-Revvâkî Cîzâvî, Ḥâşiye
ʿalâ şerḥi muḫtaṣari’l-müntehâ (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2004), 1/196.
38 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 188b.
39 Cürcânî, Şerhu’l-mevâkıf, 1/171.
40 Sonlu hâdis varlıklar arasındaki sebep-sonuç ilişkisinin daimî ve çoğunluk olması nedeniyle fizik
dünyada âdete dayalı bir irtibat söz konusudur. Her şeyi fâil-i muhtar Tanrı’ya bağlamak adına âdet
kuramı savunulmaktadır. Cîzâvî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/145. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi Temyizin ilk an-
lamı izah ve inkişaftır, söylendiği gibi “kendisi ile inkişaf olunan” değildir. Tanım
sahipleri ibarede bunu gerektirecek bir karine olmadığı halde temyiz kelimesini,
hakiki anlamdan çıkararak mecazi anlama getirmiştir. Peki bu hamleyi neden
yapmışlardır? Çünkü temyiz, izah ve inkişaf anlamında ise bilgi tasavvur ve tas-
dik ile özdeş hale geleceği, bilgi o ikisinden ayrıştırılamayacağı için geçersiz ola-
caktır. Hatibzâde’ye göre doğrusu da budur. Ancak tanım sahipleri temyize;
“kendisi ile inkişaf olunan” anlamını vermiş, böylece temyiz tasavvurda sureti,
tasdikte ise ayıklama (ikā‘) ve soyutlamayı (intizâ‘) gerektiren bir sıfat olmuştur.37
Hatibzâde’ye göre bu bir zorlamadır ve ibare bunu desteklememektedir.38 Beş: Bilgi tanımında geçen “zorunlu kılan” ifadesini Cürcânî, “âdeten” zorun-
lu kılan diyerek daraltmıştır.39 Hatibzâde’ye göre zorunluluğun “âdet” ile daral-
tılması, tanımın ilâhî ilmi dışarıda bırakmasına yol açmıştır. Tüm mükemmellik-
lerin kendisinde bilfiil zorunlu halde bulunduğu Tanrı’nın ilmi, tanımdaki da-
raltma sonucu kapsam dışına çıkarılmıştır. Bilindiği üzere belirli bir ihtimal ve
potansiyel içeren “âdet”in Tanrı’da kesinlikle olmaması gerekmektedir. Dahası
Hatibzâde için Cürcânî’nin Şerhu’l-mevâkıf’ta başvurduğu daraltma hamlesi an-
lamsızdır. Çünkü bu (Şerhu’l-mevâkıf) kitapta temyize, izah ve inkişaf anlamı ve-
rilmiştir. Her ekole göre bilgi; bilinenin inkişaf etmesini hakiki olarak zorunlu kılmak-
tadır. Akıl sahibi hiç kimse bilineninin inkişaf etmediği bir bilginin elde edilme-
sini doğru bulmaz. Yani bilgi varsa kesinlikle bir bilinen olmalıdır, bilineni olma-
yan bir bilgi olmaz. Öyleyse bilgi, hakiki inkişaf halinden başka bir şey değildir. Bu hakikati “âdet” seviyesine düşürmek, bilgiyi inkişaf halinden ayrı başka bir
şeye dayandırmak anlamı taşır. Ancak bu son tümce, inkişafsız bir bilginin elde
edilmesine imkân verir ki bu yanlıştır. Öte yandan Hatibzâde, Eş‘arî öğretinin “her şey aracısız Allah’a dayanır”40 şek-
lindeki genel fizik anlayışını belirleyici ilkesine açıklık getirmektedir. Buna gö-
re ilke hakiki olsun, itibari olsun tüm mümkün varlıkları kapsamaz. İlkenin
kapsadığı mevcut mümkün varlıklardır, yoksa varlıklardan ayrışmayan imkân, www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 155 155 imtina gibi itibari varlıklar değildir. Dolayısıyla itibari varlık sayılan temyizin
bilgiye istinat etmesi, “her şey aracısız Allah’a dayanır” şeklindeki genel ilkeye
aykırı düşmez. Hatibzâde bu açıklamalarını gerekçe göstererek tanımda geçen
“zorunlu” ifadesinin açık ve hakiki anlamından “âdeten” anlamına çekilmesine
gerek duyulmadığını söylemektedir. Bilgi itibari varlık olduğu için buradaki
zorunluluğu “âdet”e indirgemeye gerek yoktur. Ayrıca o, temyizin tasavvurda
suret, tasdikte ayıklama (ikā‘) ve soyutlama (intizâ‘) anlamına getirilmesini uy-
gun görmez. 41 Cürcânî için tasdikteki temyiz, ispat ve nefydir. Fakat buna göre ispat ve nefiy bilgi değildir, aksine bilgi
o ikisini zorunlu kılandır. Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/188.
42 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a.
43 Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/187.
44 Îcî, Şerḥi muḫtaṣari’l-müntehâ, 1/184. “Tasavvurların/kavramların çelişikleri yoktur” cümlesini anlamak,
Cürcânî’nin yorumunu anlamaya bağlıdır. Çelişki; öz bakımından iki kavramın birbirini
engellenmesidir. Fakat tasavvurların birbirini arasında hiçbir engelleme olmaz. Mesela “insan” ile
“insan değil” kavramını ele alalım. Bu ikisi, kavram bakımından birbiri ile çelişmez, sadece var olan bir
referans dikkate alınırsa çelişki meydana gelebilir. Çünkü ikinci halde kavram, önermeye dönüşür ve
doğru-yanlışa konu olabilir. “Düşünen canlıdır” ile “düşünen canlı değildir” yargıları, salt yargı olarak
anlaşılmaları bakımından çelişkili olmaz, ta ki var olan dış bir referans dikkat alınırsa olur. Örneğin
“vur” veya “vurma” emirleri de emir olmak bakımından çelişkili değildir, ancak harici bir referans
dikkate alınırsa çelişki olur. Sonuç itibariyle tasavvurlar; tasavvur olması bakımından çelişik veya
çelişkili olmazlar. Sözgelimi insanın mahiyeti tasavvur edilirse, zihinde o mahiyete mutabık bir suret
canlanır. İşte burada temyiz bu suretin temyizidir. Çünkü, ancak o suretle temyiz gerçekleşir ve insanın
mahiyeti kişide inkişaf edebilir. Temyizin iliştiği yer sözü edilen bu mahiyetin kendisidir. Bu temyizin
çelişiğe ihtimali yoktur. Zaten temyizin çelişiği olmaz. Tüm bu anlatılanlardan hareket eden Cürcânî’ye
göre “insanı bildim” cümlesindeki bilgi; suret değil, sureti zorunlu kılan sıfat olmaktadır. Cürcânî,
Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/187. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi Çünkü “hakiki olarak inkişafı zorunlu kılmak” ile “hakiki olarak
kendisi ile inkişaf olunanı zorunlu kılmak” arasında fark yoktur. İnkişafı zorun-
lu kılan, kendisi ile inkişaf olunanı da zorunlu kılmaktadır. Burada ayrım gö-
zetmek nitelik kategorisindeki suret ve ikanın da zorunlu kılmak açısından bil-
gi ile özdeşleşmesini gerektirecektir.41 Oysa zorunlu kılan bilgi sıfatıdır, suret
ve ika değildir.42 Altı: Hatibzâde, bilgi tanımına Îcî ve Cürcânî’nin müdahalelerini titizlikle ta-
kip etmiştir. O ikisinin açık ifadelerine göre tanımda zikredilen “çelişki”; sıfat ve
ilişkide (taalluk) değil, temyizde olmalıdır.43 Yine o ikisi tasavvurların tamamının,
bilgi tanımında yer almasını, tasavvurların çelişiklerinin olmamasına bağlamak-
tadır.44 Hatibzâde’nin çıkarımına göre tasavvurun çelişiği yoksa suretin de çelişi-
ği olmaması gerekir. Çünkü biri diğerine bağlıdır. Ancak bu şekilde tasavvurdaki,
sureti temyizin ve tasdikteki, ispat-nefyi temyizin çelişiği olmaz.45 Tam bu nok-
tada Hatibzâde bilgi tanımına tasavvurların dâhil ediliş gerekçesine karşı çık-
maktadır. Çünkü tasavvur, çelişiği olmadığı için46 tanıma giriyorsa bu gerekçe, 41 Cürcânî için tasdikteki temyiz, ispat ve nefydir. Fakat buna göre ispat ve nefiy bilgi değildir, aksine bilgi
o ikisini zorunlu kılandır. Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/188. 42 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a. 43 Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/187. 44 Îcî, Şerḥi muḫtaṣari’l-müntehâ, 1/184. “Tasavvurların/kavramların çelişikleri yoktur” cümlesini anlamak,
Cürcânî’nin yorumunu anlamaya bağlıdır. Çelişki; öz bakımından iki kavramın birbirini
engellenmesidir. Fakat tasavvurların birbirini arasında hiçbir engelleme olmaz. Mesela “insan” ile
“insan değil” kavramını ele alalım. Bu ikisi, kavram bakımından birbiri ile çelişmez, sadece var olan bir
referans dikkate alınırsa çelişki meydana gelebilir. Çünkü ikinci halde kavram, önermeye dönüşür ve
doğru-yanlışa konu olabilir. “Düşünen canlıdır” ile “düşünen canlı değildir” yargıları, salt yargı olarak
anlaşılmaları bakımından çelişkili olmaz, ta ki var olan dış bir referans dikkat alınırsa olur. Örneğin
“vur” veya “vurma” emirleri de emir olmak bakımından çelişkili değildir, ancak harici bir referans
dikkate alınırsa çelişki olur. Sonuç itibariyle tasavvurlar; tasavvur olması bakımından çelişik veya
çelişkili olmazlar. Sözgelimi insanın mahiyeti tasavvur edilirse, zihinde o mahiyete mutabık bir suret
canlanır. İşte burada temyiz bu suretin temyizidir. Çünkü, ancak o suretle temyiz gerçekleşir ve insanın
mahiyeti kişide inkişaf edebilir. Temyizin iliştiği yer sözü edilen bu mahiyetin kendisidir. Bu temyizin
çelişiğe ihtimali yoktur. Zaten temyizin çelişiği olmaz. Tüm bu anlatılanlardan hareket eden Cürcânî’ye
göre “insanı bildim” cümlesindeki bilgi; suret değil, sureti zorunlu kılan sıfat olmaktadır. Cürcânî,
Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/187. 45 Cîzâvî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/197. 45 Cîzâvî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/197. Eskiyeni 46 (Mart/March 2022) 41 Cürcânî için tasdikteki temyiz, ispat ve nefydir. Fakat buna göre ispat ve nefiy bilgi değildir, aksine bilgi
o ikisini zorunlu kılandır. Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/188. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi 46 Cürcânî tasavvurun çelişiği ve mutabakatı olmadığına dair bir örnek vermektedir. Diyelim ki kişi
uzaktan bir silüet/karaltı/gölge görsün ve bu “at” olsun ama kişinin zihninde de “at” yerine “insan”
görüntüsü belirsin. Beliren insan görüntüsü ve o görüntünün idrak edilmesi bir surettir. Bu açıdan 46 Cürcânî tasavvurun çelişiği ve mutabakatı olmadığına dair bir örnek vermektedir. Diyelim ki kişi
uzaktan bir silüet/karaltı/gölge görsün ve bu “at” olsun ama kişinin zihninde de “at” yerine “insan”
görüntüsü belirsin. Beliren insan görüntüsü ve o görüntünün idrak edilmesi bir surettir. Bu açıdan Eskiyeni 46 (Mart/March 2022) 156 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi diğer çelişikleri olmayan şüphe (şek) ve vehim için de geçerlidir. Böylece tanıma
tasavvurlarla beraber şüphe ve vehim de dâhil edilmiştir. Her nasıl tasavvurun
çelişiği yoksa47 vehmin de yoktur. Kaldı ki şüphe ve vehim de bir tür tasavvur-
dur. Son tahlilde bilgi tanımının içine şüphe ve vehim girmiştir. Hâlbuki şüphe
etmek veya vehmetmek, bilmek demek değildir.48 Öyleyse Hatibzâde’ye göre bil-
gi tanımına tasavvurların dâhil ediliş gerekçesi yanlıştır. Yedi: Hatibzâde altıncı maddede çelişiği olmadığı gerekçesi ile “şüphe”nin
bilgi tanımına girmesi gerektiğini vurgulamıştı. Şüphe bir tasavvur türü idi. Hatibzâde’ye göre tüm tasavvurları kapsadığı iddia edilen bilgi tanımının şüphe-
yi dışarıda bırakması anlamsızdı. Nitekim bilgiyi “akıldaki form” olarak tanımla-
yan felsefe sistemi için şüphenin bilgi tanımına girmesinde bir sorun görülme-
mişti. Bu nedenle her tasavvuru kapsadığı ileri sürülen bilgi tanımı şüpheyi dışa-
rıda bırakamaz idi. Hatırlanacağı üzere tasavvurların bilgi tanımına giriş gerek-
çesi onların tamamının çelişiklerinin olmamasına bağlanmıştı. Bu gerekçe
Hatibzâde açısından, çelişiği olmayan şüphenin veya vehmin, bir tasavvur türü
olması bakımından tanım dışına atılmamasını gerektirmektedir.49 Sekiz: “Âlem hâdistir” önermesinden elde edilen bilginin elde ediliş sürecine
yakından bakıldığında burada bir, “konu ve yükleme” ilişkin tasavvur, iki, konu ve
yüklemin irtibatını tesis eden nispetin ispatı/olumlanması vardır. Hatibzâde bura-
daki ispatın bilgi sayılmamasını eleştirmektedir. Çünkü ispat/olumlama bilgi değil-
se bu, bilginin tasavvurlarla sınırlandırılmasını gerektirmektedir. Ancak bu tasav-
vurların tasdiklerden hakiki olarak başka olduğu gerçeğine aykırı bir sonuçtur.50
Bilindiği üzere tasavvur tek başına tasdiki/ispatı zorunlu kılmamaktadır. suret yanlış değildir, yanlışa ihtimali de yoktur. Ancak yanlış aklın verdiği hükümdedir, görüntüde
değildir. Dolayısıyla görüntü görüntü olması bakımından görüntüye mutabıktır, ama mutabık olmayan
aklın verdiği yargıdır. Cürcânî, Ḥâşiye ʿalâ şerḥ muḫtaṣari’l-müntehâ, 1/187. ğ y
g
, Ḥş y
ş ḥ
ḫṣ
, /
47 Îcî ve Cürcânî’ye göre tasavvurun mutabakat ve çelişiği olmaz. Çünkü mutabakat ve çelişiklik, terkip ve
hüküm/yargı için geçerlidir. 47 Îcî ve Cürcânî’ye göre tasavvurun mutabakat ve çelişiği olmaz. Çünkü mutabakat ve çelişiklik, terkip ve
hüküm/yargı için geçerlidir. Tasavvurda hüküm şaibesi yoksa çelişiğe ihtimalin anlamı da yoktur. Fakat
Teftâzânî “tasavvurların çelişikleri yoktur” ilkesinin elde ediliş sürecine ve sonucuna mesafelidir. Ömer
Mahir Alper bunu çalışmasının özellikle 91 dipnotunda belirtmiştir. Ömer Mahir Alper, “‘Eş’arîlerin
Kavâidi ve Felâsifesinin Gavâili’ Ötesinde: Osmanlı Dönemi Mâtürîdî Kelâmcısı Mevlânâ Mehmet İzmîrî’de
Bilginin Mâhiyeti Sorunu”, İmam Mâtürîdî ve Mâtürîdiyye Geleneği: Tarih, Yöntem, Doktrin Prof. Dr. Bekir
Topaloğlu Anısına, ed. Hülya Alper (İstanbul: Marmara İlâhiyat Fakültesi Vakfı Yayınları, 2018), 285.
Teftâzânî’ye göre tasdikin mutabakatla eşitlenmesi vehimdir. Mutabakat gibi bir kavramın kişide hâsıl
olması onun mutlaka vakıada tahakkuk etmesini gerektirmez. Sa‘düddîn Teftâzânî, Şerhu’l-makāsıd, thk.
İbrahim Şemseddin (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2011), 1/61. Ziya Erdinç Teftâzânî’de mutabakatın,
öznel ve nesnel olarak ikiye ayrıldığını belirtmiştir. Ziya Erdinç, Teftâzânî’de Bilgi Teorisi (Sakarya: Sakarya
Üniversitesi, Sosyal Bilimler Enstitüsü, Doktora Tezi, 2019), 88. Teftâzânî “tasavvurun çelişiği yoktur”
ilkesine bu ayrım neden ile katılmamış gözükmektedir. Zira öznel mutabakat kavramın zihindeki
örtüşmesi, nesnel mutabakat ise kavramın hariç/vakıa ile örtüşmesidir. Dolayısıyla kavramın zihin
bakımından bir çelişiğinden bahsetmek mümkündür. Müteahhir kelâm-felsefe ve mantık müktesebatı
dikkate alınarak mesele ileri araştırmalara muhtaç olduğu söylenmelidir.
48 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a.
49 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a.
50 Teftâzânî, Şerhu’l-makāsıd, 1/59. suret yanlış değildir, yanlışa ihtimali de yoktur. Ancak yanlış aklın verdiği hükümdedir, görüntüde
değildir. Dolayısıyla görüntü görüntü olması bakımından görüntüye mutabıktır, ama mutabık olmayan
aklın verdiği yargıdır. Cürcânî, Ḥâşiye ʿalâ şerḥ muḫtaṣari’l-müntehâ, 1/187. suret yanlış değildir, yanlışa ihtimali de yoktur. Ancak yanlış aklın verdiği hükümdedir, görüntüde
değildir. Dolayısıyla görüntü görüntü olması bakımından görüntüye mutabıktır, ama mutabık olmayan
aklın verdiği yargıdır. Cürcânî, Ḥâşiye ʿalâ şerḥ muḫtaṣari’l-müntehâ, 1/187. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi Tasavvurda hüküm şaibesi yoksa çelişiğe ihtimalin anlamı da yoktur. Fakat
Teftâzânî “tasavvurların çelişikleri yoktur” ilkesinin elde ediliş sürecine ve sonucuna mesafelidir. Ömer
Mahir Alper bunu çalışmasının özellikle 91 dipnotunda belirtmiştir. Ömer Mahir Alper, “‘Eş’arîlerin
Kavâidi ve Felâsifesinin Gavâili’ Ötesinde: Osmanlı Dönemi Mâtürîdî Kelâmcısı Mevlânâ Mehmet İzmîrî’de
Bilginin Mâhiyeti Sorunu”, İmam Mâtürîdî ve Mâtürîdiyye Geleneği: Tarih, Yöntem, Doktrin Prof. Dr. Bekir
Topaloğlu Anısına, ed. Hülya Alper (İstanbul: Marmara İlâhiyat Fakültesi Vakfı Yayınları, 2018), 285. Teftâzânî’ye göre tasdikin mutabakatla eşitlenmesi vehimdir. Mutabakat gibi bir kavramın kişide hâsıl
olması onun mutlaka vakıada tahakkuk etmesini gerektirmez. Sa‘düddîn Teftâzânî, Şerhu’l-makāsıd, thk. İbrahim Şemseddin (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2011), 1/61. Ziya Erdinç Teftâzânî’de mutabakatın,
öznel ve nesnel olarak ikiye ayrıldığını belirtmiştir. Ziya Erdinç, Teftâzânî’de Bilgi Teorisi (Sakarya: Sakarya
Üniversitesi, Sosyal Bilimler Enstitüsü, Doktora Tezi, 2019), 88. Teftâzânî “tasavvurun çelişiği yoktur”
ilkesine bu ayrım neden ile katılmamış gözükmektedir. Zira öznel mutabakat kavramın zihindeki
örtüşmesi, nesnel mutabakat ise kavramın hariç/vakıa ile örtüşmesidir. Dolayısıyla kavramın zihin
bakımından bir çelişiğinden bahsetmek mümkündür. Müteahhir kelâm-felsefe ve mantık müktesebatı
dikkate alınarak mesele ileri araştırmalara muhtaç olduğu söylenmelidir. 48 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a. 49 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a. 50 Teftâzânî, Şerhu’l-makāsıd, 1/59. ğ y
g
, Ḥş y
ş ḥ
ḫṣ
, /
47 Îcî ve Cürcânî’ye göre tasavvurun mutabakat ve çelişiği olmaz. Çünkü mutabakat ve çelişiklik, terkip ve
hüküm/yargı için geçerlidir. Tasavvurda hüküm şaibesi yoksa çelişiğe ihtimalin anlamı da yoktur. Fakat
Teftâzânî “tasavvurların çelişikleri yoktur” ilkesinin elde ediliş sürecine ve sonucuna mesafelidir. Ömer
Mahir Alper bunu çalışmasının özellikle 91 dipnotunda belirtmiştir. Ömer Mahir Alper, “‘Eş’arîlerin
Kavâidi ve Felâsifesinin Gavâili’ Ötesinde: Osmanlı Dönemi Mâtürîdî Kelâmcısı Mevlânâ Mehmet İzmîrî’de
Bilginin Mâhiyeti Sorunu”, İmam Mâtürîdî ve Mâtürîdiyye Geleneği: Tarih, Yöntem, Doktrin Prof. Dr. Bekir
Topaloğlu Anısına, ed. Hülya Alper (İstanbul: Marmara İlâhiyat Fakültesi Vakfı Yayınları, 2018), 285. Teftâzânî’ye göre tasdikin mutabakatla eşitlenmesi vehimdir. Mutabakat gibi bir kavramın kişide hâsıl
olması onun mutlaka vakıada tahakkuk etmesini gerektirmez. Sa‘düddîn Teftâzânî, Şerhu’l-makāsıd, thk. İbrahim Şemseddin (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2011), 1/61. Ziya Erdinç Teftâzânî’de mutabakatın,
öznel ve nesnel olarak ikiye ayrıldığını belirtmiştir. Ziya Erdinç, Teftâzânî’de Bilgi Teorisi (Sakarya: Sakarya
Üniversitesi, Sosyal Bilimler Enstitüsü, Doktora Tezi, 2019), 88. Teftâzânî “tasavvurun çelişiği yoktur”
ilkesine bu ayrım neden ile katılmamış gözükmektedir. Zira öznel mutabakat kavramın zihindeki
örtüşmesi, nesnel mutabakat ise kavramın hariç/vakıa ile örtüşmesidir. Dolayısıyla kavramın zihin
bakımından bir çelişiğinden bahsetmek mümkündür. Müteahhir kelâm-felsefe ve mantık müktesebatı
dikkate alınarak mesele ileri araştırmalara muhtaç olduğu söylenmelidir. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi 48 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a. 49 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a. 50 Teftâzânî, Şerhu’l-makāsıd, 1/59. ğ y
g
, Ḥş y
ş ḥ
ḫṣ
, /
47 Îcî ve Cürcânî’ye göre tasavvurun mutabakat ve çelişiği olmaz. Çünkü mutabakat ve çelişiklik, terkip ve
hüküm/yargı için geçerlidir. Tasavvurda hüküm şaibesi yoksa çelişiğe ihtimalin anlamı da yoktur. Fakat
Teftâzânî “tasavvurların çelişikleri yoktur” ilkesinin elde ediliş sürecine ve sonucuna mesafelidir. Ömer
Mahir Alper bunu çalışmasının özellikle 91 dipnotunda belirtmiştir. Ömer Mahir Alper, “‘Eş’arîlerin
Kavâidi ve Felâsifesinin Gavâili’ Ötesinde: Osmanlı Dönemi Mâtürîdî Kelâmcısı Mevlânâ Mehmet İzmîrî’de
Bilginin Mâhiyeti Sorunu”, İmam Mâtürîdî ve Mâtürîdiyye Geleneği: Tarih, Yöntem, Doktrin Prof. Dr. Bekir
Topaloğlu Anısına, ed. Hülya Alper (İstanbul: Marmara İlâhiyat Fakültesi Vakfı Yayınları, 2018), 285. Teftâzânî’ye göre tasdikin mutabakatla eşitlenmesi vehimdir. Mutabakat gibi bir kavramın kişide hâsıl
olması onun mutlaka vakıada tahakkuk etmesini gerektirmez. Sa‘düddîn Teftâzânî, Şerhu’l-makāsıd, thk. İbrahim Şemseddin (Beyrut: Dârü’l-Kütübi’l-İlmiyye, 2011), 1/61. Ziya Erdinç Teftâzânî’de mutabakatın,
öznel ve nesnel olarak ikiye ayrıldığını belirtmiştir. Ziya Erdinç, Teftâzânî’de Bilgi Teorisi (Sakarya: Sakarya
Üniversitesi, Sosyal Bilimler Enstitüsü, Doktora Tezi, 2019), 88. Teftâzânî “tasavvurun çelişiği yoktur”
ilkesine bu ayrım neden ile katılmamış gözükmektedir. Zira öznel mutabakat kavramın zihindeki
örtüşmesi, nesnel mutabakat ise kavramın hariç/vakıa ile örtüşmesidir. Dolayısıyla kavramın zihin
bakımından bir çelişiğinden bahsetmek mümkündür. Müteahhir kelâm-felsefe ve mantık müktesebatı
dikkate alınarak mesele ileri araştırmalara muhtaç olduğu söylenmelidir. 48 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a. 49 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189a. 50 Teftâzânî, Şerhu’l-makāsıd, 1/59. www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 157 157 Hatibzâde için bilgi tanımında yer alan çelişkinin olmaması gereken yerin be-
lirlenmesi de bir sorundur. Buna göre çelişki; temyiz, sıfat ve taalluk olmak üzere
üç bakımdan dikkate alınabilir. Cürcânî’nin belirlemesine göre çelişkinin sıfat ve
taallukta olması yanlıştır, çelişkinin olmaması gereken yer temyizin çelişkiye ih-
timali olmamasıdır.51 Hatibzâde burada Cürcânî’nin “bilgi temyizdir, çelişki tem-
yizde olmamalıdır” tespitine katılmaz. Çünkü temyizin kendisi sayılan bilginin
anlamı inkişaf ve izah olacaktır. Bilgi; izah ve inkişaf olduğunda ise çelişkinin is-
pat ve nefiyden öte yani inkişaf ve çelişiğinden öte bir şey olması gerekir. Hatibzâde bu sorunu aşmak için çelişkinin yerini sıfata kaydırılmasını teklif eder. Fakat bilgi tanımının açık dizilimi ve anlamı bunu desteklememektedir, ayrıca
çelişkinin sıfata götürülmesi bilginin içine şüphe ve vehmi alacaktır, zira o ikisi-
nin de çelişikleri yoktur. 51 Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/187.
52 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 189b.
53 Cürcânî, Ḥâşiye ʿalâ şerḥi muḫtaṣari’l-müntehâ, 1/190.
54 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 190a. 2. Hatibzâde’nin Bilgi Tanımı Eleştirisi Hatibzâde son seçenek olarak çelişkinin yerini taalluk
olarak önerir. İdrak edene kıyasla bilgi ile bilinen arası taallukun çelişme ihtimali
yoktur. 52 Dokuz: Hatibzâde bilginin değişime kapalı olduğunu gösteren gerekçeleri ir-
delemiştir. Cürcânî’ye göre bir bilgi kesinlik taşıdığı için şimdi değişmez, kanıta
dayandığı için gelecekte değişmez, mutabık olduğu için de nefsü’l-emr’de değiş-
mez.53 Burada bilginin değişmemesi, tersinin doğru olmasını geçersiz kılmak
içindir. Hatibzâde değişmemeyi kesinliğe veya kanıta (mûcib) bağlamayı yanlış
bulmaktadır. Çünkü bilmediğini bilmeyen (cehl-i mürekkep) bir kişinin bilgisi
içinde bulunduğu durum sırasında kesinlik taşır ve değişime kapalıdır. Ayrıca
bilgiyi zorunlu kılan kanıtın değeri de gelecekteki bilginin değişmeyeceğini ga-
ranti etmez. Zira kanıtın unutulma riski her daim olasıdır. Bilindiği üzere bur-
handan elde edilen her kesin bilgi gelecekteki bilgiyi elde etmek için yeter sebep
değildir. Kanıt tek başına bilgi için yeter sebep olsa idi, kesin bilgilerin unutul-
ması mümkün olmazdı. Teslim edileceği üzere insanın her türlü bilgisini unut-
ması olağandır. Sözgelimi duyu organımız aracılığıyla bir nesnenin varlığına
hükmedilir, ama bir süre sonra o şey ve aracı unutulabilir. Duyu organı tekrar
sağlığına kavuşur, nesne ile irtibat kurulur, sonunda verilmiş olan hüküm tekrar
verilebilir.54 On: Hatibzâde, Cürcânî’nin Şerhu’l-mevâkıf (i), Hâşiye-i şerhi’l-Adud (ii) ve
Hâşiye-i şerhi’t-Tecrîd (iii) üç eserini karşılaştırarak aralarındaki ince farklılıkları
tespit etmiştir. İlk kitap bilgi tanımının diğer tanımlardaki kusurlardan uzak ol-
duğunu belirtmiş ve şüphenin tanım dışında kaldığını vurgulamıştır. İkinci kitap
şüpheyi tasavvur içine almıştır. Üçüncü kitap ise şüphenin tasavvurdan öte baş-
ka bir şey olduğunu ima etmiştir. Bilindiği üzere şüphe tasavvur ise bilgi sayıl-
malıdır, oysa Cürcânî şüpheyi bilgi tanımının dışına çıkarmıştır. Hatibzâde, ikinci Eskiyeni 46 (Mart/March 2022) Eskiyeni 46 (Mart/March 2022) 158 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi (ii) kitaptaki ibarenin sorun çıkarmayacağını söylemiştir. Çünkü şüpheden elde
edilen suretin tasavvur sayılması, şüphenin kendisini bilgi kılmamaktadır. Hatibzâde’ye göre ilk (i) kitaptaki açıklama ekol ve tanım sahiplerinin değil,
Cürcânî’nin tek kaldığı bir yorumudur. Zira tanımdaki “zorunlu kılar” ibaresini
ekolün iç tutarlılığını korumak adına Cürcânî; “âdeten” zorunlu kılar, diyerek
kendince kayıt altına almıştır. Fakat bu hamle Hatibzâde’ye göre şüphenin tanım
dışına çıkarılması için yeterli olmamıştır.55 55 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 190a.
56 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 49a.
57 Teftâzânî her ne kadar duyu organları aracılığı ile de olsa tikelleri idrak edenin nefs olduğunu
filozofların aksine iddia etmektedir. Teftâzânî, Şerhu’l-makāsıd, 2/476.
58 Akledilen (makul) suretler tümel, duyumsanan (mahsus) suretler tikeldir. Cürcânî, Şerhu’l-mevâkıf, 1/545. 56 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 49a. 55 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 190a. 55 Hatibzâde, Risâle fî ta‘rîfi’l-‘ilm (Ragıp Paşa, 1459), 190a. 57 Teftâzânî her ne kadar duyu organları aracılığı ile de olsa tikelleri idrak edenin nefs olduğunu
filozofların aksine iddia etmektedir. Teftâzânî, Şerhu’l-makāsıd, 2/476. Hüsâm Çelebi, Hâşiye i def i itirâzât ı Hatîbzâde (Veliyüddin, 3244), 49 .
57 Teftâzânî her ne kadar duyu organları aracılığı ile de olsa tikelleri idrak edenin nefs olduğunu
filozofların aksine iddia etmektedir. Teftâzânî, Şerhu’l-makāsıd, 2/476.
58 Akledilen (makul) suretler tümel, duyumsanan (mahsus) suretler tikeldir. Cürcânî, Şerhu’l-mevâkıf, 1/545. filozofların aksine iddia etmektedir. Teftâzânî, Şerhu l-makāsıd, 2/476.
58 Akledilen (makul) suretler tümel, duyumsanan (mahsus) suretler tikeldir. Cürcânî, Şerhu’l-mevâkıf, 1/545. 3. Hüsâm Çelebi’nin Hatibzâde’ye Tenkitleri Bir: Hüsâm Çelebi Hatibzâde’nin her yönden haksız olduğunu söylemez. Çele-
bi’ye göre duyumsananlara ilişen tasavvurların bilgi sayılması için sözü edilen
nesnenin göz önünde bulunma (rüyet) ve bulunmama (gaybet) gibi iki durumu
ayrı ayrı değerlendirilmelidir. Nesnenin göz önünde bulunduğu durum ihsas
iken, nesnenin kendisi gözden kaybolduğunda onun suretini zihinde canlandır-
mak bilgidir. Bilindiği üzere sözü edilen bilginin tanımını yapanlara göre hârici
varlıktaki tikeli idrak bilgi değil, ihsastır. Dolayısıyla kendisi ile birebir karşıla-
şılmamış nesnenin idraki anlamdaki ihsasın bilgi dışında kalmasında bir sorun
yoktur. İhsasın ikiye ayrıldığını dikkate almadığı için Hatibzâde’nin itirazı yanlış-
tır. Çünkü ihsas iki türlüdür. Rüyet halindeki ihsas ile gaybet halindeki ihsas bir-
birinden farklıdır. İlki nesnenin kişi tarafından doğrudan idrak edilerek ihsas ha-
lini ifade ederken, nesnenin huzurdan kalkmasının ardından nesnenin ihsas
edilmesi, yeni bir idrake dönüştüğü için bilgiyi ifade etmektedir.56 Klasik İslam felsefesine göre tikeller duyu organları aracılığıyla duyumsanır,
tümeller ise akledilir.57 Akıl ve duyular birbiri yerine ikame edilmeyecek işlev
alanına sahiptir. Akıl tümelleri, duyular tikelleri idrak etmektedir. Akıl, duyu
aracılığı olmaksızın tikelleri bilmemektedir.58 Hüsâm Çelebi’nin ifadesine göre
kişinin nesne ile ilişkisi üç türlü gerçekleşmektedir. Nesne ile birebir karşılaşma
olmaksızın gözlemden önceki (i) rüyet öncesi hali, karşılaşma sırasındaki (ii) mu-
ayene hali, bir de karşılaşmadan sonraki nesnenin bulunmadığı (iii) gaybet hali-
dir. Birinci durumda akıl, nesnenin kendisi ile karşılaşmadan önce nesneyi tü-
mel açıdan bilir. Tümel açıdan bilmek, anlamları bilmektir ve tanım bunu kap-
samaktadır. İkinci durumda akıl, nesne ile karşılaşma sırasında iken bu ihsas
halidir ve bilgi değildir. Tanımın bu anlamdaki ihsası kapsamına gerek yoktur. Üçüncü durumda akıl, her ne kadar nesneyi tikel açıdan idrak ediyor desek bu
idrak türü bir bilgidir ve tanım bunu da kapsamaktadır. Nitekim üçüncü aşa-
madaki tikelin durumu hârici varlık kabilinden değildir, tam da düşünen bi- www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 159 159 reyde bulunan muhayyel anlam kabilinden bir varlık türüdür. Dolayısıyla ihsa-
sın bilgi olmasına karşı çıkan ekole göre tasavvur edilen, duyumsanan mahsu-
sun bilgi olması bu anlamdadır. İhsas, harici varlıkla ilgili, mahsus ise anlam-
larla ilgilidir. Öyleyse bilgi tanımında geçen ayırt etmenin, hariçteki tikellerin
ihsasını kapsamamasında sorun yoktur. Hariçteki tikeller, onlarla muayeneden
önce ve sonra bir şekilde zihne aktarıldığında bilgiye dönüşmektedir. Tanımın
bu kadarını kapsaması yeterlidir. Sonuç itibariyle Hüsâm Çelebi’ye göre Hatib-
zâde’nin kapsam itirazı, sözü edilen üçlü aşamayı dikkate almadığı için geçer-
sizdir.59 İki: Hüsâm Çelebi’ye göre Hatibzâde temyizi sağlayan “aracısız gerçekleş-
me”yi yanlış anlamlandırmıştır. 59 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 49b.
60 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 49b.
61 Cürcânî’ye göre bilginin tanımı ile zihni varlık irtibatı ayrılmaz değildir. Kaş, “Zihnî Varlık
Tartışmalarının Klasik Sonrası Dönemde Alımlanışı”, 55.
62 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 50a. 3. Hüsâm Çelebi’nin Hatibzâde’ye Tenkitleri Bilgi veren iki tür aracısız gerçekleşme vardır. Sözü edilenlerin tasavvuru zorunlu kılması tamamen aracısız (îcâb bi’z-zât) değil,
kısmen (îcâbü’l-evvelî) aracısızdır. Bilginin; tasdik bakımından ayıklama (ikā‘) ve
soyutlama (intizâ‘), tasavvur bakımından suret olması kısmen aracısız gerçekle-
şenler arasındadır. Dolayısıyla doğru düşünce, aklın yönelimi vb. işlemlerin her
biri aslında tamamen aracısız gerçekleşerek bilgi anlamında suret, ika vermezler. Netice itibariyle Hüsâm Çelebi’ye göre Hatibzâde’nin tanım için ileri sürdüğü iç-
lem sorunu yoktur.60 Çünkü bilginin sıfatı zorunlu kılması ile doğru düşüncenin
bilgiyi zorunlu kılması birbirinden farklıdır. Üç: Hüsâm Çelebi’ye göre Hatibzâde’nin zihnî varlık ile formun husulünü ay-
rılmaz kılması yanlıştır. Çünkü Çelebi’nin bildirimine bağlı kalınarak söylenirse,
müteahhir kelâmcılar zihnî varlık görüşünü kabul etmedikleri halde formun hu-
sulünü benimsemişlerdir. Dolayısıyla iki görüş birbirinden ayrılmaz bir yapıda
değildir.61 Nitekim Çelebi’nin tespitine göre suretin husulünü doğrulayanlardan
kimileri, mahiyetin kendisinin nefste bulunmadığını bildirmiştir. Nefste bulunan
mahiyetlerin kendisi değil, belki suretleri, gölgeleri ve similasyonlarıdır. Bunlar
da mahiyetin kendisi değildir. Açıklanan anlamda suretin zihinde elde edilmesini
kabul edenin, yorumlanarak tadil edilmesi dışında zihnî varlık kuramını olduğu
haliyle benimsemesine gerek yoktur. Hüsâm Çelebi, husul ile zihnî varlık arası ir-
tibatın anlatılan anlamda kurulmasını Cürcânî’nin yorumlarına borçlu olduğunu
söylemiştir.62 Bilgi tanımı çerçevesinde, Hatibzâde’nin ilâhî ilmi kapsamadığına yönelik
eleştirisi Çelebi tarafından teslim edilmiştir. Buna göre gerçekten yapılan tanım
ilâhî ilmi içine almamaktadır ve bu tanım için bir sorundur. Fakat Çelebi’nin
Hatibzâde’ye katılmadığı husus onun teselsül ile ilgili ifadesidir. Çünkü yanlışlığı
hakkında uzlaşı sağlanan teselsül, hârici varlık bulanlar arası teselsüldür. Yoksa Eskiyeni 46 (Mart/March 2022) 160 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi izafet/nispet ve taalluklardaki teselsül yanlış değildir. Hatibzâde’nin bilgiyi sure-
te bağlayarak ilâhî ilimde teselsül olur, kanaati yanlıştır. Çünkü bilginin izafet
olması dikkate alınırsa ilâhî ilimdeki soyut/itibarî teselsülün, realitede karşı çıkı-
lan somut/hakiki teselsülden farklı olduğu anlaşılacaktır. Dört: Hüsâm Çelebi’ye göre Hatibzâde temyiz kelimesinin türetim yapısına
dikkat etmediği için kelimenin hakiki-mecazi anlamlarını birbirine karıştırmış-
tır. Temyiz kelimesi nesne alarak geçişli (tefîl/temyîz) veya nesne almadığı için
geçişsiz (tefa‘ul/temeyyüz) bir biçimde kullanılabilmektedir. Buna göre bilgi tanı-
mında zikredilen “temyiz” nesne alarak geçişli bir formda kullanılmıştır.63 Dola-
yısıyla Çelebi’ye göre temyiz geçişli formda kullanıldığı için ilk ve hakiki anlamı;
“kendisi ile inkişaf olunan” demek olmalıdır.64 Beş: Hüsâm Çelebi, Hatibzâde’nin potansiyellik barındırdığı için ilâhî ilme ya-
kıştırmadığı “âdet” fikrinde bir sakınca görmemektedir. Çünkü Çelebi’ye göre
âdet; olabilir (imkân) ve oldu-oluyor (vukû) şeklinde ikiye ayrılmaktadır. İmkân
düzeyinde âdetin potansiyellik barındırması bilfiil potansiyelin vaki olduğunu
göstermemektedir. 63 Tanımda geçen temyiz kelimesi kavram bakımından suretin kendisi değil, çelişiği olmayan kavram
bakımından suretten elde edilen inkişaf, yargı bakımından ise olumlu ve olumsuz olabilen inkişaf
anlamındadır. Hasan Çelebi’de temyizin inkişafın kendisi olarak anlaşılmasına karşı çıkmaktadır.
Muhammed Şah Fenârî Hasan Çelebi, Hâşiye ‘ala Şerḥi’l-Mevâḳıf (Şerḥu’l-Mevâḳıf içinde) (Beyrut: Dârü’l-
Kütübi’l-‘İlmiyye, 2012), 1/92. yy
64 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 50a. y
f
y
65 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 51a. 3. Hüsâm Çelebi’nin Hatibzâde’ye Tenkitleri Eksiklik olarak görülen potansiyelin vukua gelmeden salt
imkân düzeyinde kalması Çelebi’ye göre bir eksiklik sayılmamalıdır. Âdeten bağ-
lılık eksikliğin imkânını gösterse bile vaki olduğunu göstermez. Dolayısıyla nes-
neler arası bağı temin eden zorunluluk (îcâb) ifadesinin “âdet” ile kayıt altına
alınması Tanrı’da bir eksikliğin vaki olduğunu göstermemektedir. Çelebi âdet
fikrinin, Tanrı’nın zatı ile değil, ilim sıfatı ile ilgili olduğunu düşünmektedir.65
Tüm bu açıklamalara binaen bilgi, inkişafı hakiki olarak değil, âdeten zorunlu
kılmaktadır. Altı: Hüsâm Çelebi için Hatibzâde’nin “çelişki” yorumu ve kurduğu bağlantı
bir tür zorlamadır. Ayrıca Hatibzâde’nin şüphe (şek) hakkında söylediği de doğru
değildir. Çünkü Çelebi’ye göre şüphenin çelişiği vardır. Bilindiği üzere şüphe;
yargıdaki bağıntıyı (nispet) idrakle birlikte bu idrakin, ispat ve nefye ilişmesinin
eşit seviyede kalmasıdır. Diğer deyimle anılan seviyenin birini diğerine tercih et-
tirecek bir sebebin bulunmamasıdır. Görüldüğü üzere her bir taraf için, diğer ta-
raf çelişik olmaktadır. Kişi hangi tarafa meylederse öte taraf onun çelişiği olacak-
tır. Çelebi burada, yargıdaki bağıntıyı idrak için iki ayrı durumun söz konusu ol-
duğunu bildirmiştir. Birinci durumdaki şüphe eden için ispat ve nefye ilişmek
eşittir, ikinci durumdaki için ispat ve nefye ilişmek olmaksızın idrakin kendisinin
bulunmasıdır. İlk durum sırf şüphedir, bunun çelişiği vardır ve bilgi tanımı için- www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 161 161 de yer almaz. İkinci durumdaki şüphenin çelişiği yoktur ve bu nedenle bilgi ta-
nımı içinde yer alır. Dolayısıyla Çelebi’ye göre şüphenin bu iki ayrı durumu dik-
kate alındığı için kimi zaman tasavvurların içinde, kimi zaman da dışında sayıl-
mıştır. Sözgelimi birinci durumu dikkate alan için şüphe, bir tasavvur olacaktır. Fakat buradaki tasavvur mutlak ilmin kısmı olan tasavvurdur, yoksa tanımı yapı-
lan (muhtar) bilginin kısmı değildir. Binaenaleyh iki ayrı durum göz önüne alın-
madan yapılan eleştiri doğru değildir. Öte yandan Çelebi, Hatibzâde’nin “vehim
ve şüphenin kendisine ilişen idrak” cümlesini içeriksiz ve anlamsız bulmakta-
dır.66 Yedi: Hüsâm Çelebi altıncı maddede şüpheyi çelişiği olan ve olmayan olmak
üzere ikiye ayırmış ve böylece Hatibzâde tanım eleştirisine karşılık vermişti. İd-
rakin bir türü olan şüphenin çelişiği yoktur ve bu nedenle bilgi tanımı içine gir-
mektedir. Ancak olumlu (ispat) ve olumsuz (nefy) tarafa eşit duran şüphenin çeli-
şiği olacaktır ve bu yönüyle şüphe tanım dışında kalmaktadır. Çelebi bu maddede
şüphe ile ilgili ince ayrımı hatırlatmakta ve üzerine düşünülmesi gerektiğinin al-
tını çizmektedir. Buna göre olumlu ve olumsuz taraflara eşit derecede iliştiği
halde izanın gerçekleşmediği idrakin içinde şüphe yoktur. 66 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 51b.
67 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 52b.
68 Hasan b. Muhammed Attâr (öl. 1250/1834), et-Tezhîb: Şerhu’l-Habîsî ala Tehzîbi’l-mantık ve kelâm (Kahire:
Matbaat-i bâbi’l-Halebî, 1355), 59. 3. Hüsâm Çelebi’nin Hatibzâde’ye Tenkitleri Evet, şüphe bu idrake
bir yönden bağlı olmakla birlikte diğer yönden onun dışındadır. Şüpheye idrakin
kendisi olarak bakıldığında çelişiği olmayan tasavvur kapsamındadır. İdrak ol-
ması bakımdan şüphe bilgi tanımının içindedir. Çelebi özelleştirilmiş bilgi anlayışına sahip kelâm ile genelleştirilmiş anlayış-
taki felsefe sisteminin farkına değinmiştir.67 Kelâm için bilgi kısıtlanmış, daral-
tılmış ve preslenmiş özel bir sıfat iken, felsefe için bilgi kuşatıcı, geniş ve kap-
samlıdır. Kelâm terminolojisi bilgiyi “kesinlik” seviyesine tahsis etmiş, kesin ol-
mayan bir şeyi bilgi saymamıştır.68 Felsefede bilgi “bir suretin akılda husulü” ol-
duğu için tanım zan, cehil, taklit ve hatta şüphe ve vehmi içine almaktadır. Bu
tanımda önemli olan formun zihne alınmasıdır, kesinlik dikkate alınmamaktadır. Kelâm için bilgi tanımının bu sayılanları içermesi eksikliktir ve bilginin dil, din
ve gelenekteki kullanımına aykırıdır. Bilginin; “çelişiğine ihtimali olmayarak
temyizi zorunlu kılan bir sıfat” tanımı sözü edilen kusurlardan uzak olduğu için
takdire şayan bulunmuştur. Hüsâm Çelebi, Hatibzâde’nin kelâm ve felsefe ekolünün bilgi tanımı arasında
kurduğu bağlantıyı tuhaf bulmuştur. Bu çerçevede Hatibzâde felsefenin bilgi ta-
nımında sorun çıkarmayan şüphe ve vehmin, kusursuz olduğu iddia edilen ta-
nımda neden sorun olarak görüldüğünü sormuştur. Çelebi kelâmın bilgi tanı-
mında gözettiği “kusursuzluk ve kesinlik” hedefinin, felsefedeki kapsayıcı bilgi Eskiyeni 46 (Mart/March 2022) 162 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi tanımı gerekçe gösterilerek Hatibzâde tarafından göz ardı edildiğini söylemiş-
tir.69 Sekiz: Hüsâm Çelebi’ye göre Hatibzâde’nin kurduğu bağlantılar yanlış ve böy-
lece ileri sürdüğü değerlendirmeler kusurludur. Mesela ispatın bilgi sayılmama-
sı, bilgilerin tasavvurlarla kısıtlanmasını ve sınırlandırılmasını gerektirmez. Çünkü bilginin içinde ispatı/olumlamayı zorunlu kılan bir unsur vardır. Bu hu-
sus dikkate alındığında “âlem hâdistir” önermesinin tasavvur düzeyinde kalma-
yarak, yapısı gereği yargıyı zorunlu kıldığı anlaşılmaktadır. Çelebi, çelişkinin yeri ile ilgili Cürcânî’nin tespitlerini doğrulayarak,
Hatibzâde’nin yorumlarına katılmadığını bildirmiştir.70 Çelebi altıncı maddede
şüphenin; olumlu ve olumsuza ilişmedeki eşitlik bakımından çelişkiye ihtimali
olacağını, ancak idrak bakımından çelişkinin olmayacağını izah ettiğini hatırlat-
mıştır. Buna göre pekâlâ şüphenin çelişiği olabilir. Çelebi’ye göre şüphe
dolayımındaki sorun çözüldüğünde Hatibzâde’nin çelişki yerini temyizden ala-
rak sıfat veya taalluka kaydırma tekliflerinin zaten bir anlamı kalmayacaktır. Ayrıca Hatibzâde’ye göre tasavvurun çelişiği olmadığı için, çelişkinin yerini
temyiz olarak belirlemek de yanlıştır.71 Çünkü bilgi; tasavvur, tasavvur da temyiz
ise o zaman bilginin temyizi gerektiren bir sıfat olması yanlış olacaktır. Benzer
bir durum bilginin tasdik olarak alınmasında da kendini gösterecektir. Çelebi’ye
göre Hatibzâde’nin bilgiyi tasavvur ve temyiz ile eşitleyerek yaptığı eleştirisi ku-
surludur. 69 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 52b.
70 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 53b.
71 Aynen Teftâzânî’ye göre çelişkiye ihtimali olmayan yerin temyiz olarak belirlenmesinin pek anlamı
yoktur. Ona göre çelişikinin olmaması gereken yer, temyizin taalluku olmalıdır. Teftâzânî, Şerhu’l-
makāsıd, 1/58.
72 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 53b.
73 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 54a.
74 Teftâzânî, Şerhu’l-makāsıd, 1/59. 69 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 52b. 70 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 53b. 3. Hüsâm Çelebi’nin Hatibzâde’ye Tenkitleri Çünkü bilgi tanımı etrafında yazılan ibareler, sureti temyiz etmenin çe-
lişiği yoktur, denilerek yorumlanırsa sorun ortadan kalkacak ve tasavvurun
temyiz ile özdeş kılınması yanlış olacaktır.72 Dokuz: Hüsâm Çelebi literatürde “zorunlu kılan”ın (delil/mûcib) mutlak ve
mukayyet olmak üzere iki anlamda kullanıldığını söylemiştir. Ona göre
Hatibzâde’nin yanlışı bu iki kullanım farkına dikkat etmemiş olmasıdır.73 Evet,
zorunlu kılanın mutlak anlamda alınması, bilmediğini bilmeyenin bilgisini içi-
ne almaktadır. Ama mukayyet anlamdaki zorunlu kılanın içine onun bilgisi
girmemektedir. Bilgi tanımındaki zorunlu kılan, mukayyet anlamda kullanıl-
mıştır. Bilgiyi zorunlu kılan; sağlıklı duyu, bedahet, âdet ve burhan olabilir.74
Dolayısıyla bilmediğini bilmeyenin zorunlu kılana bir yönden dayanıp, diğer
yönden dayanmaması bununla açıklanabilir. Buradan yola çıkarak zorunlu kı-
lan sebep devam ettiği sürece bilgi şimdi değişmez ve çelişiğine ihtimali olmaz. www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 163 163 Bilginin değişmemesi onu zorunlu kılan sebebe bağlıdır. Yine bilginin gelecek-
te çelişiğine ihtimal olmaması onu zorunlu kılanın hatırlanmasına bağlıdır. Kişi
zorunlu kılan sebebi hatırladığı müddetçe gelecekteki bilgi değişmeyerek de-
vam edecektir. Zorunlu kılan sebebin ortadan kalkması ve müstakbelde unut-
manın gerçekleşmesi ile bilginin kesinliği kaybolabilir. Ama bu kayboluş bilgi-
nin güvenilirliğine zarar vermez. Gelecekte zorunlu kılan sebep hatırlansa ve
kesin, değişmez bilgi elde edilmeyecek olsa işte geri gelmeyiş bilgiye güveni
sarsacaktır. Dahası bilgiyi zorunlu kılan yakın sebep hatırlanır (geometri prob-
lemleri) ve uzak sebep (geometri ilkeleri) göz ardı edilse bile şu hal, bilginin
kesinliğini zedelemeyecektir.75 Bilmediğini bilmeyen kişinin bilgisini zorunlu kılan husus vardır ve bu husus
sayesinde kişi hâlihazırda bilgisinden emindir. Burada sorun bilgiyi zorunlu kı-
lan hususun yanlış olmasıdır. Nihai aşamada kişi ileride bu yanlış hususun realite
ile örtüşmediğini anladığı anda bilgisi çelişiğine doğru değişecektir. On: Hüsâm Çelebi yedinci maddede “şüphe” ile ilgili açıklamalarını tekrar ha-
tırlatarak, bu açıklamalar sayesinde sayılan üç kitap arasındaki farkın çözümle-
nebileceğini söylemiştir. Ayrıca Cürcânî’nin üçüncü (iii) kitaptaki “sanki” ifadesi,
şüphenin tasavvur olduğu hakkında tereddütü değil, kesinliği belirtmek içindir.76
Cürcânî’nin sözü edilen (iii) kitabındaki açıklamalarına bakılırsa şüphe ve ve-
himde hüküm/yargı olmadığı için tasavvur kapsamındadır. Zira yargı; en az
yüzde ellinin üstünde olan bir önerme için geçerlidir. Şüphe yüzde elli sınırının
tam ortasında kaldığı için, şüphenin içinde yargının bulunmadığı söylenmelidir. Vehim ise yüzde ellinin altına düştüğü için zaten, onun şüpheye oranla yargıdan
oldukça uzaklaştığı daha açıktır.77 Öte yandan Cürcânî tarafından tanımın mez-
hebe tatbik edilmesi onun tek kaldığı bir görüş değildir. 75 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 54b.
76 Seyyid Şerif Cürcânî, Tesdîdü’l-kavâid fî şerhi Tecrîdi’l-akāid (Hâşiye-i Tecrîd içinde), thk. Altaş Eşref
(İstanbul: Türkiye Diyanet Vakfı Yayınları, 2020), 2/26.
77 Cürcânî, Hâşiye-i Tecrîd, 2/27.
78 Hüsâm Çelebi, Hâşiye-i def‘i ‘itirâzât-ı Hatîbzâde (Veliyüddin, 3244), 55a. 3. Hüsâm Çelebi’nin Hatibzâde’ye Tenkitleri Çelebi için ekolün genel
duyarlılıklarına göre bilgi tanımına getirilen birtakım kayıtlar, Cürcânî’nin kendi
görüşünü değil, ekolün görüşünü yansıtır.78 Sonuç Kelâm tarihini dönemlendirirken Osmanlı ulemasının üretimde bulunduğu,
kabaca 14. yüzyıldan başlayan 19. yüzyıla kadar devam eden zaman aralığı bakı-
mından o geniş evreye “Cem ve tahkik devri” denilmektedir. “Cem ve tahkik”
adlandırması dönem içinde yazılan eserlerin içerikleri ayrıntılı araştırmalara
konu edilmeksizin verilmiştir. Genel olarak İslâm düşünce tarihi ve özel olarak
kelâm ilmi açısından bu çağ bir duraklama ve gerileme evresi olarak nitelendi- Eskiyeni 46 (Mart/March 2022) 164 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi rilmiştir. Duraklamanın ve gerilemenin muhtevası kendinden önceki eserlerin
birebir tekrar edilmesi ve yeni bir şeyin getirilmemesi olarak açıklanmıştır. Osmanlı düşünce tarihine tekabül eden zaman aralığında bir yazım tarzı ola-
rak şerh-hâşiye veya müstakil risâle yazmak bir gelenek haline gelmiştir. Her ne
kadar bir yazım türü olarak şerh ve hâşiyelerin sadece metni anlaşılır kılmak için
yazıldığı ileri sürülmüş olsa da bu yargılar çok kısıtlı verilerden hareketle veril-
miş ve aslında her bir yazıdaki hedefin farklı olabileceği gözden kaçırılmıştır. Osmanlı düşünce dünyasını genişliği ve derinliği bakımından bir derya olarak ni-
telendirmek yanlış olmayacaktır. Bu makalede deryadan bir katre mesabesindeki
Hatibzâde ve Hüsâm Çelebi’nin sadece bir bilgi tanımı etrafındaki karşılıklı eleş-
tirilerine yer verilmiştir. Bu iki yazının içeriğine bakıldığında şerh-hâşiye-risâle
yazımının tekrarlardan ibaret olmadığı görülecektir. İki risâle karşılıklı mütalaa
ve müzareke edildiğinde karşılıklı fikirlerin çarpıştırıldığı ve yarıştırıldığı anla-
şılmıştır. Risâleler dikkatlice incelendiğinde, Osmanlı ulemasının kendi soruları-
nı soran ve yanıtlamaya çalışan eleştirel düşünceye sahip olduklarına kanaat ge-
tirilmiştir. Osmanlı düşünce kültürü kendi iç sorunlarını, yine iç ve dış muhasebe yön-
temiyle yeniden revize etmeyi başarmış gözükmektedir. Paradigmaya yöneltilen
soru ve sorunlar gözden geçirilmiş ve aksaklıklar sorgulama yolu ile giderilmeye
çalışılmıştır. Açık söylemek gerekirse öz eleştiriden yoksun bir Osmanlı dünyası
olmadığına dair küçük bir örneği, Hatibzâde ve Hüsâm Çelebi’nin bilgi tanımı ile
ilgili risâleleri oluşturmaktadır. Elbette bu çok genellemeci bir yaklaşımdır, ay-
rıntıları açığa çıkarmak için farklı disiplinlere ait malzemenin üzerine derinlikli
araştırmaların yürütülmesi gerekmektedir. Makalede incelemesi yapılan bilgi
tanımı üzerine yazılan risâleler bu bağlamda temsil gücü yüksek büyük bir biri-
kimden damıtılarak elde edilmiş yalnızca mütevazı bir örnekten ibarettir. Hatibzâde üzerine yapılan akademik araştırmalar, onun bilhassa ahlâkî de-
ğerlerin kaynağı (hüsün-kubuh) ve insan fiilleri gibi hem birbirleri hem de Tanrı
ve tarih tasavvuru ile yakından irtibatlı kelâmî-felsefî problem çerçevesinde
Eş‘arî çizgide yer aldığını belirtmektedirler. Fakat bu husus, Hatibzâde’nin Eş‘arî
düşünceyi bütünüyle kabul ettiği anlamına gelmemektedir. Nitekim anılan
kelâmî meseleler etrafında Hatibzâde’nin Eş‘arî pozisyona yakın durması
Eş‘arîlerce benimsenen ve sorunsuz sayılan bilgi tanımını eleştirmesine mani
olmamıştır. Sonuç Hâlbukî müteahhir Eş‘arî kelamcılar, sözü edilen bilgi tanımını man-
tık ilminin tanım kuralları açısından kusursuz bulmuşlardı. Öyleyse
Hatibzâde’nin tümüyle Eş‘arî sayılması pek mümkün gözükmemektedir. Fatih
Sultan Mehmet dönemi ulemalarından sayılan Hatibzâde’de ve en azından de-
vamında yetişen Hüsâm Çelebi örneğindeki ulema profilinde bilinçli bir seçicilik
tavrının geliştirilmiş olduğu, herhangi bir kelâmî veya felsefî ekole tümüyle yas-
lanılmadığı söylenmelidir. www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 165 165 Hatibzâde ile Hüsâm Çelebi arasında bilginin tanımına dönük gerçekleşen
eleştiri yazıları önemli bir noktayı gün yüzüne çıkarmaktadır. Buna göre Osmanlı
düşüncesi ve bu düşüncenin oluşturucu, geliştirici ve taşıyıcı konumundaki bü-
yük zihin haritası eleştirilerle yeniden tesis ve tahkim edilmiştir. Burada araş-
tırması yapılan sayfa bakımından hacimsiz, ama içerik bakımından derinlikli iki
risâle, farklı fikirlerin öylece bırakılmadığını gösteren birer numune-i misaldir. Osmanlı düşünce dünyası yaklaşık yedi asırlık kurulu İslam düşüncesini tevarüs
etmesi bakımından otorite isimlerin kitaplardaki ibarelere kimi zaman bağlı ka-
larak bir fikir inşa etme yoluna gitmiştir. Aynı yazarın farklı kitaplarındaki ibare
farklılıklarına büyük bir titizlik gösterilmesi bunu göstermektedir. Bu husus, bü-
yük resimdeki orman yerine küçük ağaçlara takılı kalınmasına sebebiyet vermiş-
tir. Öte yandan Hatibzâde’nin bilgi tanımını on açıdan eleştirmesine rağmen yeni
bir bilgi tanımı önerisinde bulunmaması bir eksiklik olarak görülebilir. Hüsâm
Çelebi’nin Hatibzâde’ye karşı eleştirilerine bakıldığında daha çok kavramların
katmanlarını ve ayrımlarını dikkat-i nazara vererek tanımın zayıflıklarını gi-
dermeye çalıştığı görülmektedir. Mesela Çelebi’nin saf idrak ve eşitlik durumun-
da kalmak gibi şüpheye farklı açılardan bakılabileceğine işaret etmesi, yine zo-
runluluk (mûcib) kavramının mutlak ve sahih olarak ikiye ayrılmasını göstermesi
önemli katkılar olarak görülmelidir. Son tahlilde herhangi bir tanımın olumlu cümle yapısı ile bitirilmemesi ger-
çekten bir sorundur. Çünkü bir şeyin ne olmadığını göstermek onun ne olduğu-
nu göstermek için hiçbir zaman yeterli gelmez. Eleştiriler çerçevesinde söylene-
cek olursa “çelişiğe ihtimali olmayan” ibaresi bilgi tanımını zor duruma düşür-
müş gözükmektedir. İntihal Taraması
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that scientific and ethical principles have been followed while carrying out and writing
this study and that all the sources used have been properly cited. Finansman
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Yazar(lar) | Author(s): Mustafa Bilal Öztürk Bu çalışmanın hazırlanma sürecinde bilimsel ve etik ilkelere uyulduğu ve ya-
rarlanılan tüm çalışmaların kaynakçada belirtildiği beyan olunur. | It is declared
that scientific and ethical principles have been followed while carrying out and writing
this study and that all the sources used have been properly cited. i Bu makale, Creative Commons Atıf-GayriTicari 4.0 Uluslararası Lisansı (CC BY
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NonCommercial 4.0 International License Yazar(lar) | Author(s): Mustafa Bilal Öztürk Yazar(lar) | Author(s): Mustafa Bilal Öztürk Eskiyeni 46 (Mart/March 2022) 166 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi (İstanbul: Beyazıt Devlet Kütüphanesi, Veliyüddin, 3244), 41b. Ek Ek
Mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i ‘itirâzât-ı Mevlânâ Hatîbzâde Adlı Eserin İlk Varağı, Ek
Mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i ‘itirâzât-ı Mevlânâ Hatîbzâde Adlı Eserin İlk Varağı, Ek
Mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i ‘itirâzât-ı Mevlânâ Hatîbzâde Adlı Eserin İlk Varağı, Ek
Mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i ‘itirâzât-ı Mevlânâ Hatîbzâde Adlı Eserin İlk Varağı, Ek
Mâ yeteallak bi-ta‘rîfi’l-‘ilm min def‘i ‘itirâzât-ı Mevlânâ Hatîbzâde Adlı Eserin İlk Varağ www.dergipark.org.tr/tr/pub/eskiyeni Öztürk, Critique of The Criticism: Ḥusām Çelebi’s Criticism of Khaṭībzāde in the Context of The … • 167 Kaynakça Eskiyeni 46 (Mart/March 2022) 168 • Öztürk, Eleştirinin Eleştirisi: Bilgi Tanımı Bağlamında Hüsâm Çelebi’nin Hatibzâde Tenkidi Hatibzâde, Muhyiddin Mehmed. Risâle fî ta‘rîfi’l-‘ilm. İstanbul: Süleymaniye Kütüphanesi, Ragıp
Paşa, 1459, 188b-190a. Hatibzâde, Muhyiddin Mehmed. Risâle fî ta‘rîfi’l-‘ilm. İstanbul: Süleymaniye Kütüphanesi, Ragıp
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Composition of waste water from poultry factories and their suitability for irrigation of agricultural crops (as an example of Tashkent province, Uzbekistan)
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E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124401018 Composition of waste water from poultry
factories and their suitability for irrigation of
agricultural crops (as an example of Tashkent
province, Uzbekistan) Z. Artukmetov1,*, B. Nasirov1, J. Aliev1, and N. Kamolova1 Z. Artukmetov1,*, B. Nasirov1, J. Aliev1, and N. Kamolova1
1Tashkent State Agrarian University, University str., 2, Tashkent province, Uzbekistan, 100140 1Tashkent State Agrarian University, University str., 2, Tashkent province, Uzbekistan, 100140 Abstract. This article highlights the composition of wastewater from
poultry farms and their suitability for irrigation of agricultural crops. It has
been established that the chemical demand of wastewater from poultry
farms is 480–850 mg О2/l, which indicates a high level of contamination
with organic products. Coarse and finely dispersed substances vary within
430–720 mg/l, and biochemical oxygen consumption on the fifth day
(BOD5) within 0.39–0.74 g О2/l, and at the time of mass effluent
emissions it reached 15–1.6 g О2/l. The titer of Escherichia coli was equal
to 10-6, the number of microbes ranged from 48.5 x 106 to 61.6 x 106. Determination of the suitability of wastewater based on generally accepted
methods showed that they are quite suitable for irrigation of crops without
additional reclamation measures. * Corresponding author: tuag-info@edu.uz © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction Water for irrigation in the arid zone of Central Asia, including the Republic of
Uzbekistan, is a necessary factor in agriculture. In the Republic of Uzbekistan, the main
factor of food security is irrigated agriculture. To expand the area of irrigated agriculture,
free water resources are needed, although the available water resources in the republic can
expand the area of irrigated agriculture 4.8 million hectares. With the existing level of
irrigation technologies, there is a strong shortage of irrigation water [1-3]. Anthropogenic water transformations in the region have already reached a global scale:
the intensive development of irrigated agriculture in the second half of the 20th century led
to a significant increase in water intake from the Syr Darya and Amu Darya basins, which
caused the Aral Sea to become shallow [4, 11]. At present, the volume of the sea is only
28% of the indicator in 1960. A. Kurtov argues that the lack of water for the Republic of
Uzbekistan is a disaster in the truest sense of the word: due to the lack of water in a number
of regions of the republic, especially in Karakalpakia, one can already say about the social
and economic crisis [5, 8]. There is not enough water not only for agricultural purposes, but E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124401018 also for purely bit needs. If before 2000 years of low water supply in the region were
observed every 6–8 years, now this phenomenon is repeated every 3–4 years. In 2018 and
2019, water availability decreased significantly [6]. also for purely bit needs. If before 2000 years of low water supply in the region were
observed every 6–8 years, now this phenomenon is repeated every 3–4 years. In 2018 and
2019, water availability decreased significantly [6]. y
g
y
The urgent problem of the current stage of economic and social development of the
republic is the protection of the environment and, in particular, the elimination of the
danger of the negative impact on it of the growing volume of wastewater from poultry
farms. Intensively developing poultry farms and enterprises are one of the pollutants of the
republic's water bodies [5]. 1 Introduction Applied artificial treatment facilities often turn out to be
ineffective, retaining only 50-60% of the ingredients in the wastewater, because the
technological process of mechanical and biological treatment at wastewater treatment
plants functions poorly due to their unsatisfactory operation, imperfection of the
technological cleaning process itself, and they work with greater overload. Therefore, very
promising from the national economic and hygienic point of view is soil disposal of
wastewater and their use for irrigation of forage and grain crops, which allows increasing
water availability, productivity of land and is a powerful means of increasing soil fertility,
as well as an effective way of additional treatment in natural conditions [5]. In general,
sewage from poultry farms in the republic can irrigate and fertilize 10-12 thousand hectares
of land [7]. However, the problem of using wastewater for irrigation of crops requires a
regional approach, i.e. it is necessary to take into account the peculiarities of the soil and
the hydrogeological conditions of the territory [5, 10]. In conditions of a strong shortage of irrigation water in the Tashkent State Agrarian
University, special attention is paid to conducting research work on more careful and
productive use of the available water resources, including local runoff. In the CIS republics, including the former All-Union Scientific and Production
Association VNPO "Progress" (All-Union Scientific Research Institute for Agricultural
Crops), Volgograd Agricultural and Ukrainian Hydro-reclamation Institutes, Uzbek
Research Institute of Sanitation, Hygiene and Occupational Diseases and other institutions
have carried out numerous studies to study the suitability of household and livestock
wastewater for irrigation of crops. However, scientific research on the disposal of
wastewater from poultry farms has been almost non-existent [5-7]. Considering all the above, the purpose of the research is to study the composition and
suitability of wastewater from poultry farms for irrigation of agricultural crops in various
soil and hydrogeological conditions of the republic as an effective measure to prevent
pollution of water resources and save river water, determine their impact on the growth,
development and productivity of cultivated crops, sanitary and hygienic condition of
irrigation fields. The objects of research are waste waters of poultry farms and fodder and grain crops
irrigated by them on meadow and serozem soil conditions of the Tashkent region.
The materials of the research carried out are waste waters of poultry farms in
Uzbekistan and Urtachir-chikparranda, located on sierozem and meadow soils of the
Tashkent region, and fodder and grain crops irrigated by them. The suitability of
wastewater for irrigation of agricultural crops was determined by Budanov, Mozheiko and
Vorotnik, Kelly, Eaton and Stebler, by the US Department of Defense (SAR) method [5]. 2 Materials and methods The objects of research are waste waters of poultry farms and fodder and grain crops
irrigated by them on meadow and serozem soil conditions of the Tashkent region. The materials of the research carried out are waste waters of poultry farms in
Uzbekistan and Urtachir-chikparranda, located on sierozem and meadow soils of the
Tashkent region, and fodder and grain crops irrigated by them. The suitability of
wastewater for irrigation of agricultural crops was determined by Budanov, Mozheiko and
Vorotnik, Kelly, Eaton and Stebler, by the US Department of Defense (SAR) method [5]. 2 2 E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124401018 3 Results Long-term studies to determine the quality of wastewater and their suitability for use for
irrigating agricultural crops have shown the following: wastewater from poultry farms is
characterized by a specific (often ammoniacal) odor, cloudy color, slightly alkaline reaction
of the medium (pH = 7.1-7.3) , carbonate-sulphate, calcium-magnesium composition,
according to the classification of Yu. P. Lebedev and had coarse and finely dispersed
substances, which were contained in an amount of 430-720 mg/l on average, therefore,
reuse wastewater in the production process or dispose of it in the opening reservoirs
without thorough cleaning is unacceptable, since the concentration of most of the
ingredients in them exceeds the maximum allowable for reuse and dumping them into water
bodies [9]. The great importance in assessing the quality of water for irrigation of agricultural crops
is oxidizability (COD - chemical absorbed oxygen) - an indicator characterizing the total
content of organic and inorganic substances in water that react with strong oxidants [7]. A
high COD value (480–850 mg О2/l) indicates a large pollution of wastewater (maximum
permissible concentration for a reservoir is 30 mg/l) with organic products. The degree of
wastewater pollution with organic matter contained in the form of non-settling suspended
and colloidal particles can also be determined by the oxygen content consumed for
biochemical oxidation of these substances in 1 liter of the sample during the life of aerobic
bacteria. This indicator is called BOD - biochemical oxygen demand - and it varied in the
discharged waters of poultry farms in the range of 0.39-0.74 mg/l (the maximum
permissible concentration for a reservoir is 3 mg/l) on the fifth day (BOD5), and in the
moment of burst effluent emissions reached 1.5–1.6 g О2/l, ie the quality of the waste water
has deteriorated almost twofold (Table 1). Table 1. Characteristics of wastewater from poultry farms and river water to irrigate of agricultural
crops
#
Indicators
Unit
‘Uzbekistan’ poultry farm
‘Urtachirchikp
arranda’
poultry farm
2018
2019
2020
1
2
3
4
5
6
7
Wastewater from poultry farms
1
рН
–
7.3
7.2
7.2
7.1
2
Dissolved solids
mg/l
430
650
720
580
3
COD
mg/l
480
750
800
850
4
BOD5
mg/l
1,100
390
1,500
680
1,430
610
1,200
760
5
Total N
mg/l
70.7
61.4
56.5
66.4
6
Total P
mg/l
0.2
0.2
0.4
0.6
7
Gross K
mg/l
14.5
11.1
12.6
23.7
8
Ca
mg/l
58.5
50.0
53.5
69.2 able 1. 3 Results Suitability assessment of wastewater from poultry farms for irrigation of forage crops 4 4 E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124401018 + Мg) < 2,5
6
Stabler: (К > 6)
К = 288 : (rNa + 4rCl)
61.94–100
66.0 In addition to the above ingredients, sewage and river water contains a large number of
microorganisms, which require careful study when using water for irrigation of agricultural
crops. The results of sanitary and bacteriological studies indicate that the wastewater used
for irrigation of agricultural crops was significantly contaminated according to the studied
indicators. So, the titer of Escherichia coli was 10-6, the number of microbes ranged from
48.5 x 106 to 61.6 x 106. At the same time, with each cubic meter of wastewater, the field
received from 48.5 x 1,012 to 61.6 x 1,012 pcs microorganisms [7]. After cleaning in artificial structures, these indicators were respectively equal: the titer
of E. coli - 10-4, the number of microbes in 1 ml of water - from 40.9 x 106 to 52.0 x 106
pcs. River water was characterized by the following indicators: the number of microbes in 1
ml of water - from 3.7 x 106 to 4.5 x 106, and the coli-titer - 0.0004. Summing up, it should be noted that wastewater from poultry farms is quite suitable for
irrigating agricultural crops, but the final suitability of wastewater for mineralization must
be clarified in the process of constant monitoring of the accumulation of salts in the soil
during the cultivation of crops. In addition to the accumulation of salts in the soil, wastewater also has a fertilizing
value, which depends on the presence of nutrients and beneficial microorganisms in them. During cleaning on artificial structures, a significant amount of nutrients is lost, when they
are discharged into open water bodies, thousands of tons of fertilizers are carried away,
they would play a big role in soil fertility, increasing the productivity of cultivated crops. As a result of the intake of a large volume of nutrients and beneficial microorganisms
during irrigation of land with wastewater, microbiological processes are significantly
increased, as a result of which soil fertility is improved. For irrigation of corn in our experiments, we used wastewater, requesting mechanical
cleaning. During water supply, the amount of nutrients (total nitrogen, phosphorus, total
potassium) was determined. 3 Results Characteristics of wastewater from poultry farms and river water to irrigate of agricultural
crops 3 3 https://doi.org/10.1051/e3sconf/202124401018 E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 9
Mg
mg/l
68.4
75.6
54.3
30.6
10
Na
mg/l
21.8
20.9
14.7
37.9
11
Cl
mg/l
16.7
32.8
22.4
23.6
12
SO4
mg/l
95.4
99.8
73.2
78.7
13
Bicarbonates
mg/l
434.3
410.7
373.5
326.1
River water
1
рН
-
7.3
7.5
7.4
7.3
2
Dissolved solids
mg/l
236.3
208.5
310.6
274.4
3
COD
mg/l
23.4
14.3
18.8
28.6
4
BOD5
mg/l
960
280
1,120
430
1,080
390
980
280
5
Total N
mg/l
5.9
4.5
4.0
5.1
6
Total P
mg/l
0.05
0.03
0.04
0.04
7
Gross K
mg/l
3.3
2.5
2.6
3.5 In terms of nitrogen content, the effluents are characterized by a high fertilizing value -
from 51 to 87 mg/l, the potassium content varied in the range of 10–32 mg/l. Qualitative
assessment of wastewater by the total concentration of dissolved salts, the content of
chlorides, hydrocarbons, biogenic elements, toxic salts, the reaction of the medium (pH),
anionic-cationic composition and the sodium adsorption coefficient (SAR), that the
wastewater of poultry farms is quite suitable for irrigating corn , cultivated for grain and
silage, winter wheat, alfalfa, combined crops of alfalfa + oats, corn + soybeans + sunflower
without dilution and diluted with river water in ratios of 1:1 and 1:2, fodder beet on
meadow and sierozem soils without additional reclamation activities (Table 2). Table 2. Suitability assessment of wastewater from poultry farms for irrigation of forage crops
#
Methods and approaches
Poultry farms
Uzbekistan
Urtachirchikparranda
1
Budanov:
1) Na : (Ca + Mg) ≤ 0,7
2) Na : Ca ≤ 1
0.09–0.11
0.24–0.37
0.27
0.48
2
Mozheyko and Vorotnik:
(К + Na) ∙ 100 : (Ca + Mg+ +
Na) ≤ 65%
11.87–13.47
27.26
3
US department (SAR):
Na : √(Ca + Mg) : 2 ≤ 8
0.34–0.46
27.26
4
Kelly: Сa ∙100 : (Ca + + Mg +
Na + K) ≥ 35%
24.95–32.68
41.50
5
Etone:
(СО3 + НСО3) – (Са +
1.5–2.1
0.68 Table 2. 3 Results The data presented in Table 1 show that the concentration of
total nitrogen varied widely and ranged from 51.3 to 86.8 mg/l during the irrigation period,
phosphorus was contained in insignificant amounts - 0.2–0.8 mg/l, and potassium - 10.2–
31.8 mg/l. Or: from every 1000 m3 of wastewater, 50–90 kg of nitrogen, about 1 kg of
phosphorus and 10–32 kg of potassium per hectare were supplied to the field [10]. In terms of nitrogen content, the wastewaters of the Tashkent poultry farm and the
poultry plant of Urtachirchikparrand, according to the classification of V.T. Dodolina
(1975), refer to wastewater of average fertilizing value, and low in terms of potassium and
phosphorus content [5]. These nutrients are in a dissolved and available form, getting into the soil, they are
easily absorbed by plants, while simultaneously solving the problems of water and
nutritional regime for corn plants. In addition, many organic substances and
microorganisms enter the soil with wastewater, as a result of which microbiological
processes are activated and the potential and effective soil fertility increases. The calculations performed to determine the amount of nutrients supplied to the field
with irrigation water showed that when corn was irrigated with sewage at an irrigation rate
of 3,650–3,900 m3/ha, 179–220 kg of nitrogen and 42–92 kg of potassium were supplied
per hectare of crops (taking into account the coefficient taking into account the loss of
nutrients from wastewater during irrigation for nitrogen, which is 0.85, for phosphorus and
potassium - 1 [5]. With an irrigation rate of 4,400–4,650 m3/ha, respectively, 218–264 and
51–110 kg for each hectare; under dilution conditions at an irrigation rate of 3,700–3,930 5 E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124401018 m3/ha - 98–122 kg of nitrogen and 16–53 kg of potassium, and at an irrigation rate of
4,550–4,620 m3/ha at 118–150 and 31–63 kg per hectare Phosphorus was supplied with
irrigation water in insignificant quantities. m3/ha - 98–122 kg of nitrogen and 16–53 kg of potassium, and at an irrigation rate of
4,550–4,620 m3/ha at 118–150 and 31–63 kg per hectare Phosphorus was supplied with
irrigation water in insignificant quantities. g
g
q
The use of poultry wastewater for feeding and vegetation irrigation during the
cultivation of winter wheat on gray soil contributed to the improvement of plant growth and
development significantly increases the yield of grain and straw. 3 Results If during feeding and
vegetation irrigation with river water the grain yield averaged 45.2 quintal (q)/ha and straw
56.5 q/ha for 3 years, in the variant with feeding irrigation with river water, and vegetation
irrigation with waste waters, respectively, amounted to 51.0 and 64.2 q/ha. In the variant
where recharge irrigation was carried out with waste water, and vegetative irrigation with
river water, 3.7 quintals of grain and 7.6 quintals of straw were obtained more than in the
control variant. The highest yield - 55.5 q/ha of grain and 70.2 q/ha of straw was obtained
in the variant with feeding and vegetation irrigation with waste waters. When carrying out
recharge irrigation with river water, and vegetative irrigation with diluted with river
wastewater and vegetative irrigation with wastewater, 40.2 q/ha of grain and 50.9 q/ha of
straw were obtained, which, respectively, by 5.0 and 5.6 q/ha less than in the control
variant. Diluted from the river and undiluted waste water from poultry farms when used for
irrigation in combination with mineral fertilizers on meadow soils contributed to obtaining
high yields of silage mass, grain and air-dry leafy corn mass. The harvest of silage mass
reached 530.6-608.3 and 564.2-641.1 q/ha, respectively, against 508.4-564.3 q/ha and grain
- 77.8-82.9 and 80.6-86 , 4 q/ha versus 68.6-74.6 q/ha; air dry leafy mass - 191.4–208.8 and
204.1–255.3 q/ha versus 182.3-198.4 q/ha in the control variant. It has been established that in the hot climate of Uzbekistan, soils irrigated with
wastewater become normatively clean on the 10-15th day after irrigation. With proper
organization and implementation of surface irrigation with wastewater, pollution of
aboveground plant organs and groundwater is completely excluded. In case of accidental
contamination of individual parts of plants, they can be considered normatively clean 10-15
days after watering. E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 On meadow soils with groundwater at a level of 2–2.5 m of the Chirchik-Angren valley,
it is advisable to use wastewater from poultry farms diluted with river water in a 1:1 ratio
for irrigation of corn, cultivated as a re-crop after winter and the use of mineral fertilizers
(norm N - 180, P2O5 - 100 K2O - 90 kg/ha). The timing of irrigation should be determined
by the lower threshold of the moisture content of the active soil layer, 70–75–65% of the
soil FWL. The irrigation rate should be in the range of 4.4-4.6 thousand m3/ha, between
irrigation periods of 15-20 days. Vegetation watering should be completed 15–20 days
before harvest. Irrigation and irrigation rates for cultivated crops are determined by their total water
consumption, and the timing of irrigation is determined by the moisture deficit in the active
soil layer. Irrigation rates for corn for silage are calculated at the lower threshold of soil
moisture of 75–80% of the PPV, for corn for grain - 70–75-65%. Irrigation rates in meadow
soils should be 3.4–4.0 and 4.4–4.6 thousand m3/ha, respectively. For fodder alfalfa of the
first year of standing with four mows on meadow soils, the irrigation scheme should be 1-1-
1-1, and for alfalfa of the second year and subsequent years with five and six mows 0-1-1-
1-1 and 0-1 -1-1-1-1 irrigation rates 1,000-1,100 m3/ha and irrigation rates from 4.0-4.4 to
5.0-5.5 thousand m3/ha. The irrigation scheme for alfalfa of the first year of standing on sierozem soils with
deep groundwater should be 1-2-2-1 with irrigation rates of 900-1,100 m3/ha and irrigation
rates of 5.4-6.6 thousand m3/ha, and for alfalfa of the second year of standing and
subsequent years - 0-1-2-2-1 with irrigation rates of 1,000–1,200 m3/ha and irrigation rates
- 6.0–7.0 thousand m3/ha. The irrigation regime for fodder beet consists of 5 irrigations with irrigation rates of
800–1300 thousand m3/ha and irrigation rates of 6.0–7.0 thousand m3/ha. In the conditions of meadow soils with a groundwater level of 1.5-1.8 m after sowing
wheat, it is necessary to carry out recharge irrigation with wastewater at a rate of 700-730
m3/ha and vegetative irrigation with wastewater diluted with river water in a ratio of 1: 1 at
a rate of 700 –850 m3/ha and an irrigation rate of 3,080 (including recharge irrigation
3,810) m3/ha. E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 Moisture-charging and fertilizing irrigation on sierozem soils should be carried out in
deep furrows according to a dead-end scheme with a rate of 800-1,000 m3/ha, and stop
them 2-3 weeks before sowing. Control over the sanitary and hygienic state of the soil
when using wastewater for irrigation is carried out on the 15th day after irrigation. When
irrigating with waste water, it is necessary to comply with sanitary and anti-epidemic rules,
especially in the first 20 days. When using wastewater for irrigation, the inter-irrigation
periods should be at least 15–20 days in order to ensure the normal course of soil self-
cleaning processes. In case of a delay in the process of self-cleaning of soils from
pathogenic microorganisms, irrigation with river water should be carried out. Vegetation
irrigation with waste water is completed 15–20 days before harvesting. 2. Presidential Decree No. 5853 of the Republic of Uzbekistan on “Measures for efficient
use of land and water resources in agriculture” on October 23 (2019) 1. Presidential Decree No. 3405 of the Republic of Uzbekistan on “The state program for
the development of irrigation in 2018–2019 and improvement of the reclamation state
of irrigated lands” on November 27 (2017) 4 Discussion and conclusion Our long-term studies confirm the feasibility and high efficiency of purification and
disposal of wastewater in agriculture by using them for irrigation of agricultural land,
especially in the arid zone of Uzbekistan. Wastewater from poultry farms is quite suitable
for irrigation of forage and industrial crops without additional reclamation measures. g
g
p
When irrigated with wastewater, a significant amount of nutrients enters the field, which
plays a large role in providing plants and improving soil fertility. It is advisable to apply the
following wastewater disposal scheme: sewerage - mechanical treatment facility - storage
ponds - irrigation fields. Waste water from poultry farms is quite suitable for irrigation of
forage crops used for the production of vitamin flour, haulage, silage, fodder, and forage
root crops. The most acceptable option for irrigation is the use of diluted wastewater from
the river in a 1: 1 ratio in combination with mineral fertilizers, since the soil self-cleaning
process begins on the 15th day after irrigation. Under the same conditions, for irrigation of
alfalfa for the 2nd and 3rd years of standing with diluted sewage water from poultry
factories with river water in the ratio of 1:1 or 1:2 (one part is waste and 1 or 2 parts is river
water). In this case, the irrigation rate should be set according to the water demand of
alfalfa. In this regard, the irrigation pattern is 1-2-2-1. One-time irrigation rate is equal to
900–1,100 m3/ha. 6 6 https://doi.org/10.1051/e3sconf/202124401018 E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 References 1. Presidential Decree No. 3405 of the Republic of Uzbekistan on “The state program for
the development of irrigation in 2018–2019 and improvement of the reclamation state
of irrigated lands” on November 27 (2017) 2. Presidential Decree No. 5853 of the Republic of Uzbekistan on “Measures for efficient
use of land and water resources in agriculture” on October 23 (2019) 7 7 E3S Web of Conferences 244, 01018 (2021)
EMMFT-2020 https://doi.org/10.1051/e3sconf/202124401018 3. Presidential Decree No. 6024 of the Republic of Uzbekistan about “Approval of the
Concept of development of water economy of the Republic of Uzbekistan for 2020-
2030” on June 10 (2020) 3. Presidential Decree No. 6024 of the Republic of Uzbekistan about “Approval of the
Concept of development of water economy of the Republic of Uzbekistan for 2020-
2030” on June 10 (2020) 4. M. Saidan, H. J. Khasawneh, M. Tayyem, M. Hawari, Journal of Chemical Technology
& Metallurgy 56(1), 163-171 (2018) 4. M. Saidan, H. J. Khasawneh, M. Tayyem, M. Hawari, Journal of Chemical Technology
& Metallurgy 56(1), 163-171 (2018) 5. Z. A. Artukmetov, Caspian Research Institute of Arid Agriculture Press, 65-70 (2017 6. A. V. Nazarova, O. I. Sergienko, N. R. Molodkina, V. Y. Efremova, In IOP
Conference Series: Earth and Environmental Science 613(1), 012088 (2020) 7. F. Zhang, Y. B. Man, W. Y. Mo, M. H. Wong, Reviews in Aquaculture 12(2), 582-599
(2020) 8. P. Song, G. Huang, C. An, X. Xin, P. Zhang, X. Chen, X. Yang, Water Research 188,
116480 (2021) 9. T. Hülsen, K. Hsieh, S. Tait, E. M. Barry, D. Puyol, D. J. Batstone, Water research
144, 665-676 (2018) 10. K. Meiramkulova, Z. Jakupova, D. Orynbekov, E. Tashenov, A. Kydyrbekova, T. Mkilima, V. J. Inglezakis, Sustainability 12(1), 5110 (2020) 11. R. Kulmatov, A. Taylakov, S. Khasanov, Envi. Science and Pollution Research, 1-11
(2021) 8 8
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Review of: "Counting Processes with Multiple Randomness: Examples in Queuing Theory"
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Qeios, CC-BY 4.0 · Review, September 29, 2023
Review of: "Counting Processes with Multiple Randomness:
Examples in Queuing Theory"
Petr Volf1
1 The Czech Academy of Sciences
Potential competing interests: No potential competing interests to declare.
Some remarks to Qeios manuscript "Counting Processes with Multiple Randomness:
Examples in Queuing Theory"
This manuscript is based on quite interesting reflection, that pure observation of a
counting process does not sometimes satisfy to its full description. It is shown on examples of simple queuing systems. In
fact the problem arises due rather insufficient choice of basal probability space (a space of departure processes). The
models of queues are standardly analyzed in the framework of random walks, where the main characteristics is the
number of items in the system (including the served item). Such a description is richer and based on transition
probabilities (or intensities) of both arrivals and departures. Hence, from this point of view, the notion of "counting process
with multiple randomness" is interesting from, say, a formal point of view, however has no high significance (in my opinion)
from the points of view of modeling and analysis.
One can consider also a case when the arrival process is neither observed nor known,
just the departures are available. I expect that there exists a number of works dealing with such cases, solving the
problem of system identification at least in certain special cases (as M|M|1).
Some other rmarks:
- In Lemma 2.1., point b) does not hold in the case of exponential arrivals.
- The discussion regarding the Burke's Theorem seems to me rather superficial.
Resulting variable - the inter-departures time, is really a mixture of two cases, however with such weights that the mean
time is 1/lambda. And the basic observation, also formulated in Burke's Theorem, saying that "At time t the number of
customers in the queue is independent of the departure process prior to time t", leads to the conclusion on Poisson
process of departures.
Extremely easy it is seen in a discrete time version, using geometric distribution instead exponential.
- The discussion concerning the Jackson network suffers from the same defect.
- What about references, are there some more recent papers dealing with similar or connected questions?
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Qeios ID: PMDQ7Y · https://doi.org/10.32388/PMDQ7Y
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Can You Play with Fire and Not Hurt Yourself? A Comparative Study in Figurative Language Comprehension between Individuals with and without Autism Spectrum Disorder
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RESEARCH ARTICLE OPEN ACCESS Citation: Chahboun S, Vulchanov V, Saldaña D,
Eshuis H, Vulchanova M (2016) Can You Play with
Fire and Not Hurt Yourself? A Comparative Study in
Figurative Language Comprehension between
Individuals with and without Autism Spectrum
Disorder. PLoS ONE 11(12): e0168571. doi:10.1371/journal.pone.0168571 Sobh Chahboun1¤a, Valentin Vulchanov1¤a, David Saldaña2¤b, Hendrik Eshuis1¤a,
Mila Vulchanova1¤a* Sobh Chahboun1¤a, Valentin Vulchanov1¤a, David Saldaña2¤b, Hendrik Eshuis1¤a,
Mila Vulchanova1¤a* 1 Language Acquisition and Language Processing Lab, Department of Language and Literature, Norwegian
University of Science and Technology, Trondheim, Norway, 2 Language, Cognition and Individual
Differences Lab, Department of Educational Psychology, Faculty of Psychology, University of Seville, Seville,
Spain ¤a Current address: Department of Language and Literature, Norwegian University of Science and
Technology, Trondheim, Norway
¤b Current address: Departamento de Psicologı´a Evolutiva y de la Educacio´n. Universidad de Sevilla, Avda. Camilo Jose´ Cela s/n, Seville, Spain
* mila.vulchanova@ntnu.no ¤a Current address: Department of Language and Literature, Norwegian University of Science and
Technology, Trondheim, Norway
¤b Current address: Departamento de Psicologı´a Evolutiva y de la Educacio´n. Universidad de Sevilla, Avda. Camilo Jose´ Cela s/n, Seville, Spain
* mila.vulchanova@ntnu.no Abstract Individuals with High functioning autism (HFA) are distinguished by relative preservation of
linguistic and cognitive skills. However, problems with pragmatic language skills have been
consistently reported across the autistic spectrum, even when structural language is intact. Our main goal was to investigate how highly verbal individuals with autism process figurative
language and whether manipulation of the stimuli presentation modality had an impact on
the processing. We were interested in the extent to which visual context, e.g., an image cor-
responding either to the literal meaning or the figurative meaning of the expression may
facilitate responses to such expressions. Participants with HFA and their typically develop-
ing peers (matched on intelligence and language level) completed a cross-modal sentence-
picture matching task for figurative expressions and their target figurative meaning repre-
sented in images. We expected that the individuals with autism would have difficulties in
appreciating the non-literal nature of idioms and metaphors, despite intact structural lan-
guage skills. Analyses of accuracy and reaction times showed clearly that the participants
with autism performed at a lower level than their typically developing peers. Moreover, the
modality in which the stimuli were presented was an important variable in task performance
for the more transparent expressions. The individuals with autism displayed higher error
rates and greater reaction latencies in the auditory modality compared to the visual stimulus
presentation modality, implying more difficulty. Performance differed depending on type of
expression. Participants had more difficulty understanding the culturally-based expressions,
but not expressions grounded in human experience (biological idioms). This research
highlights the importance of stimulus presentation modality and that this can lead to differ-
ences in figurative language comprehension between typically and atypically developing Editor: Philip Allen, University of Akron, UNITED
STATES Editor: Philip Allen, University of Akron, UNITED
STATES Introduction Figurative language is a pervasive phenomenon in daily communication, and is reflected in a
range of expressions, such as metaphors, idioms, cliche´s, irony, jokes. Such expressions vary in
structure and degrees of transparency and familiarity, and can fluctuate from a single word to
a complex sentence. A common feature that unites the categories of figurative language is the
fact that reference in such expressions is indirect. More importantly, the interpretation of figu-
rative expressions is non-compositional (non-literal). This deviation from compositional on-
line computation and the heterogeneity of figurative uses makes figurative language more
demanding in terms of processing [1]. In language development, the ability to interpret figura-
tive language develops gradually, and comprehension of such expressions can be especially
demanding in deficit populations where language is affected. Individuals with High Functioning Autism (HFA) are distinguished by relative preserva-
tion of language and cognitive skills. Intelligence in this group is at normal or above average
levels, and quite often such individuals demonstrate specific advantages in certain cognitive
areas. The term high functioning captures the level of adequate cognitive functioning in that
group, while often, when referring to the level of language competence (i.e., intact structural
language), they are described as highly verbal. However, problems with pragmatic language
have been consistently reported across the autistic spectrum, even when structural language is
intact [2–6]. In addition, some studies have found evidence of weaknesses in the figurative and
idiomatic language domains, contrasting with clear strengths in areas of grammar [4, 6]. Several behavioral and imaging studies have shown that there are many issues to take into
account regarding the interpretation of figurative language. It depends on the type of expres-
sion, its degree of compositionality/transparency, its linguistic structure, its source domain of
knowledge, and not least, its novelty/conventionality. Thus, the main question is how these
factors impact on the comprehension of the categories of interest (idioms, metaphors, irony),
and whether the dissociations between structural language skills and figurative language com-
petence reported in highly verbal individuals with autism are still maintained with a careful
choice of experimental paradigm. The current study is designed to answer these questions by focusing on figurative language
processing in highly verbal individuals with autism. Introduction The main goal was to investigate whether
individuals with autism and preserved language competence understand idiomatic expressions
and novel metaphors at the same level as carefully matched controls, and, specifically, whether
in this process they benefit from visually presented context and can integrate information
from multiple sources (e.g. visual modality and oral language presented simultaneously). Can You Play with Fire? Figurative Language Comprehension in Autism individuals. The current study also contributes to current debates on the role of structural
language in figurative language comprehension in autism. Competing Interests: The authors have declared
that no competing interests exist. Editor: Philip Allen, University of Akron, UNITED
STATES Received: July 7, 2016
Accepted: December 2, 2016
Published: December 30, 2016
Copyright: © 2016 Chahboun et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Received: July 7, 2016
Accepted: December 2, 2016
Published: December 30, 2016 Copyright: © 2016 Chahboun 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: Due to confidentiality
restrictions and personal data protection, data are
available upon request. Requests for data may be
sent to: mila.vulchanova@ntnu.no/sobh. Funding: This research has received funding from
the European Union’s Seventh Framework
Program for research, technological development
and demonstration under grant agreement n˚
316748. 1 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Idiomatic expressions and their processing The mechanisms underlying the processing of figurative language have been widely debated. Idiomatic expressions are one of the most common and popular forms of figurative language
used in daily interaction. Idioms are multi-word expressions whose interpretation is non-lit-
eral, meaning it cannot be derived by using regular compositional processes. As such, they
require the language user to go beyond what is stated literally in order to infer the communica-
tive intent of the speaker [7]. In addition, idioms have attracted attention both on theoretical 2 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism and empirical grounds. The reason is that they cannot be placed immediately within a gram-
mar-lexicon dichotomy of language. On the one hand, it can be assumed that they belong in,
and are retrieved from, the mental lexicon, following the assumption that they need to be
acquired and stored in the same way as lexical items. On the other, their (complex) structure
suggests that they may be processed like structures generated by grammar [8–9]. Idiom processing has been addressed extensively in recent research, but mainly, two dif-
ferent types of theoretical approaches have emerged regarding the processing and comprehen-
sion of idioms [10]. Representative of the first type of approach is the Lexical Representation
Hypothesis. Originally proposed by Swinney & Cutler [11] (see also Bobrow & Bell [12] and
Gibbs [13]), this hypothesis claims that idioms are stored as lexical items, and understanding
an idiom involves two parallel processes, a retrieval process, and a literal compositional
computation process based on decomposing every element separately [8–10]. Further, it is
assumed that the retrieval process is faster, while the compositional process demands longer
time. In a further development, Hamblin & Gibbs [14] suggest that idioms are essentially de-
composable. For these authors, idiomatic interpretation rests on identifying the individual
constituents, mainly because of the decomposability of the idiomatic expressions. Along simi-
lar lines, some authors have claimed that the processing and understanding of idioms cannot
be reduced to lexical access or lexical retrieval only, and an additional interpretation process
might be necessary [15–17]. Thus, Gibbs [16] suggests that idioms invoke complex meanings
which are motivated by independently existing conceptual metaphors which are part of every-
day thought. The second type of approach, labelled the Configuration Hypothesis, aims to capture the
dynamic process involved in idiom interpretation. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Figurative language in High Functioning Autism (HFA) Despite the long-standing interest in idioms, their nature and processing in typical popula-
tions, research on idiom comprehension in atypical populations overall, and in autism, in par-
ticular, has been surprisingly scanty. Mashal & Kasirer [24] conducted a study where individuals with HFA, children with learn-
ing disorder and typically developing controls were compared. The authors used 11 subtests,
ranging from interpretation of figurative expressions, including visual metaphors, idioms, con-
ventional metaphors, novel metaphors, to comprehension of homophones and semantic tests
(synonymy, similarity). Their results showed that children with learning deficit scored signifi-
cantly lower than typically developing peers on comprehension of conventional metaphors
and idioms, but no significant differences were observed between the two groups on compre-
hension of visual and novel metaphors. Furthermore, both deficit groups displayed a dissocia-
tion between the visual modality and the auditory modality, supporting the assumption that
modality is a salient factor in figurative language processing. In contrast, the control group
demonstrated an association between idioms and conventional metaphors. This result sup-
ports the idea that idioms and conventional metaphors share a common ground, in that both
are relatively frozen conventionalised expressions and most likely rest on similar mechanisms
for their processing. This is consistent with findings in a recent study of metaphor processing
in highly verbal individuals with autism [25]. Vogindroukas & Zikopoulou [26] conducted a study aimed at the developmental trajectory
of idiom comprehension in Asperger’s syndrome (AS) and HFA. The authors compared chil-
dren with HFA/AS to their typically developing peers and to control adults. They found that
children with AS/ HFA performed more poorly on the idiom task compared to the other two
groups, while the two typically developing groups did not display any differences in terms of
performance. In addition, the authors did not find a significant correlation between IQ scores
and performance on the task for the HFA group, but IQ did correlate with idiomatic compre-
hension and age for the two control groups. It is important to mention that this study used idi-
oms out of context. The fact that the idioms were presented without a supportive context means
that the participants had to rely exclusively on a semantic analysis or retrieval. Thus, the study
design might have introduced an additional problem and a potential confound between expres-
sion familiarity/frequency and decomposability. Idiomatic expressions and their processing The authors in favour of this position argue
that idioms are represented in a concrete and distributed way and are processed as complex
expressions [15]. This latter approach can be described as a “compositional” approach whereby
each constituent is assumed to contribute semantically to the idiom interpretation. In a comprehensive review of approaches to idioms, Titone & Connine [7] suggest that
either view fails to account adequately for findings in research or capture the nature of idioms. These authors thus suggest a “hybrid” model, which acknowledges both the arbitrary “word”
nature and the compositional aspect of idioms. Their model takes into account an important
parameter on which idioms vary: degree of decomposability. A similar view, albeit from a
slightly different perspective, is defended in Sanford [18] who describes idioms as the intersec-
tion of conceptual and syntactic schemas. Another factor which has been shown to impact on
idiom processing is the mode of presentation. Tabossi, Fanari & Wolf [19] found that idiom
identification differs depending on whether the expression was presented auditorily or visually
(as text). This clearly implies that the different modalities in which the idioms are presented,
demand different forms of processing. Another related figurative and formulaic language category are proverbs. Proverbs are fixed
sentential expressions which invoke well-received wisdom, social or moral norms [20–21]. They reflect themes from everyday life and often refer to general principles of reasonable
human behaviour and action [21–22], and, as such, may seem to express self-evident truths. However, like idioms, proverbs display a wide range of interpretations depending on the type
of mechanism involved: they can be metaphorical, as You can’t judge a book by its cover, or
based on personification, hyperbole or a paradox (No news is good news). Unlike idioms, how-
ever, proverbs are typically based on some kind of cause-effect reasoning (Once bitten, twice
shy) or reasoning by analogy, but may also reflect a certain type of cultural practice (Never look
a gift horse in the mouth). Although proverbs may have prototypical figurative meanings, they
are very much vital due to the necessity to draw precise inferences in each specific context of PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 3 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism encounter [21]. Idiomatic expressions and their processing Thus, the correct interpretation of a proverb requires the ability to navigate
between their concrete, literal meaning and their figurative meaning by establishing a meta-
phorical connection between these two interpretations. These properties of proverbs make
them an interesting category to study, especially in comparison to idioms. In the current study
we refer to this type of figurative expression as “instructive expressions” reflecting their com-
municative role in language use. Unlike idioms and proverbs, metaphors are a category which is often less conventionalised
and more transparent. It is often assumed that metaphors are a device which maps concepts
from two distinct domains, which are not otherwise conceptually linked to each other [23]. Like idioms, however, metaphors vary in degree of novelty/conventionality which affects the
way they are processed. In the current study we use novel metaphors as a category which does
not depend on stored vocabulary knowledge, but rather invites active processing based on
analogy. Thus, the processing of novel metaphors can act as a base-line for the processing of
novel expressions, in contrast to all other idiomatic categories included in the study. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism Whyte et al [27] addressed what factors predict performance on idiom comprehension in
highly-verbal children with autism in comparison to an age-matched control group and a
-control group matched on grammar-age equivalence. This study focused specifically on the
contribution of syntax competence and advanced Theory of Mind (ToM) abilities to idiom
comprehension in autism. The results document that children with autism indeed perform
more poorly compared to age-matched controls, but that there is no significant difference with
the syntax competence matched group. Furthermore, while advanced ToM ability did not pre-
dict performance in the control groups, it correlated with idiom comprehension in the partici-
pants with autism. Lee et al [28] tested Korean high-functioning children with autism compared to similarly
aged children with Attention Deficit Hyperactivity Disorder (ADHD) and a control group of
typically developing children. The test design included matching idioms to a picture, which
either described the figurative meaning of the expression or the literal interpretation. Non-fig-
urative sentences paired with their literal interpretation were used as a base-line condition. This study found that both the children with autism and the ADHD group performed signifi-
cantly worse than controls on the two idiom conditions, and especially in the condition where
the idiom was paired with its literal interpretation. No differences were observed on the non-
figurative expressions. Norbury [29] investigated idiom comprehension in context in children with communica-
tion disorders and age-matched peers. Children belonging to the communication disorders
group were selected to represent a spectrum of communication difficulty, including Specific
Language Impairment (SLI), Pragmatic Language Impairment (PLI) (with or without presence
of autistic symptoms), autistic disorder, and Asperger syndrome. An idiom definition task was
chosen to measure idiom comprehension in context. The main finding of that study was that
children with language and communication impairments benefit from context in interpreting
unfamiliar idioms. However, children with deficits in structural language did not benefit from
context as much as typically developing peers. Furthermore, children with autism spectrum
disorder (ASD) symptoms and pragmatic impairments, but without concomitant structural
language deficits, were not impaired to the same degree in their idiom understanding and
were able to use context as effectively as their peers. Importantly, language ability was one of
the most significant predictors of idiom understanding. These findings highlight the role of
language skills as a key determinant in the use of context to aid idiom comprehension, and fur-
ther suggest that contextual cues can provide sufficient information to derive at least a cursory
understanding of new idiomatic expressions. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Figurative language in High Functioning Autism (HFA) In that study, Vogindroukas & Zikopoulou
[26] confirm that children with AS/HFA have a tendency to make literal interpretations, but
that the difficulty in idiom comprehension is definitely not due to an intellectual deficit. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 4 / 24 Factors in idioms processing Previous research has documented that idioms are more easily processed in the presence of
supportive context ([16]; see Vulchanova et al [10] for a review). Findings from several studies
suggest that the main role of context is to provide semantic support for decoding the target
(appropriate) meaning of a sentence or an expression [17, 30–33]. In contrast, it is widely
argued that individuals with autism are impaired in processing ambiguous linguistic informa-
tion in context [34–36]. In addition, they often fail to attach context to their memories, and are
specifically impaired in processing the social aspects of contextual information [37], while
Ozonoff & Miller [38] demonstrate deviant use of context. However, the evidence concerning
a contextual deficit is somewhat controversial. Thus, other studies have shown that compe-
tence at making use of context depends on language status, and children with higher verbal
age can perform at the level of controls [39]. Also, success in that group may depend on the
exact position of the ambiguous word (homograph) in the sentence. Lo´pez & Leekam [36] PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 5 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism found that, even though children with ASD performed worse than controls, they were more
successful with homographs occurring later in the sentence (middle or end), suggesting sensi-
tivity to context also in that group. A study by Brock, Norbury, Einav & Nation [40] showed that participants with autism dis-
play a tendency similar to controls to use predictions based on the meaning of the lexical verb,
as reflected in an increase in looks to the picture matching the object of the verb. At the same
time both groups were less likely to be distracted by a phonological competitor of the object
word. This tendency, however, was mediated by language status in the autism group. In that
study context was restricted to the level of the sentence, with a focus on the information
encoded in the lexical verb and the ability to use that information to successfully orient to the
possible object. Also, Frith [41] argues that individuals with autism need a concrete context to
be able to understand words and expressions, and Saldaña & Frith [42] document that children
with autism have a normal reduction in reading times for expressions which are congruent
with previous events. Factors in idioms processing Given this evidence, we expect that context will not place any additional
burden on the interpretation of the stimuli used in the current design. Idiomatic expressions vary in degree of transparency/decomposability, and it seems clear
that the more transparent the expression, the easier it is to understand, and conversely, the
more opaque, the more difficult. A number of studies [43–44] demonstrate that the lexical
makeup of idioms is a matter of degree and largely depends on the idiom’s compositional
properties, and that idioms are accessed differentially according to their degree of syntactic
frozenness. These authors thus claim that idioms do not form a unique class of linguistic
items, but rather share properties with “more” literal language. Following this tradition, and
with Titone & Connine [7], we expect that comprehension difficulty will be a direct function
of the transparency/decomposability of the idiom [31, 33]. It can be assumed that the ability to understand idioms relies on the competence to process
these expressions beyond the literal interpretation of individual words. This means that under-
standing idiomatic expressions demands making inferences, as well as integrating contextual
information from both verbal and non-verbal sources. In our daily life, our knowledge about
the world around us is based on the semantic and conceptual information gathered through
our senses, implying that we perceive the world through different modalities [45]. Conse-
quently, it seems logical to expect that some of the difficulties that individuals with autism
demonstrate in figurative language may be due to a more generic deficit in multimodal inte-
gration. Indeed, problems with multi-sensory information have been documented in autism,
and specifically at speech-specific multisensory integration [46]. Irwin, Tornatore, Brancazio
& Whalen suggest that there are fundamental differences in audio-visual integration in chil-
dren and adolescents with autism in comparison to typical peers [47]. Furthemore, individuals
with autism have been shown to present with a reduced McGurk effect in the processing of
audio-visual speech [48]. It has also been suggested that the neurodevelopmental abnormalities
in autism may be due to poor information integration across brain regions [49]. Van Der
Smagt, Engeland & Kemner [50] further suggest that any problems arising from integrating
auditory and visual information must stem from higher processing stages, as is the case in pro-
cessing complex linguistic material, and not low-level integration processes (e.g., at the level of
individual sound/visual stimulus). Can You Play with Fire? Figurative Language Comprehension in Autism of participants within the broad group of individuals with autism by focusing on the higher
end of the spectrum (high functioning individuals with intact structural language) in two
homogeneous chronological age samples. Thus, we are interested in establishing a potential
developmental trajectory, albeit through a cross-sectional design. Consistent with the evidence from previous research, we expected to find effects of age and
group. In particular, we hypothesized that the two groups with autism will perform significantly
worse than the age- and verbal abilities-matched control groups. Specifically, we expected to
find higher reaction times (RTs) and error means in the participants with autism compared to
their typically developing peers, despite an improvement with age, as observed in the case of
metaphors in a recent study (Chahboun et al., [25]). Furthermore, it can be expected that, since
individuals with HFA have a tendency for literal interpretation, they will have difficulties in
appreciating the figurative nature of non-literal language and more often select interpretations
that match the literal meaning of the expression. Given that figurative language skills develop
constantly through childhood, we also expect that younger participants (the two children
groups) will perform at a lower level than the young adult groups. While cross-modal tasks, which require the integration of information from different
modalities may not present a problem for typically developing participants, they may tax pro-
cessing in individuals with autism and make the task more demanding. Concerning the pres-
ence of visually supported meaning (pictures that matched the interpretation of the idioms), it
can be expected that visual context may facilitate the processing of the figurative meaning, or,
at least, will not present an additional problem, as suggested by Lo´pez & Leekam [36] and indi-
cated by the results in Brock et al [40], also consistent with the findings in Norbury about the
facilitatory role of context [29]. Alternatively, it may be the case that the presence of both audi-
tory and visual information might rather present a problem, as a result of the demand for
information integration in the face of documented problems in that domain in autism. Thus,
we anticipated higher error rates and RTs in the condition when stimuli were presented in a
cross-modal fashion (auditory stimulus/visual target) in the groups with autism, and the
absence of such an effect in the control groups. One of the most important criteria in the processing of figurative language is expression
transparency. We expected that degree of transparency would impact both on reaction laten-
cies and accuracy in both participants with autism and controls. More specifically, the most
transparent category, the one labelled biological idioms would be easiest to process, while cul-
tural idioms, which are more highly conventionalised and less transparent would, in compari-
son, present greater difficulty, especially for the child participants and participants with
autism. We also expected novel metaphors and instructive idioms to be more easily processed
than the less transparent cultural idioms. Hypotheses In this study, we investigate the interaction of linguistic and visual context in the interpretation
of figurative expressions in highly-verbal individuals with autistic spectrum disorder compared
to typically developing peers. The aim of this research is to test a more homogeneous sample 6 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 local
background (local cultural practices), to account for the grounding of language in human
Table 1. Mean (and standard deviation) of background measures for each age and group. HFA (high functioning autism), FSIQ (intelligence quotient,
Full Scale score), VCI (verbal comprehension index), RG (receptive grammar scores), RC (reading comprehension scores). Note: p < .05 for scores with the
same superscript. Children
Adults
HFA
Control
HFA
Control
n
25
19
20
20
Age (months, SD)
11,3 ±0,96
11,9±0,89
18,1±1,65
18,3±1,92
Gender (M)
21
17
17
14
FSIQ
110,71±14,58
105,76±11,56
108,3±13,39
118,34±8,46
VCI
118,7±17,47
107,4±15,03
122,62±16,71
117,63±12,97
RG
72,72±5,56
74,31±4,30
74,40±3,53a
78,42±1,74 a
RC
0,58±0,17
0,59±0,16
0,63±0,15
0,72±0,12
doi:10.1371/journal.pone.0168571.t001 Table 1. Mean (and standard deviation) of background measures for each age and group. HFA (high functioning autism), FSIQ (intelligence quotient,
Full Scale score), VCI (verbal comprehension index), RG (receptive grammar scores), RC (reading comprehension scores). Note: p < .05 for scores with the
same superscript. Children
Adults
HFA
Control
HFA
Control
n
25
19
20
20
Age (months, SD)
11,3 ±0,96
11,9±0,89
18,1±1,65
18,3±1,92
Gender (M)
21
17
17
14
FSIQ
110,71±14,58
105,76±11,56
108,3±13,39
118,34±8,46
VCI
118,7±17,47
107,4±15,03
122,62±16,71
117,63±12,97
RG
72,72±5,56
74,31±4,30
74,40±3,53a
78,42±1,74 a
RC
0,58±0,17
0,59±0,16
0,63±0,15
0,72±0,12
doi:10.1371/journal.pone.0168571.t001 Table 1. Mean (and standard deviation) of background measures for each age and group. HFA (high functioning autism), FSIQ (intelligence quotient,
Full Scale score), VCI (verbal comprehension index), RG (receptive grammar scores), RC (reading comprehension scores). Note: p < .05 for scores with the
same superscript The individuals with HFA and their typically developing peers were matched on age, gen-
der and the verbal comprehension based on the Wechsler scale (WISC-IV [52] or WAIS-IV
[53] were used, depending on the chronological age of the participant). The Verbal Com-
prehension Index (VCI) was used as an overall measure of verbal reasoning and includes
measures of semantic/conceptual reasoning (similarities and vocabulary), and verbal compre-
hension. The descriptive statistics are given in Table 1. The matching of the groups was based
on the Wilcoxon Test and the smallest p value was p = .222 suggesting that there were no sig-
nificant differences observed between the groups (See Table 1). We also considered reading
comprehension scores and receptive grammar (based on CEG, which is the Spanish adaptation
of TROG [54]) as background measures to ensure our participants display scores within the
norm (see Table 1). No differences were observed between participants for reading compre-
hension. However, despite all our participants being within the norm for receptive grammar,
differences were observed between the two groups of young adult participants. For this reason,
we included receptive grammar as a co-variate in the analyses of the data. Participants Two age groups of HFA (n = 45) and typically developing (TD) controls (n = 39) were
recruited, all native speakers of Spanish. The first age group included children in the age range
ten to twelve years. The second age group included young adults from sixteen to twenty-two
years old (see Table 1 for the participants’ descriptive and demographic data). Participants and
their legal guardians (usually the parents) provided written informed consent for entry into
the study according of the declaration of Helsinki principles. The study was approved by the
Andalusian Biomedical Research Ethics Committee. The diagnosis of HFA was confirmed
with the Autism Diagnostic Observation Schedule (ADOS) [51] (Children, x = 10,8; Young
adults, x = 12,3). 7 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism The individuals with HFA and their typically developing peers were matched on age, gen-
der and the verbal comprehension based on the Wechsler scale (WISC-IV [52] or WAIS-IV
[53] were used, depending on the chronological age of the participant). The Verbal Com-
prehension Index (VCI) was used as an overall measure of verbal reasoning and includes
measures of semantic/conceptual reasoning (similarities and vocabulary), and verbal compre-
hension. The descriptive statistics are given in Table 1. The matching of the groups was based
on the Wilcoxon Test and the smallest p value was p = .222 suggesting that there were no sig-
nificant differences observed between the groups (See Table 1). We also considered reading
comprehension scores and receptive grammar (based on CEG, which is the Spanish adaptation
of TROG [54]) as background measures to ensure our participants display scores within the
norm (see Table 1). No differences were observed between participants for reading compre-
hension. However, despite all our participants being within the norm for receptive grammar,
differences were observed between the two groups of young adult participants. For this reason,
we included receptive grammar as a co-variate in the analyses of the data. Apparatus and stimuli
In a pilot study with 50 adults, all native speakers of Spanish, we determined the degree of
familiarity and frequency of the figurative expressions to be used in the main study. We
selected 124 idioms and instructive expressions and 20 metaphors, for which participants in
the pilot study had to determine: 1. “Do you know this expression?” 2. “If yes, do you know
what it means?” 3. “Do you use this expression yourself?”. Participants were asked to answer
truthfully, and to take into account that there were no wrong answers. We established ratings
for each expression by assigning numerical values to the answers (1 for yes and 0 for no) and
averaging the scores from the 3 questions. The idiomatic expressions which received a rating
of over 0.80 were selected for the study and classified into three main categories: a/ biological
idioms; b/ cultural idioms; and c/ instructive expressions, as used in Vulchanova et al. [8]. For
the first two categories they followed the typology adopted in Penttila¨, Nenonen & Niemi [55],
in turn inspired by Searle’s idea of deep background (the human biological nature) vs. Apparatus and stimuli In a pilot study with 50 adults, all native speakers of Spanish, we determined the degree of
familiarity and frequency of the figurative expressions to be used in the main study. We
selected 124 idioms and instructive expressions and 20 metaphors, for which participants in
the pilot study had to determine: 1. “Do you know this expression?” 2. “If yes, do you know
what it means?” 3. “Do you use this expression yourself?”. Participants were asked to answer
truthfully, and to take into account that there were no wrong answers. We established ratings
for each expression by assigning numerical values to the answers (1 for yes and 0 for no) and
averaging the scores from the 3 questions. The idiomatic expressions which received a rating
of over 0.80 were selected for the study and classified into three main categories: a/ biological
idioms; b/ cultural idioms; and c/ instructive expressions, as used in Vulchanova et al. [8]. For
the first two categories they followed the typology adopted in Penttila¨, Nenonen & Niemi [55],
in turn inspired by Searle’s idea of deep background (the human biological nature) vs. local
background (local cultural practices), to account for the grounding of language in human
experience and practice. The stimuli in these two categories were selected by 2 independent
expert linguist raters (91% interrater agreement). Examples of biologically-based idioms are
e.g., estar con el agua hasta el cuello (to be with water up to the neck); or venir como anillo al
dedo (to come as ring to the finger), and of culturally-based idioms, e.g estar como una cabra
(to be like a goat). The difference between the two categories is that, while biological idioms
are derived from human (bodily) experience and interaction with the environment, and as PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 8 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism such, tend to be more transparent and available to compositional parsing, cultural idioms are
more idiosyncratic, tend to vary from one culture to another, and are, in general, less transpar-
ent. Our third category were instructive expressions (i.e., proverbs), whose meaning can be
computed on-line, by drawing inferences on the basis of their individual constituent phrases
(e.g., Las mentiras tienen las patas cortas—Lies have short legs—). Apparatus and stimuli The inclusion of this cate-
gory was dictated by the need to compare such expressions, whose interpretation may be more
directly available in appropriate context, to the comprehension of idioms. Finally, novel meta-
phors were included as a category that may be assumed to be less frozen/fixed, and as such
more readily interpretable. From the pilot study, we selected for inclusion in the novel meta-
phors category those metaphors which earned a rating below .20 on the familiarity and fre-
quency scale (e.g.: Estas flotando en el aire—You are floating in the air). The study was designed as a sentence-picture matching task (38 visual—presented as text—
and 38 auditory expressions (a total of 76 expressions—20 biological idioms/20 cultural idi-
oms/16 instructive expressions/20 metaphors) randomized and counterbalanced between
groups and participants). Each expression was linked to four possible images: One reflecting
the figurative target meaning of the expression, a second image reflecting the literal meaning, a
third image corresponding to a figurative meaning, but not the target one, and finally, a dis-
tractor as forth image (Cf. Fig 1). Each expression was presented in a short appropriate context
(See S1 Appendix for examples). Thus, the study design included both verbal (presented as
text/discourse) and visual (pictures) context. Matlab (R2014b) (matrix laboratory) was used as a multiparadigm numerical programming
language to design, build and run the experiment. Results The data of both the control and experimental groups (n = 84) were analysed with R [56, 57];
and the lme4, afex and lmerTest packages [58]. We used two performance measures in the
study; reaction time which reveals the ease with which the figurative expression is processed,
and accuracy which essentially measures the ability to link the figurative expression to its target
image. We ran linear mixed models on reaction times and generalized linear mixed models on
accuracy, including the variables of Group (ASD vs. control), Age (Children vs. Young
Adults), Modality (Visual vs Auditory), and Type of Expression (Biological, Cultural, Proverbs,
and Novel Metaphors). We report relevant F and p-values below (based on the Satterthwaite
approximation for degrees of freedom) for reaction times and Chi square and p-values for
accuracy. We ran four analyses. The first one, the reaction time analysis, provided us with a general
overview of the ease with which the participants responded to the different experimental
manipulations in the study. The next analyses explore the ability to identify the correct target
compared to other options. Thus, a second model provided us with participants’ accuracy and
compared overall target and non-target responses. The third and fourth specifically explore
correct to other specific non-appropriate responses. The third analysis, called “comparison
between figurative target responses and literal responses”, provides a comparison between
responses to target images and images corresponding to the literal meaning. Finally, our fourth
model is based on a comparison between figurative target responses and figurative non-target
responses. This model only includes responses to images reflecting target meanings and
responses to images which reflected a figurative albeit not target meaning. Procedure In this experiment, we used visual stimuli (images and sentences) that reflected literal mean-
ings and idiomatic meanings. First, a fixation point (+) appeared for 500ms. Participants then
saw on the screen the idiom or metaphor. Depending on the experimental block, the partici-
pant heard the expression in context via loud-speakers or read it in context in a short text pre-
sented on the computer screen orthographically. Thus, the condition in which stimuli were
presented auditorily presented a cross-modal environment whereby participants had to select
a visual target which matched a figurative expression they had heard via the loud-speakers. In
the auditory experimental block, first the figurative expression appeared on the computer
screen followed by a blank screen, while the expression in appropriate context were being pre-
sented auditorily. In the text (orthographic) condition both stimulus (expression and context)
and target picture were all in the same (visual) modality. First the expression appeared on the
screen, and was then followed by the context. Then, in both experimental conditions a fixation Fig 1. Images for the idiom “estar con el agua hasta el cuello!” (lit.: be with water up to the neck/fig.: drown in work)
doi:10.1371/journal.pone.0168571.g001 Fig 1. Images for the idiom “estar con el agua hasta el cuello!” (lit.: be with water up to the neck/fig.: drown in work)
doi:10.1371/journal.pone.0168571.g001 mages for the idiom “estar con el agua hasta el cuello!” (lit.: be with water up to the neck/fig.: drown in work) Fig 1. Images for the idiom “estar con el agua hasta el cuello!” (lit.: be with water up to the neck/fig.: drown in work)
d i 10 1371/j
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0168571 001 doi:10.1371/journal.pone.0168571.g001 9 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism point (+) reappeared in the middle of the screen followed by four images in each corner, each
one reflecting the figurative meaning (target) meaning, the literal meaning, figurative, but not
target, meaning, and a distractor image. The position of presenting the images was counterbal-
anced between participants and between the modality of presenting the context and the order
of the expressions. The participant’s task was to click on the stimulus he/ she considered most
appropriate for the meaning of the expression. Accuracy and reaction times were collected to
determine the ease of processing. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Reaction time analyses Reaction times were analysed using Log(RT) (ms) as a measure rather than reaction times to
correct for right skewedness of the reaction time data. Only correct responses are included in
the analyses. We also excluded extremely fast or slow responses. We excluded two trials with
reaction times longer than 18s and we calculated the Z value of Speed (Log(RT) (ms)) for each
participant (Zp) and for each item per age and group (Zi). If the sum of the squares of the Z val-
ues was smaller or equal than 8 (Zp
2+Zi
2 8), the trial was included, and values bigger than 8
were excluded. An overall linear mixed model analysis on Log (RT) (ms) performance has been done with
R, which provided us with the best fitting model by step-wise backward selection based on the
Akaike Information Criterion (See S2 Appendix). We were left with a model including a three-
way interaction of group, modality and type of expression, a two-way interaction of age and
type of expression, and all lower effects. Also included in the model were the by-subject ran-
dom intercepts and slopes for modality and by-item random intercepts and slopes for Age and
Group. The overall model reveals that there are differences in reaction latency for Age (children/
young adults) (F (1, 82.64) = 20.38, p < .001) (Satterthwaite approximation for degrees of 10 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism Fig 2. Average reaction times (ms) for each age (children/young adults) and group (control/HFA). Error bars
denote one standard error of the mean. doi:10 1371/journal pone 0168571 g002 Fig 2. Average reaction times (ms) for each age (children/young adults) and group (control/HFA). Error bars
denote one standard error of the mean. d i 10 1371/j
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0168571 002 Fig 2. Average reaction times (ms) for each age (children/young adults) and group (control/HFA). Error ba
denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g002 freedom) and Group (control/HFA) (F (1, 86.85) = 10.64, p = .001), with slower responses by
children and individuals with autism (see Fig 2). Furthermore, an interaction between age and
type of expression was found (F (3, 66.11) = 2.903, p = .041) (see Fig 3). A multiple comparison
with Tukey contrasts showed that the interaction is due to differences between different types
of expressions in children, but not adults. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Reaction time analyses Here, a significant difference was observed between
novel metaphors and instructive expressions (p = .0014). In addition, a marginally significant
difference between cultural idioms and novel metaphors was observed (p = .07) showing that
instructive expressions were the most demanding and difficult to process for the children. freedom) and Group (control/HFA) (F (1, 86.85) = 10.64, p = .001), with slower responses by
children and individuals with autism (see Fig 2). Furthermore, an interaction between age and
type of expression was found (F (3, 66.11) = 2.903, p = .041) (see Fig 3). A multiple comparison
with Tukey contrasts showed that the interaction is due to differences between different types
of expressions in children, but not adults. Here, a significant difference was observed between
novel metaphors and instructive expressions (p = .0014). In addition, a marginally significant
difference between cultural idioms and novel metaphors was observed (p = .07) showing that
instructive expressions were the most demanding and difficult to process for the children. As we have pointed out, a three-way interaction between type of expression, group and
modality was observed (p < .001) (see Fig 4A and 4B). In order to establish the origin of the
three-way interaction, additional multiple comparisons with Tukey contrasts were run. For
the HFA group in the auditory modality, no significant differences were observed between dif-
ferent types of expressions. However, for the visual modality, an almost significant difference
was observed between instructive expressions and novel metaphors (p = .0501), with novel
metaphors processed faster than instructive expressions. For the control group in the auditory
modality, significant differences were observed between instructive expressions and biological
idioms (p = .013), with slower responses in the instructive expressions category, and between
novel metaphors and cultural idioms (p = .033), with faster responses in the novel metaphor
category, and between novel metaphors and instructive expressions (p < .001), with faster
responses for novel metaphors. No significant differences were observed in the visual modality PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 11 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism Fig 3. Average reaction times (RTs) for each age (children/young adults) and group (control/HFA) for
each type of expression. Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g003 Fig 3. Average reaction times (RTs) for each age (children/young adults) and group (control/HFA) for
each type of expression. Comparison between figurative target responses and non-target
responses An overall generalized linear mixed model analysis on ACC (mean errors) performance has
been done with R, which provided us with the best fitting model by step-wise backward selec-
tion based on the Akaike Information Criterion. We were left with a model including a two-
way interaction of age and group and main effects. Also included in the model were the by-
subject random intercepts and slopes for modality and by-item random intercepts and slopes
for Age and Group. The overall model thus reveals differences in accuracy in Age (children/young adults)
(χ2 (1, 23) = 5.73, p = .0016) and Group (control/HFA) (χ2 (1, 23) = 11.21, p < .001), with
more errors by children and individuals with autism (see Fig 5). In addition, a main effect of
type of expression was observed as well (χ2 (1, 21) = 8.49, p = .036). Multiple Comparisons of
Means with Tukey contrasts (See S3 Appendix) revealed that this effect was due to marginally
significant differences between instructive expressions and biological idioms (p = .07), and be-
tween instructive expressions and novel metaphors (p = .06), with more errors in the instruc-
tive expressions category. Moreover, a two-way interaction between age and group was observed (χ2 (1, 23) = 4.98,
p = .025). Multiple Comparisons of Means with Tukey contrasts (See S3 Appendix) revealed
that the interaction was due mainly to the differences between young adults (HFA and control)
(p < .001), and between the two control groups (children and young adults) (p = .01). No dif-
ferences were observed between the two HFA groups (p = 0.99) or children (HFA and control)
(p = 0.74). These results suggest that participants with autism perform at the same level regard-
less of age. Can You Play with Fire? Figurative Language Comprehension in Autism more transparent expressions processed faster, this pattern is absent in participants with
autism. Moreover, we explored the differences between groups and modalities for each type of
expression. For biological idioms, while the control group was faster in the auditory modality,
the HFA group was faster in the visual modality (p = .026), but always significantly slower than
controls (p = .001). For cultural idioms, the HFA group was significantly slower than controls
(p = .001). In addition, no modality differences were observed in the HFA group for this type
of expression, while the control group were faster in the visual modality (p = .02). For instruc-
tive expressions, the control group was significantly slower in the auditory modality (p = .01),
while the HFA group showed no modality preference in this category and was slower in com-
parison to the control group overall in this type of expression (p = .015). Finally, for the novel
metaphor category, again the HFA group was much slower overall (p < .001), and showed no
modality differences, while the control group showed faster responses in the auditory modality
(p = .01). To sum up those results, with increase of expression non-transparency, the advantage
of modality changes only for controls. Thus, for controls transparent expressions are processed
faster in the oral modality and less transparent expressions are processed faster in the ortho-
graphic modality. No such trend is observed for the participants with autism. These results
suggest a different processing pattern in autism. Reaction time analyses Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g003 doi:10.1371/journal.pone.0168571.g003 in the control group between different types of expressions. These results suggest a different
interaction pattern between participants with autism and controls. While for controls there is
differential processing depending on type of expression in the oral language modality, with Fig 4. 4a, 4b. Average reaction times (ms) group (control/HFA) type of expression and modality. Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g004 Fig 4. 4a, 4b. Average reaction times (ms) group (control/HFA) type of expression and modality. Error bars denote one standard error of the mean. d i 10 1371/j
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0168571 004 Fig 4. 4a, 4b. Average reaction times (ms) group (control/HFA) type of expression and modality. Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g004 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 12 / 24 Comparison between figurative target responses and literal responses In order to establish responses to target figurative meaning contra literal interpretations, we
re-ran the model, while excluding non-target figurative, and distractor responses (See S4
Appendix). In this model, Target responses were scored with 1 and literal responses were
scored with 2. The model includes a significant three-way interaction between modality, age PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 13 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism Fig 5. Average response accuracy (mean errors) for each age (children/young adults) and group
(control/HFA). Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g005 Fig 5. Average response accuracy (mean errors) for each age (children/young adults) and group
(control/HFA). Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g005 doi:10.1371/journal.pone.0168571.g005 and group (control/HFA), and all lower interactions and main effects. Also included in the
model were the by-subject random intercepts and slopes for modality and by-item random
intercepts and slopes for Age and Group. This model reveals a main effect of group (control/HFA) ((χ2 (1, 26) = 5.22, p = .022), with
more literal responses by children and young adults with autism and a marginally significant
difference in accuracy between Age (children/young adults) ((χ2 (1, 26) = 3.51, p = .06) (see
Fig 6). In addition, a main effect of type of expression was observed (χ2 (1, 24) = 10.37, p =
.015). Multiple Comparisons of Means with Tukey contrasts (See S4 Appendix) revealed that
this effect was due to a difference between instructive expressions and novel metaphors (p =
.02), with more literal responses in the instructive expressions category, and to a marginally
significant difference between cultural idioms and novel metaphors, with more literal re-
sponses in the cultural idioms category. These results suggest that less transparent expressions
are likely to trigger more literal responses. Furthermore, a two-way interaction between age and group was observed (χ2 (1, 26) = 4.89,
p = .02). We ran multiple comparisons of means with Tukey contrasts which revealed that this
interaction was due to a significant difference between control young adults and HFA young
adults (p = .015), with the young adults with autism giving more literal responses than their
respective typically developing peers. Also, the difference between both ages of control partici-
pants was significant (p = .03), where the young adults detected the figurative target image
more accurately. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Comparison between figurative target responses and literal responses No differences were observed between the two HFA groups (p = 0.99) or chil-
dren (HFA and control) (p = 0.99). These results suggest that children and participants with
autism are more likely to provide literal responses. 14 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism Fig 6. Average literality for each age (children/young adults) and group (control/HFA). Target responses
were scored with 1 and literal responses were scored with 2. Higher means indicate more literal responses. Error bars denote one standard error of the mean. Fig 6. Average literality for each age (children/young adults) and group (control/HFA). Target responses
were scored with 1 and literal responses were scored with 2. Higher means indicate more literal responses. Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g006 A three-way interaction between age, group and modality was observed (χ2 (1, 26) = 3.97,
p = 0.046) (see Fig 7). Again, a multiple comparison of means with Tukey contrasts showed no
significant effects in the visual modality in any of the age groups, but clear differences in the audi-
tory modality between the control young adults and the young adults with autism (p = 0.001),
where the older groups of participants had inverted patters of responses. The young adults with
HFA were less literal in the visual modality than the auditory modality, while the control young
adults were better in the auditory modality, suggesting a differential pattern in response. More-
over, a marginally significant difference was observed between the control children and the con-
trol young adults (p = .055), where the children did not show modality differences, but the older
group was better in the auditory modality. Comparison between figurative target responses and figurative non-
target responses The final part of the analyses compared figurative target responses and figurative non-target
ones. Therefore, we ran the model again without responses to the literal image and responses to
the distractor (See S5 Appendix). From the overall model it is clear again, that there are differ-
ences in accuracy between groups (control/HFA) (χ2 (1, 11) = 6.49, p = .01), with more errors
by individuals with autism. Additionally, a main effect of age (children/young adults) was found
as well (χ2 (1, 11) = 12.92, p < .001 and (see Fig 8), with more errors in the two younger groups. These results suggest that figurative language comprehension is not yet stabilized and still 15 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism developing in the child participants. No other significant effects were found in this comparison. Despite this, figurative non-target responses only appeared in very few instances (4.5% of the
responses in comparison with 81.8% for figurative target ones). Consequently, the comparison
between figurative target and figurative non-target responses shows a very small error range. General linear model—Receptive grammar as a covariate We were also interested in the extent to which the difference between the two young adult
groups on receptive grammar influenced these groups’ performance on the tasks. Repeated
measures ANOVA were run in SPSS [59], where we considered age and group as between sub-
ject factors, modality and type of expression as within subject variables and receptive grammar
as a covariate. Bonferroni confidence interval adjustment was considered and observed power
was calculated in this analysis (See S6 Appendix). We followed the same procedure for all the
previously considered dependent variables (reaction time, overall accuracy, scores for target
and literal responses, and scores for target and figurative non target responses). Receptive grammar as a covariate did not affect in any way any of the models and none of
the main effects or interactions changed with the inclusion of receptive grammar scores as a
covariate in the regression model. We report the power effects analyses in S7 Appendix. Discussion Our main hypothesis was that we would find poorer performance on figurative language pro-
cessing by children and the participants with autism compared to their peers, carefully Fig 7. Average literality for each age (children/young adults) and group (control/HFA) and modality
(visual/auditory). Target responses were scored with 1 and literal responses were scored with 2. Higher
means indicate more literal responses. Error bars denote one standard error of the mean. doi:10 1371/journal pone 0168571 g007 Fig 7. Average literality for each age (children/young adults) and group (control/HFA) and modality
(visual/auditory). Target responses were scored with 1 and literal responses were scored with 2. Higher
means indicate more literal responses. Error bars denote one standard error of the mean. doi:10.1371/journal.pone.0168571.g007 doi:10.1371/journal.pone.0168571.g007 16 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism Fig 8. Average accuracy in figurative meanings for each age (children/young adults) and group
(control/HFA) and modality (visual/auditory). Target responses were scored with 1 and literal responses
were scored with 0. Higher means indicate more accurate responses. Error bars denote one standard error of
the mean. Fig 8. Average accuracy in figurative meanings for each age (children/young adults) and group
(control/HFA) and modality (visual/auditory). Target responses were scored with 1 and literal responses
were scored with 0. Higher means indicate more accurate responses. Error bars denote one standard error o
the mean. doi:10.1371/journal.pone.0168571.g008 doi:10.1371/journal.pone.0168571.g008 matched on age, non-verbal IQ, and verbal comprehension. Consistent with our hypotheses,
the analyses of reaction times in the current study found a main effect of age and group, with
slower responses for children and participants with autism. While no significant difference
was observed between the two children’s groups on a test of receptive grammar, there was a
difference between the two young adult groups of participants, and oral language differences
could have explained the different performance between these groups. However, the fact that
our results were not affected by the inclusion of receptive grammar scores as a co-variate in
the analysis, ensures that the obtained results were not due to a structural language problem
(e.g., in receptive grammar), but to the experimental manipulation. These analyses also revealed that two types of expressions were particularly difficult for chil-
dren, cultural idioms and instructive expressions (proverbs), while novel metaphors were pro-
cessed much faster. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Discussion More importantly, both the reaction
latencies and accuracy analyses reveal a different processing pattern in the interaction between
modality (auditory/visual) and type of expression, especially evident in the young adult groups. This pattern of responses is suggestive of different processing or perhaps alternative strategies
for the comprehension of figurative language in autism, despite overall high accuracy. We consider that this different pattern of processing is due, in part, to a delayed trajectory
in developing the necessary skills to process figurative language. This is clearly seen in the sim-
ilar response pattern of the young adults with autism and the control children on both accu-
racy and reaction speed. This may suggest that rather than an inability to process figurative
language or idioms altogether, in this case, there is a delay in the ability to detect such expres-
sions, and to differentiate between the different categories of figurative language. These results
support the findings from another study with the same participants on metaphorical language
[25]. Yet, even though participants with autism seem to improve with age in terms of reaction
time, suggesting faster processing, they still do not reach the same levels of performance as
control matched peers. The improvement with age does not only apply to the individuals with
HFA, but also to the control groups, which is consistent with Nippold & Duthie [63], Levorato
& Cacciari [64] and Vulchanova et al [8], and our original hypothesis, but not with Vogindrou-
kas & Zikopoulou [26], who, surprisingly, did not find any reliable difference in performance
between the younger and older controls. Furthermore, our study explored the impact of the nature and the transparency of the type
of figurative expression. In the study we included biological idioms, which have been argued
to be more closely grounded in human experience and interaction with the environment
(Searle’s deep background). We expected such expressions to be more readily accessible and
more decomposable, despite their idiomatic nature, compared to cultural idioms, also for the
participants with autism. In line with Chahboun et al [25], we also expected that novel meta-
phors would be easier to process. These expectations were confirmed. Even though the partici-
pants with autism were significantly delayed in comparison to controls, they displayed similar
patterns of difficulties and ease across the test categories as the control groups. Biological idi-
oms and novel metaphors were the easiest to process. Discussion These results suggest that the impact of expression transparency tends to
diminish with age and as figurative skills stabilize in young adulthood. The analyses of accuracy revealed a similar pattern: more errors were found in the two
child groups and in the two groups with autism. Here a main effect of type of expression was
also observed, whereby instructive expressions emerged as the most difficult category to pro-
cess in comparison to both novel metaphors and biological idioms. The analyses of accuracy
also revealed that participants with autism were significantly more likely than controls to select
the literal interpretation of the figurative expression, as represented in a visual image. 17 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism In this study, we hypothesized that the participants with autism would display a relative
weakness in performance on idiomatic language, despite intact structural language skills. Although individuals with autism obtain high accuracy scores and can obviously solve the
task, the current findings confirm that the adult individuals with autism are not reaching an
equivalent level of performance to typically developing matched controls, both with regard to
accuracy and reaction time. These results are consistent with results from the few previous
studies of idiom comprehension in autism, where the participants with HFA performed more
poorly and more slowly than their typically developing matched peers, suggesting a different
pattern of processing figurative language in general, and idiomatic competences, in particular. These results are also consistent with the finding that the contrast in ability in autism in com-
parison to controls is more evident on the higher end of the spectrum [60]. The current results, however, contradict findings of comparable semantic and language
processing skills in studies where the controls and participants with autism are matched on
structural language skills [27,40]. The participants in the current study were highly-verbal indi-
viduals with autism carefully matched to the control groups on IQ and verbal skills, including
semantic, and oral and reading comprehension skills. The current results thus challenge the
idea that the problems in figurative language in autism arise exclusively from verbal status and
language skills, also consistent with results in Eigsti, de Marchena, Schuh & Kelley [61] and
Olofson et al. [62] and the review in Vulchanova et al. [10]. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Discussion We reason that these two categories are
the most transparent, and their meaning can be easily inferred from the context, while cultural
idioms are less transparent and more “frozen” [65]. 18 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism These findings are consistent with Vulchanova et al. [8] and Vega-Moreno [17] who claim
that inferential skills are not only involved, but mandatory in idiom comprehension. In our
study, the figurative expressions were elicited based on a pilot, where we selected the most
familiar and frequent expressions for the idioms category, and the least frequent (most novel)
for the metaphors category. The current results confirm that novel metaphors were easier to
process compared to less transparent expressions, such as idioms. Our findings are partly con-
sistent with Giora, Gazal, Goldstein, Fein, & Stringaris [66], who found that familiarity/novelty
of the expression were reliable predictors of performance in both typically developing individ-
uals and individuals with HFA. The current results are also consistent with the findings in
Norbury [29], where participants with autism were facilitated by the presence of context in the
comprehension of novel idioms and performed at levels similar to controls. The current find-
ings, however, contradict the results obtained by Mashal & Kasirer [24]. These authors found
that the most novel figurative expressions were the most difficult to process in both adults and
children with HFA. However, this difference might be due to experimental design and choice
of experimental stimuli, as is often the case in research on figurative language. Contrary to our expectations, instructive expressions (proverbs) were the most difficult cat-
egory to process, along with cultural idioms. We included this category in the design because
of the expectation that they may not be stored in the mental lexicon, but rather rely on the
immediate activation of richer “memory organization packages” [21,67], thus allowing more
readily for interpretations on the fly when encountered in appropriate context. This expecta-
tion was not confirmed, however. Moreover, both children and participants with autism were
significantly more likely to select the literal interpretations of both instructive expressions and
cultural idioms. These results support the findings in Honeck, Welge & Temple [68] concerning the pro-
cessing of proverbs, where participants were faster to make appropriateness judgements when
the proverb was related literally to the context compared to when it was related figuratively. PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Discussion These authors interpret the findings as supporting a multi-stage model of proverb comprehen-
sion, with literal interpretations becoming available faster than figurative ones. The current
results suggest that instructive expressions (proverbs) indeed require some ability to think
abstractly [20] and cannot be interpreted so readily based on their constituent words. We also
find evidence that this skill takes time to acquire, as evident in the child participants. We also
speculate that the more culturally-based an expression is, the more reliant it is on learning it in
social interaction, thus consistent with the limitations documented in this domain in autism. Regarding the effect of modality, we hypothesized that the control group would not display
dissociations between the visual and auditory presentation modalities, while for the partici-
pants with autism we expected certain problems when the expressions were presented audito-
rily and had to be matched to a visual stimulus (a picture) in the cross-modal condition. This
was confirmed, but only for the most transparent expressions (biological idioms and novel
metaphors). The control groups performed better when biological idioms and novel meta-
phors were presented in the auditory modality. Thus, it appears that the control group have an
advantage for oral language, and can easily integrate oral stimuli with visual contextual support
(the images). This was not the case for the participants with autism, and it could suggest a a dif-
ficulty even in highly-verbal individuals with autism in multimodal information integration,
even when the idiomatic expressions are decomposable (cf. also Ozonoff & Miller, [38]). These
differential responses to modality in the autism group could be indicating the involvement of
different processes in the resolution of the idioms, or different approaches when attempting to
respond to this task. These findings point to the possibility of a problem of simultaneous processing in both
modalities in this population. An additional possibility may be that there are residual oral PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 19 / 24 Can You Play with Fire? Figurative Language Comprehension in Autism language problems also in individuals with intact structural language skills. This is consistent
with Eigsti [69], where the problems in ASD are attributed to low-level impairments in generic
cognitive and processing mechanisms. Alternatively, it can be assumed that “seeing” text is an
advantage, especially in the groups with autism. Discussion Indeed, there is evidence that orthographic
decoding is a strength in autism and this may, in turn, lead to better comprehension outcomes,
if a simple view of reading is adopted [70]. Another interesting result was the difference in degree of literalness observed in response
accuracy. As expected, the younger participants and participants with autism interpreted the
stimuli more often literally than the older and the control groups. Figurative language compe-
tence takes longer to develop and requires a number of other skills (inferencing, discourse
skills; [71]), and the older we are, the more resources for processing we have. In the case of par-
ticipants with autism, both children and young adults were very literal and did not appear to
improve in this respect with age (the two age groups in the study performed similarly). This
was not the case for the control groups. For children presentation modality did not affect
response literalness, while for the older participants there was an advantage in the auditory
modality, suggesting that idiom comprehension most probably resides in mature oral language
skills. The results of the participants with autism are also consistent with other research sug-
gesting a tendency for literal (compositional) interpretation in autism [26,72]. The overall results of this study support our hypothesis that idiomatic expressions require
more competences than just good structural language. These clearly provide a necessary condi-
tion, but may not be sufficient. This is evidenced by the findings supporting the presence of a
delay in the two groups with HFA. Additionally, the current study contributes to research
studying the processing of idioms specifically, and figurative language, more broadly, in pro-
viding empirical evidence on how different categories of figurative expressions are processed
in both highly verbal individuals with autism and typically developing individuals. We find
support that expressions, which are more firmly grounded in human bodily experience (bio-
logical idioms), and are also more transparent (novel metaphors) are processed more readily,
despite their idiomatic nature, regardless of participant group. Finally, we find evidence that
young adult controls have a clear advantage in processing auditory language stimuli, most
likely as a result of subtle differences in oral language skills. Discussion The problems observed in the par-
ticipants with autism are suggestive of either multi-sensory information integration difficulty
or simply a residual problem in oral language and need to be addressed in future research
potentially leading also to better target intervention for that group. Acknowledgments We would like to thank the participants and the families for their effort and time in participat-
ing in this study. We are also grateful to Martina Micai for help in the data collection. Last but
not least, we would like to thank schools and associations for putting us in contact with both
the HFA groups and the TD groups. Conceptualization: MV VV DS SC. Funding acquisition: MV VV DS. Investigation: SC. Methodology: MV VV DS SC. Project administration: MV VV DS. Resources: MV VV DS HE SC. Software: HE SC. Supervision: MV VV DS. Validation: MV VV HE SC. Visualization: MV VV DS SC. Writing – original draft: SC. Writing – original draft: SC. Writing – review & editing: MV VV DS HE SC. Author Contributions Conceptualization: MV VV DS SC. Data curation: HE SC. Formal analysis: MV VV HE SC. Funding acquisition: MV VV DS. Investigation: SC. Methodology: MV VV DS SC. Project administration: MV VV DS. Resources: MV VV DS HE SC. Software: HE SC. Supervision: MV VV DS. Validation: MV VV HE SC. Visualization: MV VV DS SC. Writing – original draft: SC. Writing – review & editing: MV VV DS 6.
Vulchanova M, Talcott JB, et al. Morphology in autism spectrum disorders: Local processing bias and
language. Cognitive Neuropsychology 2012; 29, 7–8, 584–600. doi: 10.1080/02643294.2012.762350
PMID: 23521055 Supporting Information Supporting Information
S1 Appendix. Examples of expressions in context. (DOCX)
S2 Appendix. Overall model (Reaction times). (RTF)
S3 Appendix. Overall model (Accuracy analysis: Target vs non-target). (RTF)
S4 Appendix. Overall model (Accuracy analysis: Target vs literal responses). (RTF)
S5 Appendix. Overall model (Accuracy analysis: Figurative target vs Figurative non-targ
responses). (RTF) S1 Appendix. Examples of expressions in context. (DOCX) S1 Appendix. Examples of expressions in context. (DOCX) S2 Appendix. Overall model (Reaction times). (RTF) S3 Appendix. Overall model (Accuracy analysis: Target vs non-target). (RTF) S3 Appendix. Overall model (Accuracy analysis: Target vs non-target). (RTF) S4 Appendix. Overall model (Accuracy analysis: Target vs literal responses). (RTF) S5 Appendix. Overall model (Accuracy analysis: Figurative target vs Figurative non-target
responses). (RTF) 20 / 24 PLOS ONE | DOI:10.1371/journal.pone.0168571
December 30, 2016 Can You Play with Fire? Figurative Language Comprehension in Autism PLOS ONE | DOI:10.1371/journal.pone.0168571
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The Epigenetic Regulation of HCC Metastasis
|
International journal of molecular sciences
| 2,018
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cc-by
| 11,699
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Received: 14 November 2018; Accepted: 7 December 2018; Published: 10 December 2018 Received: 14 November 2018; Accepted: 7 December 2018; Published: 10 December 2018 Abstract:
Epigenetic alterations,
such as histone modification,
DNA methylation,
and
miRNA-mediated processes, are critically associated with various mechanisms of proliferation
and metastasis in several types of cancer. To overcome the side effects and limited effectiveness of
drugs for cancer treatment, there is a continuous need for the identification of more effective drug
targets and the execution of mechanism of action (MOA) studies. Recently, epigenetic modifiers have
been recognized as important therapeutic targets for hepatocellular carcinoma (HCC) based on their
reported abilities to suppress HCC metastasis and proliferation in both in vivo and in vitro studies. Therefore, here, we introduce epigenetic modifiers and alterations related to HCC metastasis and
proliferation, and their molecular mechanisms in HCC metastasis. The existing data suggest that the
study of epigenetic modifiers is important for the development of specific inhibitors and diagnostic
targets for HCC treatment. Keywords: epigenetics; miRNA; DNA methylation; histone methylation; HCC; metastasis The Epigenetic Regulation of HCC Metastasis 1
Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea; tshan@kribb.re.kr (T.-S.H.);
banhs@kribb.re.kr (H.S.B.)
2
Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University,
Daegu 41944, Korea
*
Correspondence: KeunHur@knu ac kr (K H ); chohs@kribb re kr (H S C ); Tel : +82 42 879 8152 *
Correspondence: KeunHur@knu.ac.kr (K.H.); chohs@kribb.re.kr (H.-S.C.); Tel.: +82-42-879-8152
†
These authors contributed equally to this work. ulation of HCC Metastasis
, Keun Hur 2,*
and Hyun-Soo Cho 1,*
e and Biotechnology, Daejeon 34141, Korea; tshan@kribb.re.kr (T.-S.H.);
l Biology, School of Medicine, Kyungpook National University,
kr (K.H.); chohs@kribb.re.kr (H.-S.C.); Tel.: +82-42-879-8152
this work.
7 December 2018; Published: 10 December 2018
such as histone modification,
DNA methylation,
and
itically associated with various mechanisms of proliferation
ncer. To overcome the side effects and limited effectiveness of
a continuous need for the identification of more effective drug
m of action (MOA) studies. Recently, epigenetic modifiers have
eutic targets for hepatocellular carcinoma (HCC) based on their
metastasis and proliferation in both in vivo and in vitro studies.
netic modifiers and alterations related to HCC metastasis and
chanisms in HCC metastasis. The existing data suggest that the
ortant for the development of specific inhibitors and diagnostic
A methylation; histone methylation; HCC; metastasis
a common primary liver cancer, is the second leading cause of
d by chronic hepatitis B and C virus (HBV and HCV) infections
iabetes, and aflatoxin exposure [1,2]. In addition to surgical
b, a multikinase inhibitor, is recognized as an effective treatment
both in vivo and in vitro studies, suppressed cell proliferation,
tivation of VEGFRs, Raf-1, and B-Raf [3]. However, sorafenib
ered a serious problem that must be overcome for HCC therapy.
herapeutic targets and the development of specific inhibitors
veral papers reported that various epigenetic modifiers and
), histone modification, and DNA methylation) participate in
pigenetic regulation of oncogenes and tumor suppressor genes,
re important therapeutic targets for the development of specific
roduce HCC-related epigenetic modifiers (miRNA, DNA
d histone methyltransferases/demethylases) and suggest their
rticular, we summarize the molecular mechanisms and functions
astasis, suggesting their potential as therapeutic targets and ulation of HCC Metastasis
, Keun Hur 2,*
and Hyun-Soo Cho 1,*
e and Biotechnology, Daejeon 34141, Korea; tshan@kribb.re.kr (T.-S.H.);
l Biology, School of Medicine, Kyungpook National University,
kr (K.H.); chohs@kribb.re.kr (H.-S.C.); Tel.: +82-42-879-8152
this work. 7 December 2018; Published: 10 December 2018
such as histone modification,
DNA methylation,
and
itically associated with various mechanisms of proliferation
ncer. To overcome the side effects and limited effectiveness of
a continuous need for the identification of more effective drug
m of action (MOA) studies. Recently, epigenetic modifiers have
eutic targets for hepatocellular carcinoma (HCC) based on their
metastasis and proliferation in both in vivo and in vitro studies. netic modifiers and alterations related to HCC metastasis and
chanisms in HCC metastasis. The existing data suggest that the
ortant for the development of specific inhibitors and diagnostic
A methylation; histone methylation; HCC; metastasis
a common primary liver cancer, is the second leading cause of
d by chronic hepatitis B and C virus (HBV and HCV) infections
iabetes, and aflatoxin exposure [1,2]. In addition to surgical
b, a multikinase inhibitor, is recognized as an effective treatment
both in vivo and in vitro studies, suppressed cell proliferation,
tivation of VEGFRs, Raf-1, and B-Raf [3]. However, sorafenib
ered a serious problem that must be overcome for HCC therapy. herapeutic targets and the development of specific inhibitors
veral papers reported that various epigenetic modifiers and
), histone modification, and DNA methylation) participate in
pigenetic regulation of oncogenes and tumor suppressor genes,
re important therapeutic targets for the development of specific
roduce HCC-related epigenetic modifiers (miRNA, DNA
d histone methyltransferases/demethylases) and suggest their
rticular, we summarize the molecular mechanisms and functions
astasis, suggesting their potential as therapeutic targets and International Journal of
Molecular Sciences 1. Introduction Hepatocellular carcinoma (HCC), a common primary liver cancer, is the second leading cause of
death in cancer patients, and is caused by chronic hepatitis B and C virus (HBV and HCV) infections
and other factors, such as alcohol, diabetes, and aflatoxin exposure [1,2]. In addition to surgical
removal, chemotherapy using sorafenib, a multikinase inhibitor, is recognized as an effective treatment
for HCC. Treatment with sorafenib, in both in vivo and in vitro studies, suppressed cell proliferation,
metastasis, and angiogenesis via inactivation of VEGFRs, Raf-1, and B-Raf [3]. However, sorafenib
resistance in advanced HCC is considered a serious problem that must be overcome for HCC therapy. Therefore, the identification of new therapeutic targets and the development of specific inhibitors
for HCC is required [4]. Recently, several papers reported that various epigenetic modifiers and
alterations (e.g., microRNA (miRNA), histone modification, and DNA methylation) participate in
HCC proliferation and metastasis via epigenetic regulation of oncogenes and tumor suppressor genes,
suggesting that epigenetic modifiers are important therapeutic targets for the development of specific
inhibitors of HCC [5–7]. Thus, in this review, we introduce HCC-related epigenetic modifiers (miRNA, DNA
methyltransferases/demethylases, and histone methyltransferases/demethylases) and suggest their
clinical utility in HCC treatment. In particular, we summarize the molecular mechanisms and functions
of epigenetic modifiers in HCC metastasis, suggesting their potential as therapeutic targets and
diagnostic markers in HCC. Int. J. Mol. Sci. 2018, 19, 3978; doi:10.3390/ijms19123978 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 3978 2 of 17 2. MicroRNA miRNAs regulate gene expression at the posttranscriptional level by inhibiting the translation
or decay of transcripts, depending on the target sequence matching ratio. The biogenesis of
miRNAs has been extensively studied. In brief, primary miRNAs (pri-miRNAs) are generated
by RNA polymerase II and processed into precursor miRNAs (pre-miRNAs) by the RNase III
enzyme Drosha and the DiGeorge critical region 8 (DGCR8) microprocessor complex in nucleus [8,9]. Subsequently, pre-miRNAs are exported to the cytoplasm by exportin 5 [10], where they are
processed by Dicer and TAR RNA-binding protein (TRBP) into 21–24 nucleotide miRNA duplexes
that are unwound and incorporated into the RNA-induced silencing complex (RISC) to mature
miRNA [11,12]. However, defects in miRNA biogenesis machinery are frequently observed in various
cancers [13–15]. Furthermore, abnormal regulation of miRNA expression is linked to tumor progression
and metastasis [16,17]. In this miRNA section, we will discuss epithelial–mesenchymal transition
(EMT)-related miRNAs in HCC and their oncogenic or tumor suppressive functions (Table 1). Recent studies have indicated that abnormal regulation of miRNA-mediated TGF-β/Smad
signaling pathways induce malignant tumor development. Low expression of miR-542-3p and miR-142 2.1.1. TGF-β Signaling Pathway-Related miRNAs Transforming growth factor (TGF)-β signaling has been shown to play an important role in EMT. TGF-β induces cells to lose their epithelial characteristics and to acquire migratory behavior through
activating Smad (mothers against decapentaplegic homolog) signaling [18]. The major downstream
genes of TGF-β signaling are Smad2/3 and Smad4, which act as transcription factors to change the
expression of EMT-related genes, including those in the Snail and zinc-finger E-box-binding homeobox
(ZEB) families [19,20]. Table 1. Epithelial–mesenchymal transition (EMT)-related miRNAs and their target genes in
hepatocellular carcinoma (HCC). Signaling Pathway
miRNA
Regulation
Expression
Target Genes
Functions
References
TGF-β
miR-542-3p
-
Down
TGFB1
Proliferation, EMT
[21]
miR-142
DNA methylation
Down
TGFB1
Proliferation, EMT
[22]
miR-216/217
-
Up
Smad7, PTEN
Migration, EMT
[23]
miR-300
-
Down
FAK
Invasion, EMT
[24]
miR-199b-5p
-
Down
N-cadherin
EMT, Metastasis
[25]
miR-155
-
Up
E-cadherin
Invasion, EMT
[26]
miR-181a
TGF-β
Up
BIM
EMT
[27]
miR-630
TGF-β
Down
Slug
EMT
[28]
WNT
miR-122
-
Down
Wnt1
Migration,
Invasion
[29]
miR-148a
-
Down
Wnt1
Migration, EMT
[30]
miR-25
-
Up
RhoGDI1
Proliferation, EMT
[31]
Twist1
miR-26b-5p
Twist1
Down
Smad1
Invasion, EMT
[32]
miR-27a-3p
Twist1
Down
VE-cadherin
EMT
[33,34]
Snail
miR-1306-3p
FOXM1
Up
FBXL5
EMT, Metastasis
[35]
miR-122
-
Down
Snail1, Snail2
Proliferation, EMT
[36]
miR-30a, b
-
Down
Snail1
EMT, Metastasis
[37,38]
miR-153
-
Down
Snail1
Invasion, EMT
[39]
Slug
miR-140-5p
HBV/Unigene56159
Down
Slug
EMT
[40]
miR-506
KLF4
Down
Slug
EMT
[41]
Other
miR-345
-
Down
IRF1
EMT
[42]
Exosomal miRNAs
miR-103
-
Up
VE-Cad, p120,
ZO1
Migration,
Metastasis
[43]
miR-1247-3p
-
Up
B4GALT3
Metastasis
[44]
miR-320a
-
Down in
CAFs
PBX3
Proliferation,
Metastasis
[45]
Recent studies have indicated that abnormal regulation of miRNA-mediated TGF-β/Smad
signaling pathways induce malignant tumor development. Low expression of miR-542-3p and miR-142 Table 1. Epithelial–mesenchymal transition (EMT)-related miRNAs and their target genes in
hepatocellular carcinoma (HCC). Recent studies have indicated that abnormal regulation of miRNA-mediated TGF-β/Smad
signaling pathways induce malignant tumor development. Low expression of miR-542-3p and miR-142 Recent studies have indicated that abnormal regulation of miRNA-mediated TGF-β/Smad
signaling pathways induce malignant tumor development. Low expression of miR-542-3p and miR-142 3 of 17 Int. J. Mol. Sci. 2018, 19, 3978 is frequently found in HCC; these two miRNAs directly regulate the TGFB1 transcript by binding to
the 3’-UTR [21,22] and, therefore, negatively regulate the TGF-β/Smad signaling pathway, implicating
them in EMT and metastasis in HCC. 2.1.1. TGF-β Signaling Pathway-Related miRNAs However, it has been reported that Smad7, an inhibitor of
TGF-β signaling, is also regulated by miRNAs. High expression of miR-216a/217 promoted EMT, cell
migration, stem-like properties, and HCC recurrence by targeting the Smad7, and phosphatase and
tensin homolog (PTEN) tumor suppressor genes [23]. Several studies have shown that miRNAs regulate various downstream genes of TGF-β signaling,
but this does not occur through Smad signaling. For example, TGF-β1-induced EMT was suppressed
by miR-300 through targeting focal adhesion kinase (FAK), which modulates EMT [24]. Another
study found an inverse correlation between miR-199b-5p and the expression of its target gene,
N-cadherin, in HCC. Restoration experiments showed that miR-199-5p overexpression in HCC
suppressed TGF-β1-induced Akt phosphorylation, and inhibition of the PI3K/Akt pathway blocked
TGF-β1-induced N-cadherin overexpression [25]. Some studies have reported that miRNAs can be positively or negatively regulated by TGF-β
signaling. For example, treatment with recombinant TGF-β1 increased miR-155 and miR-181a
expression levels in HCC [26,27]. Overexpression of these miRNAs by TGF-β signaling led to increased
EMT, with similar phenotypes observed after TGF-β treatment in vitro, by blocking E-cadherin (CDH1),
and promoting Snail and ZEB1 in HCC. 2.1.2. WNT Signaling Pathway-Related miRNAs There are several lines of evidence that indicate that the Wnt/β-catenin signaling pathway plays
important roles in EMT [46]. In particular, the nuclear localization of β-catenin increases the expression
of target genes, such as fibronectin and matrix metalloproteinase-7 (MMP-7); consequently, cells
adopted mesenchymal-like phenotypes [47]. Wang et al. found that the downregulation of miR-122 enhanced the proliferation, migration, and
invasion of HCC. However, they showed that miR-122 overexpression decreased cell proliferation,
migration, and invasion by targeting Wnt1, and inhibiting EMT-related gene expression [29]. miR-148a
also has a binding site in the Wnt1 3’-UTR, and negatively regulates Wnt1 expression in HCC [30]. Therefore, the expression of miR-122 and miR-148a attenuates the Wnt signaling pathway by inhibiting
Wnt1 expression, thereby suppressing progression through EMT and cancer stem cell (CSC)-like
properties in HCC. The Wnt/β-catenin signaling pathway can regulate miRNA expression levels. For example,
miR-25 is significantly upregulated in human HCC tissues compared with normal liver tissues. The
functions of miR-25 include stimulating HCC cell growth and activating EMT by targeting Rho GDP
dissociation inhibitor alpha (RhoGDI1) [31]. miR-25 is upregulated by the Wnt/β-catenin signaling
pathway. miR-25 expression levels are positively correlated with β-catenin levels, and negatively
correlated with RHOGDI1 levels in HCC. 2.2. Metastasis-Related Exosomal miRNAs 2.2. Metastasis-Related Exosomal miRNAs Exosomes, a type of extracellular vesicle, are small vesicles less than 200 nm in diameter [49]. In
the human body, exosomes secreted by cells exist in body fluids, such as serum and urine, and contain
DNA, mRNA, noncoding RNA, and protein. Exosomes move to other cells and communicate via
their contents. A previous study showed that a high level of miR-103 was associated with a higher metastasis
potential of HCC. Exosomal miR-103 increases vascular permeability and promotes metastasis by
directly targeting endothelial junction proteins, including VE-cadherin (VE-Cad), p120-catenin (p120),
and zonula occludens 1 (ZO1). Therefore, miR-103 is a therapeutic target and metastasis marker
of HCC [43]. Another study showed that highly metastatic liver cancer cells secreted exosomal
miR-1247-3p, which affected cancer-associated fibroblasts (CAFs). The functions of miR-1247-3p
include suppressing B4GALT3 expression and promoting lung metastasis [44]. A previous report
showed that CAF-derived miR-320a is related to HCC progression and metastasis. A low level of
exosomal miR-320a of CAF promotes HCC progression and metastasis by targeting the PBX3-Erk1/2
pathway in HCC [45]. Thus, exosomal miRNAs might be used not only as diagnostic or prognostic
markers, but also as therapeutic targets for the treatment of malignant HCC. 2.1.3. Snail-, Slug-, and Twist1-Related miRNAs Snail1 is the critical point of convergence in EMT regulation and represses CDH1 expression at
the transcriptional level [48]. A previous report showed that miR-1306-3p activation by Forkhead box
protein M1 (FOXM1) directly inhibits the F-box/LRR-repeat protein 5 (FBXL5) gene, which regulates
Snail protein stability by binding to its 3’-UTR. Upregulation of miR-1306-3p by FOXM1 suppresses
FBXL5, resulting in increased Snail stability. Therefore, high expression of miR-1306-3p promoted EMT
progression [35]. miR-122 downregulation is associated with tumor invasion and metastasis in HCC. Functional studies have shown that the upregulation of miR-122 inhibits cell proliferation and EMT
by targeting the expression of Snail1 and Snail2 [36]. miR-30a and miR-30b have been reported to
negatively regulate Snail1 in HCC, but the downregulation of miR-30a and miR-30b in HCC affects
EMT by increasing Snail expression levels [37,38]. Furthermore, Snail1 is also negatively regulated by Int. J. Mol. Sci. 2018, 19, 3978 4 of 17 miR-153 in HCC. Ectopic expression of miR-153 inhibited migration, invasion, and EMT, and decreased
EMT marker levels [39]. Previously, miR-140-5p and miR-630 were found to directly bind to Slug and negatively regulate
its expression in HCC [28,40]. Another study showed that miRNAs upregulated by KLF4 included
miR-153, miR-506, and miR-200b, which target Snail1, Slug, and ZEB1 mRNA, respectively [41]. Recent reports have shown that Twist1 induces EMT and promotes metastasis in HCC because it
regulates various EMT-associated genes. Significant downregulation of miR-26b-5p and miR-27a-3p
has been found in HCC. Mechanistically, Twist1 could suppress these miRNAs by binding to their
promoter regions [32–34]. Previous studies showed that overexpression of miR-26b-5p and miR-27a-3p
suppressed EMT and invasion by targeting Smad1 and VE-cadherin, respectively. pp
y
g
g
p
y
In addition, high expression of miR-345 inhibited EMT and cell mobility by targeting
IRF1-mediated mTOR/STAT3/AKT signaling, and genes downstream of these pathways, including
Snail, Slug, and Twist, are related to EMT in HCC [42]. 3.1. DNMT1 To date, several studies on the roles of DNMT1 in HCC metastasis have been reported. In
CD133+/CD44+ cells, a subpopulation of HCC with CSC properties, a noncollagenous bone matrix
protein osteopontin (OPN), enhances HCC metastasis by regulating DNA methylation. Knockdown
of OPN in CD133+/CD44+ cells suppressed sphere formation and migration by inhibiting DNMT1
expression, which reduced the methylation of tumor suppressor genes such as RASSF1, GATA4, and
CDKL2 [56]. The axis governed by hepatocyte growth factor (HGF) and its receptor c-Met plays important
roles in cell proliferation, survival, and migration in the liver [65]. Within the HCC microenvironment,
epigenetic upregulation of c-Met is associated with tumor progression and metastasis [66]. Epigenetic
analysis of the c-Met promoter clarified that a significant reduction in DNA methylation is correlated
with increased c-Met expression in circulating tumor cells (CTCs) during hematogenous metastasis of
HCC [66]. Another study on the role of HGF in HCC metastasis reported that the induction of DNMT1
expression by HGF resulted in DNA hypermethylation of tumor suppressor genes, including MYOCD,
PANX2, and LHX9 [57]. It has been reported that reactive oxygen species (ROS) are involved in tumor
metastasis, migration, invasion, and tumor angiogenesis [67]. In HCC cells, ROS inhibit the expression
of E-cadherin, a regulator of EMT, by inducing epigenetic changes in the promoter [58]. Moreover, the
regulatory mechanism was clarified as follows: ROS upregulate Snail expression by activating the
PI3K/Akt/GSK3β pathway and, then, Snail induces CpG methylation of the E-cadherin promoter by
recruiting DNMT1 [58]. 3. DNA Methylation DNA methylation catalyzed by DNA methyltransferases (DNMTs) is a chemical modification
of DNA that occurs by conjugation of a methyl group to the 5’ carbon position of the cytosine ring,
which is crucial for regulating gene expression [50]. In cancer, the expression of tumor suppressor
genes is frequently silenced by CpG island hypermethylation in promoter regions [51]. Therefore, the
upregulation of DNMTs promotes cancer development [52]. For example, the mRNA levels of DNMTs,
including DNMT1, DNMT3a, and DNMT3b, are significantly higher in HCC than in nonneoplastic
liver tissues [53]. In addition, the increased expression of DNMTs is recognized as a predictor of poor
survival in HCC [54]. To date, numerous studies have demonstrated that DNMT-mediated epigenetic
changes regulate HCC metastasis, invasion, progression, and development [55]. This section provides
information on the regulation of HCC metastasis by DNMTs and their molecular mechanism (Table 2). Int. J. Mol. Sci. 2018, 19, 3978 5 of 17 Table 2. DNA methyltransferases in HCC metastasis. Table 2. DNA methyltransferases in HCC metastasis. Table 2. DNA methyltransferases in HCC metastasis. Genes
Function
References
DNMT1
Osteopontin induces HCC metastasis through increasing DNMT1 expression and
the hypermethylation of RASSF1, GATA4, and CDKL2
[56]
HGF-mediated HCC metastasis is associated with the induction of DNMT1
expression and the hypermethylation of MYOCD, PANX2, and LHX9
[57]
ROS induces E-cadherin promoter methylation through Snail-dependent
DNMT1 recruitment
[58]
DNMT3
Increased DNMT3b expression in HCC patients is associated with poorer overall
survival and a shorter metastasis-free survival interval
[59]
HBx induces DNMT3a and 3b recruitment and MTA1 promoter
hypermethylation, which interferes with the DNA binding of p53
[60]
DNMT3b reduces the expression of the metastasis suppressor MTSS1 via a DNA
methylation-independent mechanism
[61]
Undefined DNMTs
Inhibition of DNMT by Aza restores downregulated PCDH10 expression in HCC
[62]
Promoter hypomethylation upregulates CD147 expression by increasing
Sp1 binding
[63]
The reduction in SLIT2 expression by promoter methylation correlates with
lymph node metastasis in HCC
[64] 3.3. Undefined DNMTs To date, several studies on the epigenetic regulation of tumor suppressor gene expression by
undefined DNMTs have been reported in HCC. PCDH10 was recently demonstrated to be a tumor
suppressor gene, and is frequently silenced in HCC [70]. An analysis of the clinicopathologic features
of HCC patients found a significant correlation between the methylation status of PCDH10 and
metastasis [62]. Treatment with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (Aza)
restored PCDH10 mRNA expression by inhibiting promoter methylation, indicating that decreased
PCDH10 expression is related to the promoter methylation status in HCC [62]. The transmembrane glycoprotein CD147 has been implicated in HCC progression and metastasis,
and CD147 gene silencing reduced MMP secretion and the invasive potential of HCC cells [71]. Within HCC patient tissues, an inverse correlation between CD147 expression and methylation
status has been reported, and a mechanism study with Aza revealed that promoter hypomethylation
enhances CD147 expression by increasing Sp1 binding [63]. In addition, SLIT2 has been proposed
to be a tumor suppressor gene that is epigenetically silenced in various cancers [72–74]. In HCC, a
reduction in SLIT2 expression by promoter methylation correlated with lymph node metastasis [64]. Moreover, overexpression of SLIT2 in the SMMC-7721 HCC cell line reduced cell growth, invasion,
and migration [64]. Therefore, DNA methylation status and DNMT levels may be potential biomarkers of HCC and
attractive therapeutic targets for HCC treatment. 3.2. DNMT3 Several recent studies have shown that DNMT3 regulates HCC invasion and metastasis. A
clinicopathological study by Oh et al. reported significant correlations between DNMT expression
and overall survival and metastasis-free survival in HCC patients [59]. Among the DNMTs, DNMT3b
showed a greater than 4-fold increase in mRNA levels in HCC compared to nonneoplastic livers,
and these increased levels were associated with poorer overall survival and a shorter metastasis-free
survival interval [59]. In HCC metastasis and invasion, DNMT3 is involved in the epigenetic regulation of the
metastasis-associated protein 1 (MTA1) gene [68]. MTA1 is known as a modulator of cancer-promoting
processes, including invasion, angiogenesis, metastasis, and survival [69]. In HBV-associated HCC, the 6 of 17 Int. J. Mol. Sci. 2018, 19, 3978 HBV X (HBx) protein enhances MTA1 expression by epigenetic regulation. A molecular mechanism
has been clarified in which HBx induces MTA1 transcription by increasing promoter methylation and
releasing p53 through the recruitment of DNMT3a and DNMT3b [60]. In addition to gene silencing
by DNMT3b through epigenetic modification, a DNA methylation-independent mechanism in HCC
metastasis has also been identified. A study by Fan et al. demonstrated that metastasis suppressor 1
(MTSS1) is a novel target gene of DNMT3b, and its expression was negatively associated with DNMT3b
overexpression in clinical HCC specimens [61]. The mechanism of action (MOA) by which DNMT3b
inhibits MTSS1 expression was determined to involve direct binding to the MTSS1 promoter region
rather than DNA hypermethylation [61]. 4.1. Histone Lysine Methyltransferase Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group complex and plays
an important role in the proliferation and metastasis of various cancers via methylation of histone
H3K27 [92]. EZH2 mRNA is highly expressed in HCC cell lines and primary HCC tumors; moreover,
clinicopathological analysis of EZH2 revealed a statistically significant difference in portal vein invasion
between the high expression group and the low expression group [93]. Regarding the epigenetic
regulation of tumor suppressor genes, the upregulation of chromodomain helicase DNA binding
protein 5 (CHD5), which acts as a tumor suppressor in several types of cancer [94], by shEZH2, led to
a reduction in cell migration and invasion, and a negative correlation between EZH2 and CHD5 was
observed in HCC patients [79]. Additionally, the upregulation of tumor suppressor deleted in liver
cancer 1 (DLC1) and four and a half LIM domains 1 (FHL1) by DZNep, an EZH2-specific inhibitor, also
suppressed the proliferation and migration of HCC cell lines [80,81]. Recently, several papers have
reported on the regulation of EZH2 and miRNA in HCC metastasis. miR-173 suppressed migration
and invasion by downregulating the EZH2-STAT3 signaling pathway [95], and the upregulation of
miR-22 and miR-203 by epigenetic regulation of EZH2 decreased HCC metastasis [82,83]. Moreover,
miR-124 and miR-26a decreased EZH2 levels to reduce HCC metastasis in vivo and in vitro [96,97]. SETDB1 (KMT1E) is a methyltransferase that targets histone H3K9 methylation to repress gene
expression [98]. SETDB1 knockdown and overexpression studies, in vivo and in HCC cell lines,
clearly established its involvement in cell proliferation. In addition, downregulation of T-lymphoma
invasion and metastasis gene (Tiam1), by SETDB1 knockdown in HCC cell lines, reduced EMT and cell
migration/invasion. Finally, a positive correlation between SETDB1 and Tiam1 was observed in HCC
tissues [84]. The transcription factor SP1 is involved in the positive regulation of SETDB1 in HCC, and
overexpression of miR-29a significantly suppressed SETDB1 expression [85]. Euchromatic histone lysine methyltransferase 2 (G9a, EHMT2) and suppressor of variegation
3-9 homolog 1 (SUV39H1) predominantly methylate histone H3K9 to induce the formation of
heterochromatin, and are overexpressed in several types of cancer [99–101]. In HCC, G9a upregulation
was significantly associated with aggressive clinicopathological features of HCC, and G9a knockdown
suppressed HCC cell metastasis and proliferation via the induction of retinoic acid receptor
responder protein 3 (RARRES3) [86]. 4. Histone Modifications Histone modifications, such as histone methylation, acetylation, and ubiquitination, critically
control oncogenes and tumor suppressor genes at the transcriptional level during tumor
progression [75,76]. Among the histone modifying enzymes, histone methyltransferases and
demethylases are the primary focus in studies on molecular targets for anticancer drug
development [77]. In HCC, several recent papers have reported that histone methylation is tightly
connected to the up- and downregulation of metastasis- and proliferation-related genes. To initiate
HCC metastasis from primary tumors, various methyltransferases and demethylases methylate histone
H3 lysine (K) 4 and K36, which causes conformational changes affecting the balance and distribution
of euchromatin and heterochromatin, leading to the upregulation of mesenchymal–epithelial transition
(MET)-related genes. In contrast, these genes are involved in the downregulation of EMT-related genes
after the formation of heterochromatin through methylation of histone H3K9 and K27 [78]. Therefore,
here, we introduce the overexpression of histone methyltransferases and demethylases in HCC, and
discuss the role of these histone modifying enzymes in HCC metastasis and proliferation (Table 3). 7 of 17 Int. J. Mol. Sci. 2018, 19, 3978 Table 3. Histone methyltransferase/demethylase in HCC metastasis. Function
Reference
Histone methyltransferase
Upregulation of CHD5, DLC1, FHL1, miR-22, and miR-203 by EZH2 knockdown-reduced
HCC metastasis
[79–83]
Downregulation of Tiam1 and SP1 by SETDB1-decreased EMT and cell migration/invasion
[84,85]
Induction of RARRES3 by EHMT2 knockdown-suppressed HCC metastasis and
proliferation
[86]
Knockdown of SUV39H1 by miR-125b-induced cell senescence and metastasis
[87]
Histone demethylase
Epigenetic silence of BMP7 and PTEN by KDM5C and JARID1B knockdown-reduced cell
invasion/migration and wound healing analysis
[88,89]
miR-615-5p downregulation knocked down KDM4B levels, which increased RAB24
expression and induced HCC growth and metastasis
[90]
miR-941 decreased KDM6B levels to reduce cell migration and invasion in vitro and
in vivo
[91] Table 3. Histone methyltransferase/demethylase in HCC metastasis. 5. Clinical Application of Epigenetic Alterations as Hepatic Metastasis Biomarkers and
Epigenetic Modifiers as Therapeutic Targets Cancer metastasis, the spread of cancer cells from the primary site, is the major cause of morbidity
and mortality in various cancers. HCC metastasis is defined as either intrahepatic metastasis (IHM)
via portal vein dissemination, or extrahepatic metastasis (EHM) to other organs, including the lungs,
lymph nodes, bones, and adrenal glands [105–107]. Although IHM is the most effective predictor of
the risk of recurrence after curative resection [108], approximately 80% of EHM patients present at a
more advanced intrahepatic tumor stage at diagnosis [106,109], and EHM has also been detected in
patients with early stage intrahepatic HCC [110]. Therefore, early detection of metastasis is crucial
to make adequate treatment plans and to avoid unnecessary therapy. Recently, advanced imaging
techniques have improved the detection rate of small tumors and the characterization of hepatic
lesions. Nonetheless, there are still limits on the prediction and early diagnosis of hepatic metastases. p
y
g
p
EMT and MET have been proposed to be engines of metastasis in various cancers, including
HCC [111–113]. During EMT, E-cadherin is repressed, and β-catenin undergoes nuclear translocation;
these events are significantly correlated with IHM and poor survival of HCC patients. Similarly,
E-cadherin is strongly expressed in nonmetastatic HCC tissue, whereas it is weakly expressed in
metastatic tissues [114]. In analyses of EMT-related markers’ expression using the 123 HCC tissue
cohort, overexpression of Snail and Twist was correlated with the downregulation of E-cadherin
expression, and was related to a worse disease prognosis [115]. Another study of EMT-related genes
in a cohort of 128 HCC patients revealed that four candidate genes (E-cadherin (CDH1), inhibitor
of DNA binding 2 (ID2), matrix metalloproteinase 9 (MMP9), and transcription factor 3 (TCF3))
were independent prognostic factors for the overall survival of HCC patients [116]. Moreover, low
E-cadherin and high vimentin expression were significantly correlated with poor tumor differentiation,
vascular invasion, and extrahepatic recurrence in 150 HCC patients [117]. Intriguingly, EMT also
plays critical roles during liver cell fibroproliferative wound healing, which can lead to fibrosis, and is
associated with chronic inflammation and repair of adult tissues after injury. Several previous studies
demonstrated that human hepatocytes and cholangiocytes can be induced to undergo EMT by TGF-β
or hedgehog, and they actively contribute to liver cell fibrogenesis [118–120]. Since EMT is a crucial event in hepatocyte progression and metastasis, epigenetic alterations
have potential as clinically applicable biomarkers and therapeutic targets in HCC. 4.2. Histone Lysine Demethylases KDM5C and JARID1B are histone demethylases in the family of JmjC domain-containing
proteins that mainly demethylate histone H3K4 to suppress gene expression via the formation
of heterochromatin, and are overexpressed in many types of cancer [103,104]. In HCC, KDM5C
and JARID1B are abundantly expressed in invasive human HCC cells, and are correlated with
distant metastasis in HCC. The roles of KDM5C and JARID1B in HCC have been clarified by
various experiments—for example, knockdown of KDM5C and JARID1B reduced migration, invasion,
and wound healing via epigenetic silencing of BMP7 and PTEN expression, respectively [88,89]. Demethylation of histone H3K9 and K27 leads to the formation of open chromatin structures;
knockdown of KDM4B, a H3K9 demethylase, decreased miR-615-5p expression in HCC cell lines. Subsequently, a reduction in miR-615-5p expression induced HCC growth and metastasis, in vitro,
through increased RAB24 expression [90]. The expression of KDM6B (H3K27 demethylase) is critically
regulated by miR-941 levels in HCC cells. Low KDM6B levels in response to miR-941 regulation
reduced cell proliferation, migration, and invasion in vitro and in vivo [91] 4.1. Histone Lysine Methyltransferase In addition, suppression of G9a expression by treatment with
basil polysaccharide under hypoxia also induced EMT markers and reduced MET markers [102]. Upregulation of SUV39H1 in HCC cell lines markedly enhanced cell clonogenicity. In addition, 8 of 17 Int. J. Mol. Sci. 2018, 19, 3978 SUV39H1 knockdown by the overexpression of miR-125b, a posttranscriptional regulator of SUV39H1,
induced cell senescence, and reduced cell migration and metastasis [87]. SUV39H1 knockdown by the overexpression of miR-125b, a posttranscriptional regulator of SUV39H1,
induced cell senescence, and reduced cell migration and metastasis [87]. 5. Clinical Application of Epigenetic Alterations as Hepatic Metastasis Biomarkers and
Epigenetic Modifiers as Therapeutic Targets (Figure 1) As we
described above, several transcription factors, such as SNAIL, SLUG, ZEB1, and ZEB2/SIP1, participate
in the repression of epithelial markers (E-cadherin) and the conversion of epithelial-like cancer cells 9 of 17
ial-like Int. J. Mol. Sci. 2018, 19, 3978
participate in the repres into metastatic cancer cells. In this context, multiple aspects of epigenetic regulation, including
DNA methylation, histone modification, and noncoding RNAs, are cooperatively engaged in EMT
regulation. The inverse correlation between E-cadherin expression and hypermethylation of the CDH1
gene promoter has been confirmed in many cancers [121–123]. In addition, increased methylation
of CDH1 was related to worse overall survival, which was associated with vascular invasion and
recurrence in HCC [122,123]. Notably, aberrant cytosine methylation itself was reported as a possible
molecular marker for HCC progression, and treatment with the DNMT inhibitor decitabine inhibited
the invasiveness of HCC cells [124,125]. Several EMT transcriptional factors also epigenetically regulate
CDH1 gene expression. Snail recruits the histone demethylase lysine-specific demethylase 1 (LSD1),
which removes the dimethylation of K4 on histone H3 (H2K4m2) and mediates the transcriptional
repression of CDH1 [126]. Elevated Snail levels predict the poor survival of HCC patients [127]. including DNA methylation, histone modification, and noncoding RNAs, are cooperatively engaged
in EMT regulation. The inverse correlation between E‐cadherin expression and hypermethylation of
the CDH1 gene promoter has been confirmed in many cancers [121–123]. In addition, increased
methylation of CDH1 was related to worse overall survival, which was associated with vascular
invasion and recurrence in HCC [122,123]. Notably, aberrant cytosine methylation itself was reported
as a possible molecular marker for HCC progression, and treatment with the DNMT inhibitor
decitabine inhibited the invasiveness of HCC cells [124,125]. Several EMT transcriptional factors also
epigenetically regulate CDH1 gene expression. Snail recruits the histone demethylase lysine-specific
demethylase 1 (LSD1), which removes the dimethylation of K4 on histone H3 (H2K4m2) and
mediates the transcriptional repression of CDH1 [126]. Elevated Snail levels predict the poor survival
of HCC patients [127]. Figure 1. Summary for epigenetic regulation in HCC metastasis. Figure 1. Summary for epigenetic regulation in HCC metastasis. Figure 1. Summary for epigenetic regulation in HCC metastasis. Figure 1. Summary for epigenetic regulation in HCC metastasis. Currently, noncoding RNAs have been highlighted as new regulators of various genes,
including EMT-related genes. The best-known EMT-related miRNAs are those in the miR-200 family
(miR-200a, miR-200b, miR-200c, miR-141, and miR-429) and miR-205 [113,128]. 5. Clinical Application of Epigenetic Alterations as Hepatic Metastasis Biomarkers and
Epigenetic Modifiers as Therapeutic Targets Expression of genes
in the miR-200 family is reversibly controlled by promoter CpG methylation during EMT and MET
in cancer metastasis. In addition, Bundh et al. discovered 20 HCC metastasis-specific miRNAs via
miRNA microarray analysis of 29 metastatic HCC tissues vs. 102 nonmetastatic HCC tissues [129]. This 20-miRNA signature could significantly predict HCC metastasis status with an overall accuracy
of 76%. In further analysis, miR-219-5p was found to be an important metastasis-related miRNA in
HCC [130]. CDH1 was directly regulated by miR-219-5p, and high miR-219-5p expression in HCC
tissues was associated with vascular invasion and poor prognosis in HCC patients. Currently, noncoding RNAs have been highlighted as new regulators of various genes, including
EMT-related genes. The best-known EMT-related miRNAs are those in the miR-200 family (miR-200a,
miR-200b, miR-200c, miR-141, and miR-429) and miR-205 [113,128]. Expression of genes in the
miR-200 family is reversibly controlled by promoter CpG methylation during EMT and MET in cancer
metastasis. In addition, Bundh et al. discovered 20 HCC metastasis-specific miRNAs via miRNA
microarray analysis of 29 metastatic HCC tissues vs. 102 nonmetastatic HCC tissues [129]. This
20-miRNA signature could significantly predict HCC metastasis status with an overall accuracy of 76%. In further analysis, miR-219-5p was found to be an important metastasis-related miRNA in HCC [130]. CDH1 was directly regulated by miR-219-5p, and high miR-219-5p expression in HCC tissues was
associated with vascular invasion and poor prognosis in HCC patients. tissues was associated with vascular invasion and poor prognosis in HCC patients. In terms of cancer biomarkers, the body fluid-based liquid biopsy concept has recently emerged
for the noninvasive analysis of biomarkers. Indeed, many research groups have discovered and
published on circulating HCC-derived biomolecules. Circulating cell-free methylated DNA from
cancer cells has been detected in HCC patient body fluids, such as serum, plasma, and urine. Hypermethylated DNA from the Ras association domain family protein 1A (RASSF1A) gene was
detectable in over 90% of HCC patient sera samples, and predicted a shorter relapse-free survival
interval for HCC patients [131]. Interestingly, another group demonstrated that the methylation
status of 5 tumor suppressor genes (APC, FHIT, p15, p16, and E-cadherin) in HCC tissues was
successfully reproduced in plasma circulating DNA [132]. 5. Clinical Application of Epigenetic Alterations as Hepatic Metastasis Biomarkers and
Epigenetic Modifiers as Therapeutic Targets Thus, circulating methylated DNA is
readily detectable and has potent clinical utility, such as in early HCC diagnosis and prognosis
prediction Nonetheless HCC metastasis-specific circulating DNA methylation has not yet been
In terms of cancer biomarkers, the body fluid-based liquid biopsy concept has recently emerged for
the noninvasive analysis of biomarkers. Indeed, many research groups have discovered and published
on circulating HCC-derived biomolecules. Circulating cell-free methylated DNA from cancer cells has
been detected in HCC patient body fluids, such as serum, plasma, and urine. Hypermethylated DNA
from the Ras association domain family protein 1A (RASSF1A) gene was detectable in over 90% of
HCC patient sera samples, and predicted a shorter relapse-free survival interval for HCC patients [131]. Interestingly, another group demonstrated that the methylation status of 5 tumor suppressor genes
(APC, FHIT, p15, p16, and E-cadherin) in HCC tissues was successfully reproduced in plasma circulating
DNA [132]. Thus, circulating methylated DNA is readily detectable and has potent clinical utility, such
as in early HCC diagnosis and prognosis prediction. Nonetheless, HCC metastasis-specific circulating
DNA methylation has not yet been reported. prediction. Nonetheless, HCC metastasis specific circulating DNA methylation has not yet been
reported. Circulating miRNAs are another potential noninvasive marker for HCC liquid biopsy. A nested
case-control study with prospectively collected sera from HCC patients and controls revealed
Circulating miRNAs are another potential noninvasive marker for HCC liquid biopsy. A
nested case-control study with prospectively collected sera from HCC patients and controls revealed
significantly increased serum levels of miR-29a, miR-29c, miR-133a, miR-143, miR-145, miR-192, and
miR-505 in patients with HCC [133]. In addition, Ding et al. reported the high frequency expression of Int. J. Mol. Sci. 2018, 19, 3978 10 of 17 3 miRNAs (miR-21, miR-199, and miR-122) based on a meta-analysis, and these miRNAs may be more
specific for the diagnosis of HCC [134]. Moreover, multiple circulating miRNAs have been reported as
candidate markers for the detection of HCC. HCC patients with HBV or HCV infection had relatively
high circulating levels of miR-21, miR-222, and miR-223 [135,136]. Serum miRNA-34 was proposed as
a predictive marker of bone metastasis in patients with HCC [137]. In recent years, exosomes, a type of extracellular microvesicle, have been shown to be vehicles
for miRNAs. Therefore, exosomal miRNAs may be stable in body fluids due to the protective role
of exosomes against RNase. 5. Clinical Application of Epigenetic Alterations as Hepatic Metastasis Biomarkers and
Epigenetic Modifiers as Therapeutic Targets Exosomes carrying cancer-specific miRNAs are released from cancer
cells and can circulate in body fluids. After being delivered into acceptor cells, exosomal miRNAs
play functional roles by targeting specific genes. miR-21 plays an oncogenic role in various cancer
tissues. Recently, high levels of exosomal miR-21 were reported in serum from HCC patients, and
were shown to be significantly associated with HCC patient prognosis [138]. Notably, miR-21 was
highly concentrated in exosomes compared to whole serum, which indicates that exosomal miR-21 is a
more sensitive marker. Other exosomal miRNAs have also been suggested as diagnostic markers of
HCC. For instance, the levels of exosomal miR-18a, miR-221, miR-222, and miR-224 were significantly
higher in the serum of patients in the HCC group than in the hepatitis and liver cirrhosis groups [139]. Collectively, EMT plays critical role in HCC development and metastasis. EMT is modulated
via several epigenetic changes, such as DNA methylation, histone modifications, miRNAs, indicating
each epigenetic modifier has great potential to serve as a novel diagnostic and/or therapeutic marker
for HCC metastasis. Thus, future research should be more focused on the development of specific
inhibitors, discovery of cancer metastasis biomarker, and MOA studies based on epigenetic modifiers. Acknowledgments: This study was supported by grants from the National Research Foundation of Korea (NRF)
funded by the Ministry of Science, ICT and Future Planning (2014R1A5A2009242, NRF-2018M3A9H3023077,
NRF-2016R1C1B2011739 and NRF-2017R1C1B2012268) and the KRIBB Research Initiative Program. Acknowledgments: This study was supported by grants from the National Research Foundation of Korea (NRF)
funded by the Ministry of Science, ICT and Future Planning (2014R1A5A2009242, NRF-2018M3A9H3023077,
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Direitos humanos, devido processo legal e a internação compulsória/involuntária de usuários de drogas no Brasil
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Revista Brasileira de Sociologia do Direito
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1 Doutor em Direito Público (UNISINOS). Professor dos Cursos de Direito da UNIJUÍ e da UNISINOS. Coordenador do
Programa de Pós-Graduação Stricto Sensu - Mestrado e Doutorado em Direitos Humanos - da UNIJUÍ. Líder do Grupo
de Pesquisa Biopolítica & Direitos Humanos (CNPq). Pesquisador Gaúcho – Edital FAPERGS nº 05/2019. DIREITOS HUMANOS, DEVIDO
PROCESSO LEGAL E A INTERNAÇÃO
COMPULSÓRIA/INVOLUNTÁRIA DE
USUÁRIOS DE DROGAS NO BRASIL
HUMAN RIGHTS, DUE LEGAL PROCESS AND THE
COMPULSORY/INVOLUNTARY HOSPITALIZATION OF
DRUG USERS IN BRAZIL
Revista Brasileira de Sociologia do Direito ISSN 2359-5582 Revista Brasileira de Sociologia do Direito Maiquel Ângelo D. Wermuth1
Luíza Hrynyszyn Menezes2 ABSTRACT: The text contemplates a discussion about the
adequate way of coping with the excessive use of
chemical drugs, based on the relationship between the
guarantee of Human Rights, due to the legal process and
compulsory / involuntary hospitalization of the user. The
study
analyzes
the
compulsory
/
involuntary
hospitalization of drug users as a violation of due process
and, consequently, of Human Rights. To this end, it
initially presents an overview of drug legislation in Brazil,
from a historical perspective, identifying advances and
setbacks, in order, in sequence, to link the compulsory /
involuntary hospitalization of drug users with due process
and human rights. The research problem can be
summarized in the following objection: to what extent
does compulsory / involuntary hospitalization of drug
users represent a violation of the constitutional principle
of due process and, consequently, of Human Rights? The
work was viewed from the inductive research method,
using bibliographic and documentary research techniques. It is concluded that the treatment of users and drug
addicts in Brazil requires changes, and the choices of
citizens must be respected, as a condition of possibility for
the establishment of a drug policy in line with Human
Rights. RESUMO: O texto contempla uma discussão sobre a forma
adequada de enfrentamento ao uso desmedido de drogas
químicas, a partir da relação entre garantia dos Direitos
Humanos,
devido
processo
legal
e
internação
compulsória/involuntária do usuário. O estudo analisa a
internação compulsória/involuntária de usuários de drogas
enquanto uma violação do devido processo legal e,
consequentemente, dos Direitos Humanos. Para tanto,
apresenta inicialmente um panorama da legislação sobre
drogas no Brasil, sob perspectiva histórica, identificando
avanços e retrocessos, para, em sequência, relacionar a
internação compulsória/involuntária do usuário de drogas
com o devido processo legal e os Direitos Humanos. O
problema de pesquisa pode ser sintetizado na seguinte
objeção:
em
que
medida
a
internação
compulsória/involuntária de usuários de drogas representa
uma violação ao princípio constitucional do devido
processo
legal
e,
consequentemente,
aos
Direitos
Humanos? O trabalho foi perspectivado a partir do método
de pesquisa indutivo, valendo-se de técnica de pesquisa
bibliográfica e documental. Conclui-se que o tratamento de
usuário e dependentes de drogas, no Brasil, requer
mudanças, devendo ser respeitadas as escolhas dos
cidadãos, como condição de possibilidade para o
estabelecimento de uma política de drogas em consonância
com os Direitos Humanos. Keywords: Compulsory Hospitalization. Drug Laws. Due
legal process. Human rights. 2 Especialista em Direito Penal e Processual pela UNISINOS. Advogada Criminalista 1 Doutor em Direito Público (UNISINOS). Professor dos Cursos de Direito da UNIJUÍ e da UNISINOS. Coordenador do
Programa de Pós-Graduação Stricto Sensu - Mestrado e Doutorado em Direitos Humanos - da UNIJUÍ. Líder do Grupo
de Pesquisa Biopolítica & Direitos Humanos (CNPq). Pesquisador Gaúcho – Edital FAPERGS nº 05/2019.
2 Especialista em Direito Penal e Processual pela UNISINOS. Advogada Criminalista. DIREITOS HUMANOS, DEVIDO
PROCESSO LEGAL E A INTERNAÇÃO
COMPULSÓRIA/INVOLUNTÁRIA DE
USUÁRIOS DE DROGAS NO BRASIL
HUMAN RIGHTS, DUE LEGAL PROCESS AND THE
COMPULSORY/INVOLUNTARY HOSPITALIZATION OF
DRUG USERS IN BRAZIL
Revista Brasileira de Sociologia do Direito ISSN 2359-5582 Palavras-chave: Internação Compulsória. Leis de Drogas. Devido processo legal. Direitos Humanos. 123
1 Doutor em Direito Público (UNISINOS). Professor dos Cursos de Direito da UNIJUÍ e da UNISINOS. Coordenador do
Programa de Pós-Graduação Stricto Sensu - Mestrado e Doutorado em Direitos Humanos - da UNIJUÍ. Líder do Grupo
de Pesquisa Biopolítica & Direitos Humanos (CNPq). Pesquisador Gaúcho – Edital FAPERGS nº 05/2019. 2 Especialista em Direito Penal e Processual pela UNISINOS. Advogada Criminalista. 2 Especialista em Direito Penal e Processual pela UNISINOS. Advogada Criminalista. 123 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 ■■■ ■■■ 1 CONSIDERAÇÕES INICIAIS A internação compulsória/involuntária do dependente químico é tema polêmico e
complexo devido às intensas discussões levantadas a seu respeito. Essas discussões não se
limitam ao cenário brasileiro, mas permeiam as políticas públicas voltadas ao
enfrentamento às drogas em vários outros países. Neste artigo, propõe-se uma análise da
temática sob a perspectiva da legislação vigente no Brasil (Lei nº 11.343/2006),
relacionando a internação compulsória/involuntária do usuário com o princípio do devido
processo legal e os Direitos Humanos. É sabido que as questões relacionadas ao uso de drogas não se limitam às figuras do
usuário e do traficante. Uma análise que leva em conta apenas esses dois atores, além de
incorrer em um reducionismo extremo, deixa de levar em consideração questões como,
por exemplo, as políticas públicas e os Direitos Humanos implicados nesse debate, os quais
irradiam efeitos sobre a sociedade como um todo – incluindo os não usuários. Nesse sentido, o presente estudo visa a responder à seguinte problemática de
pesquisa: em que medida a internação compulsória/involuntária de usuários de drogas
representa uma violação ao princípio constitucional do devido processo legal e,
consequentemente, aos Direitos Humanos? Como
objetivo
geral,
o
estudo
pretende
avaliar
se
a
internação
compulsória/involuntária de usuários de drogas configura uma violação do devido
processo legal e, consequentemente, dos Direitos Humanos desses sujeitos. Os objetivos
específicos do texto, espelhados em suas duas seções, consistem na apresentação do
cenário legislativo relacionado às drogas no Brasil, sob perspectiva histórica, evidenciando
os avanços e retrocessos relacionados à garantia/efetivação dos Direitos Humanos em
relação
aos
usuários,
para,
em
sequência,
apresentar
a
internação
compulsória/involuntária do usuário como uma medida que viola o devido processo legal
e, consequentemente, os Direitos Humanos dessa população. O trabalho foi perspectivado a partir do método de pesquisa indutivo, valendo-se de
técnica de pesquisa bibliográfica e documental. De acordo com Lakatos e Marconi (2007, 124 124 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes p. 86), a “indução é um processo mental por intermédio do qual, partindo de dados
particulares, suficientemente constatados, infere-se uma verdade geral ou universal, não
contida nas partes examinadas”. 1 CONSIDERAÇÕES INICIAIS Nesse sentido, o método em questão pareceu-nos
adequado à abordagem realizada, na medida em que “o objetivo dos argumentos
indutivos é levar a conclusões cujo conteúdo é muito mais amplo do que o das premissas
nas quais se basearam.” Portanto, parte-se do pressuposto de que, diferentemente do que
ocorre com a dedução, por meio da qual se chega a conclusões verdadeiras, “já que
baseadas em premissas igualmente verdadeiras, por meio da indução chega-se a
conclusões que são apenas prováveis” (GIL, 2008, p. 11). Nesse sentido, sabedores da
existência de perspectivas diversas de análise da temática discutida neste estudo, os
autores propõem respostas a partir do conjunto de bibliografias e documentos analisados. 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO O Brasil, a Argentina, a Bolívia, o Equador, o Paraguai, o Uruguai e a Venezuela, em
27 de abril de 1973, firmaram um Acordo sobre Entorpecentes e Psicotrópicos, que restou
internalizado por meio da sua aprovação pelo Decreto Legislativo nº 78, de 1973 (BRASIL,
1973). A partir do disposto no Acordo Sul-Americano, o Brasil sancionou, em 21 de outubro
de 1976, a Lei nº 6.368/1976, estabelecendo medidas de prevenção e repressão ao tráfico
e uso de substâncias entorpecentes ou que determinassem dependência física ou psíquica,
sendo norteada pelo objetivo de reprimir e punir condutas relacionadas ao porte e tráfico
de drogas (BRASIL, 1976). Esta legislação vigorou no país por vinte e seis anos, sendo a primeira Lei a abordar
a questão das drogas no ordenamento jurídico brasileiro, promovendo uma separação
entre as figuras do usuário e do traficante e tornando o laudo toxicológico imprescindível
para a comprovação do uso da substância química. No corpo desta legislação evidenciava-
se também uma nítida sobreposição dos discursos médico e jurídico, aliados frente à
necessidade de se “eliminar” da sociedade o “perigo” representado pelo traficante –
constituído, nos discursos políticos de então, como verdadeiro “inimigo”. Com isso, Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 procurava-se densificar a repressão ao comércio ilícito e aliviar a resposta penal aos
dependentes e usuários de drogas (CARVALHO, 2013). Mesmo que a Lei nº 6.368/1976 não tenha tratado expressamente o usuário de
drogas como “delinquente”, o seu art. 16, ao determinar a internação compulsória do
dependente químico3, aplicava a ele uma sanção, sem a correspondente garantia do
devido processo legal. Por seu turno, o art. 19 comutava a pena de detenção em medida
socioeducativa, caso o usuário fosse, ao tempo da infração, inteiramente incapaz de
entender o caráter ilícito do fato ou de determinar-se de acordo com esse entendimento,
em razão da dependência ou sob o efeito de substância entorpecente ou que
determinasse dependência física ou psíquica proveniente de caso fortuito ou força maior,
qualquer que tivesse sido a infração penal praticada. Nesse caso, a pena poderia ser
reduzida de um a dois terços. Entretanto, mesmo constatada tal circunstância, não havia
previsão de medidas alternativas a serem adotadas. Ou seja, a própria Lei, no §1º do art. 9º, dispunha que “enquanto não se criarem os estabelecimentos referidos neste artigo,
serão adaptados, na rede já existente, unidades para aquela finalidade” (BRASIL, 1976). 3 Dispunha o artigo 16 da Lei em questão: “Art. 16. adquirir, guardar ou trazer consigo, para uso próprio, substância
entorpecente ou que determine dependência física ou psíquica, sem autorização ou em desacordo com determinação
legal ou regulamentar. Penas – detenção, seis meses a dois anos, e pagamento de 20 a 50 dias-multa.” (BRASIL, 1976). 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO Em face de deficiências cujo curto espaço de análise do presente estudo não
recomenda destacar, e considerando as estatísticas que apontavam para um considerável
aumento da criminalidade relacionada às drogas no país, a Lei nº 6.368/1976 não se
demonstrou mais eficaz, razão pela qual foi editada uma nova Lei de Drogas, qual seja, a
Lei nº 10.409/2002 (BRASIL, 2002). O novo diploma tinha por objetivo conferir uma visão
mais atualizada sobre a temática. No entanto, a nova lei sofreu duras críticas, na medida
em que aprovada com muitas incorreções em seu texto, tendo sido aprovada com diversos
vetos, passando a vigorar completamente descaracterizada quando comparada ao seu
projeto originário. Com efeito, de um total de cinquenta e nove artigos, houve o veto de
trinta. Toda a parte dedicada à tipificação dos delitos e das penas foi vetada pela
Presidência da República, razão pela qual a legislação entrou em vigor somente no que se
refere à sua dimensão processual, gerando problemas de aplicação, uma vez que, como 126 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes decorrência dos vetos, a Lei anterior continuou em vigor, ocasionando grande divergência
doutrinária e jurisprudencial e gerando verdadeira insegurança jurídica (CARVALHO, 2013). A partir de um debate instaurado como decorrência da situação teratológica
imposta pela vigência simultânea de ambas as legislações, foi promulgada, em 23 de
agosto de 2006, a Lei nº 11.343 (BRASIL, 2006). Esta legislação foi responsável pela
instituição do Sistema Nacional de Políticas Públicas sobre Drogas (SISNAD), pela
prescrição de medidas para prevenção do uso indevido, atenção e reinserção social de
usuários e dependentes de tóxicos, bem como pelo estabelecimento de normas para
repressão à produção não autorizada e ao tráfico ilícito de drogas, definindo crimes. A Lei
nº 11.343/2006 preferiu a denominação “drogas”, abandonando a expressão “substância
entorpecente” empregada nas leis anteriores, trazendo o conceito no parágrafo único de
seu art. 1º: “Para fins desta Lei, consideram-se como drogas as substâncias ou os produtos
capazes de causar dependência, assim especificados em lei ou relacionados em listas
atualizadas
periodicamente
pelo
Poder
Executivo
da
União” (BRASIL,
2006). Complementando a regra citada, o art. 66 do mesmo diploma legal estabelece que, “para
fins do disposto no parágrafo único do art. 4 “Art. 28. Quem adquirir, guardar, tiver em depósito, transportar ou trouxer consigo, para consumo pessoal, drogas
sem autorização ou em desacordo com determinação legal ou regulamentar será submetido às seguintes penas: § 7o O juiz determinará ao Poder Público que coloque à disposição do infrator, gratuitamente, estabelecimento de
saúde, preferencialmente ambulatorial, para tratamento especializado.” (BRASIL, 2006). 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO 1º desta Lei, até que seja atualizada a
terminologia da lista mencionada no preceito, denominam-se drogas substâncias
entorpecentes, psicotrópicas, precursoras e outras sob controle especial, da Portaria
SVS/MS nº 344, de 12 de maio de 1998” (BRASIL, 2006). Como se evidencia dos dispositivos citados, bem como da política instituída, a Lei nº
11.343/2006 procurou estabelecer uma nova política nacional relacionada ao
enfrentamento do uso e tráfico de drogas, priorizando medidas preventivas à dependência
e conferindo tratamento diferenciado aos dependentes e traficantes. Para isso, revogou
expressamente as leis anteriores e trouxe muitas inovações, criando novos tipos penais e
adotando uma política criminal, em tese, não punitiva em relação ao usuário de
entorpecentes. Com o advento da nova Lei de Drogas houve uma grande discussão em torno do
teor do seu art. 284. Esse dispositivo apresenta uma política criminal extremamente Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 inovadora, na medida em que não prevê, aos usuários, pena privativa de liberdade, mas
somente a de “advertência sobre os efeitos das drogas; prestação de serviços à
comunidade; e medida educativa de comparecimento à programa ou curso educativo”. Em
outras palavras, os usuários não mais seriam punidos com privação de sua liberdade, mas
submetidos às penas restritivas de direitos previstas no dispositivo. Guilherme Nucci (2006, p. 755) observa que o art. 28 da Lei nº 11.343/2006 tipifica
uma infração penal de “ínfimo potencial ofensivo”, diferindo, portanto, da infração penal
de menor potencial ofensivo, pois não se pode aplicar a pena privativa de liberdade. Conclui, assim, que a conduta recebeu tratamento mais brando por parte do legislador, o
que não existe até mesmo em relação às contravenções penais. Na análise de Luiz Flávio
Gomes (2013, p. 111), o art. 28 da Lei seria uma infração sui generis, uma vez que o porte
de drogas para consumo pessoal não seria mais crime por ausência de pena de reclusão ou
detenção. Desde 2011, por meio do Recurso Extraordinário 635.659-SP, o art. 28 está em
debate no Supremo Tribunal Federal (STF). Referido Recurso Extraordinário foi manejado a
partir do argumento de que o referido dispositivo legal tem levado à criminalização do I - advertência sobre os efeitos das drogas; I - advertência sobre os efeitos das drogas; II - prestação de serviços à comunidade; cativa de comparecimento à programa ou curso educativo. III - medida educativa de comparecimento à programa ou curso educativo. 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO § 1o Às mesmas medidas submete-se quem, para seu consumo pessoal, semeia, cultiva ou colhe plantas destinadas à
preparação de pequena quantidade de substância ou produto capaz de causar dependência física ou psíquica. § 1o Às mesmas medidas submete-se quem, para seu consumo pessoal, semeia, cultiva ou colhe plantas destinadas à
preparação de pequena quantidade de substância ou produto capaz de causar dependência física ou psíquica. § 2o Para determinar se a droga destinava-se a consumo pessoal, o juiz atenderá à natureza e à quantidade da
substância apreendida, ao local e às condições em que se desenvolveu a ação, às circunstâncias sociais e pessoais,
bem como à conduta e aos antecedentes do agente. § 3o As penas previstas nos incisos II e III do caput deste artigo serão aplicadas pelo prazo máximo de 5 (cinco) meses. § 4o Em caso de reincidência, as penas previstas nos incisos II e III do caput deste artigo serão aplicadas pelo prazo
máximo de 10 (dez) meses. § 5o A prestação de serviços à comunidade será cumprida em programas comunitários, entidades educacionais ou
assistenciais, hospitais, estabelecimentos congêneres, públicos ou privados sem fins lucrativos, que se ocupem,
preferencialmente, da prevenção do consumo ou da recuperação de usuários e dependentes de drogas. § 5o A prestação de serviços à comunidade será cumprida em programas comunitários, entidades educacionais ou
assistenciais, hospitais, estabelecimentos congêneres, públicos ou privados sem fins lucrativos, que se ocupem,
preferencialmente, da prevenção do consumo ou da recuperação de usuários e dependentes de drogas. § 6o Para garantia do cumprimento das medidas educativas a que se refere o caput, nos incisos I, II e III, a que
injustificadamente se recuse o agente, poderá o juiz submetê-lo, sucessivamente a: § 6o Para garantia do cumprimento das medidas educativas a que se refere o caput, nos incisos I, II e III, a que
injustificadamente se recuse o agente, poderá o juiz submetê-lo, sucessivamente a: I - admoestação verbal; § 7o O juiz determinará ao Poder Público que coloque à disposição do infrator, gratuitamente, estabelecimento de
saúde, preferencialmente ambulatorial, para tratamento especializado.” (BRASIL, 2006). 128 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes porte, para uso pessoal, de substâncias psicoativas. 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO Também se questiona a sua
constitucionalidade na medida em que viola o princípio da intimidade e vida privada,
direito expressamente previsto no artigo 5º, X, da Constituição Federal e, por conseguinte,
o princípio da lesividade, valor basilar do direito penal. A votação no STF ainda não foi
finalizada, porém alguns ministros já se posicionaram pela inconstitucionalidade do art. 28
da Lei 11.343/2006, por reconhecerem que o Estado não pode interferir na vida privada
das pessoas e nem privá-las da decisão sobre usar, ou não, drogas (SUPREMO TRIBUNAL
FEDERAL, 2011). A partir de um cenário mais amplo, no âmbito internacional, a “guerra contra as
drogas” foi deflagrada a partir do ano de 1971, pelo então Presidente dos Estados Unidos,
Richard Nixon. A política de combate às drogas desenhada por Nixon partia de uma
“postura governamental dirigida à exteriorização do problema da produção de psicoativos
e à repressão interna a consumidores e organizações narcotraficantes”. Desse modo, a
política de enfrentamento às drogas de Nixon representava, por um lado, “uma
instrumentalização da proibição às drogas como artifício de política externa” e, por outro,
“um recurso para a governamentalização – disciplinarização, vigilância e confinamento –
de grupos sociais ameaçadores à ordem interna como negros, hispânicos e jovens
pacifistas” (RODRIGUES, 2003, p. 259). Desde então, tem aflorado, entre a população, o “mito da droga”, atribuindo-se aos
usuários o estigma de infratores (PASSETTI, 1991). No Brasil, o referido estereótipo foi
reforçado no período de transição do regime civil-militar, havendo uma determinação
estrutural regulada por leis de oferta e de demanda, concomitantemente a uma ideologia
emocional disseminada pela mídia e acolhida pelo imaginário social, a partir de uma
estratégia dos países capitalistas centrais. O fato é que, como assevera Guilherme (2013), a política brasileira antidrogas
transforma os traficantes em verdadeiros “inimigos” – e, consequentemente – na léxica de
Jakobs (2009) –, “não cidadãos” e “não pessoas”, dado o entendimento de que eles não se
submetem às regras do ordenamento jurídico nacional. No entanto, passa despercebido 129 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021
o fato de que a sua experiência com o Estado costuma ser negativa, cercada de
práticas arbitrárias, de indiferença e de utilitarismo por parte dos governantes. Não se trata de afirmar que tais pessoas se recusam a acatar as regras do jogo;
elas sequer são reconhecidas como incluídas (GUILHERME, 2013, p. 45). Revista Brasileira de Sociologia do Direito, v. 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO 8, n. 3, set./dez. 2021 o fato de que a sua experiência com o Estado costuma ser negativa, cercada de
práticas arbitrárias, de indiferença e de utilitarismo por parte dos governantes. Não se trata de afirmar que tais pessoas se recusam a acatar as regras do jogo;
elas sequer são reconhecidas como incluídas (GUILHERME, 2013, p. 45). O fato é que, em 1988, promulgada a Constituição Federal Brasileira, estabelece-se
como fundamento da República Federativa do Brasil a dignidade humana, o qual
consubstancia verdadeiro princípio constitucional basilar do Estado Democrático de
Direito, como propõe Sarlet (2007). Sobre o referido princípio, cumpre salientar que a
cláusula protetiva da dignidade da pessoa humana atua, segundo Haberle (2009, p. 89), em
uma dupla direção: ela é, ao mesmo tempo, “um direito público subjetivo, direito
fundamental do indivíduo contra o Estado (e contra a sociedade)” e “um encargo
constitucional endereçado ao Estado, no sentido de um dever de proteger o indivíduo em
sua dignidade humana em face da sociedade (ou de seus grupos).” Por meio dessa dupla
direção, o Estado deve criar condições para que a dignidade humana não seja violada por
terceiros (integrantes da sociedade), sendo que “esse dever constitucional pode ser
cumprido classicamente, portanto jurídico-defensivamente, mas também pode ser
desempenhado jurídico-prestacionalmente”, o que significa dizer que “ele pode ser
realizado por caminhos jurídico-materiais e por vias processuais (no sentido de um status
activus processualis), bem como por meios ideais e materiais”. Mesmo tendo na dignidade da pessoa humana um de seus sustentáculos, a
Constituição Federal, em seu art. 5º, XLIII, considerou o tráfico ilícito de entorpecentes e
drogas afins como crimes inafiançáveis e insuscetíveis de graça ou anistia (BRASIL, 1988). Em seguida, em 25 de julho de 1990, foi sancionada a Lei nº 8.072/90 – Lei de Crimes
Hediondos –, que ratificou o posicionamento da Constituição Federal, no sentido de vedar
fiança, anistia e graça nos crimes de tráfico ilícito de entorpecentes e drogas afins, bem
como proibiu a concessão de indulto e liberdade provisória, dentre outras medidas
(BRASIL, 1990). Tais dispositivos, portanto, parecem corroborar com a estratégia político-
criminal de estabelecer uma verdadeira “guerra contra as drogas” no Brasil. Desde então, em decorrência de uma política criminal cada vez mais repressiva e
punitiva, o processo de demonização do tráfico de drogas fortaleceu os sistemas de 130 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes controle social. 5 A Cannabis sativa é uma planta reconhecida e utilizada pelos seus efeitos terapêuticos desde a antiguidade, também
há registros da sua utilização em rituais religiosos e como alimento. É conhecida popularmente no Brasil como
maconha. Dentre as substâncias da planta, duas são as que apresentam efeitos terapêuticos cientificamente testados: 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO Ocorre que dependência não se confunde com vício, eis que, para
depender do tóxico, a droga precisa retirar a autodeterminação do indivíduo. No caso do
vício, retira, somente, a capacidade de entendimento da pessoa. Todavia, para ter acesso
às drogas, em diversas vezes, os usuários são obrigados a frequentar pontos de tráfico. Assim, conforme entendimento de Luiz Flávio Gomes (2011, p. 123), é preciso distinguir, prontamente, o usuário do dependente de drogas. Nem
sempre o usuário torna-se dependente. Aliás, em regra o usuário de droga não se
converte num dependente. Ser usuário de droga (ou de álcool) não significa ser
tóxico-dependente (ou alcoólatra). A distinção é muito importante para o efeito
de se descobrir qual alternativa será mais adequada a cada caso. A identidade repressiva contra as drogas é notória, evidenciando uma forte
influência da Teoria da Defesa Social, cujo objetivo seria a tutela da sociedade contra os
criminosos, por meio dos sistemas de prevenção de delitos e tratamento de delinquentes. Segundo Arlete Hartmann (1999, p. 9) a história já comprovou que a lógica de combate às
drogas assentada nos pressupostos da Defesa Social não é o método mais eficaz, sendo
que “o valor de uma lei está na sua eficácia”. O que ocorre, no Brasil, em relação à
temática, é um forte recorte social e racial, muito bem apreendido por Vera Malaguti
Batista (1998, p. 74) ao afirmar que “aos jovens de classe média, que a consomem
(cocaína), aplica-se o estereótipo médico, e aos jovens pobres, que a comercializam, o
estereótipo criminal”. A eficácia preventiva do tratamento acentua um império do proibicionismo legal,
uma vez que só a droga não leva o dependente ou usuário ao crime, mas sim a
necessidade, já que o fornecimento o torna criminoso, visto que a ilicitude se encontra no
ato de compra e venda, o que é claramente controverso. No que se refere à aplicação do art. 28 da Lei nº 11.343/2006, é curioso que o
elemento subjetivo seja a destinação da droga para o uso pessoal. E mais, quem observa a
natureza da droga é o juiz. É ele quem analisa a conduta, o local, as circunstâncias da ação,
o meio em que o agente vive, os seus antecedentes criminais e a quantidade de
entorpecente. Assim, a atual Lei mascara a descriminalização da droga, já que as penas
previstas no artigo referido devem ser chamadas de medidas socioeducativas. 5 A Cannabis sativa é uma planta reconhecida e utilizada pelos seus efeitos terapêuticos desde a antigu há registros da sua utilização em rituais religiosos e como alimento. É conhecida popularmente
maconha. Dentre as substâncias da planta, duas são as que apresentam efeitos terapêuticos cientificam 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO Porém, a 131 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 função social educativa, à qual realmente se destina, não existe. Para Salo de Carvalho
(2013, p. 329), essas medidas: acabam por destoar da própria lógica do sistema dogmático da teoria
constitucional do delito, substancialmente porque intentam absolutizar critérios
objetivos de forma a induzir a esfera subjetiva do tipo. A partir de conjunturas
fáticas que caracterizam os elementos objetivos (circunstâncias, tempo, local e
forma de agir) ou de características pessoais do autor do fato (antecedentes e
circunstâncias pessoais e sociais), são projetados dados de imputação referentes à
integralidade da tipicidade, olvidando seu aspecto mais importante, o elemento
subjetivo. acabam por destoar da própria lógica do sistema dogmático da teoria
constitucional do delito, substancialmente porque intentam absolutizar critérios
objetivos de forma a induzir a esfera subjetiva do tipo. A partir de conjunturas
fáticas que caracterizam os elementos objetivos (circunstâncias, tempo, local e
forma de agir) ou de características pessoais do autor do fato (antecedentes e
circunstâncias pessoais e sociais), são projetados dados de imputação referentes à
integralidade da tipicidade, olvidando seu aspecto mais importante, o elemento
subjetivo. ndo o entendimento de Salo de Carvalho, Samuel Miranda Arruda (2007,
que se cria, dessa forma,
uma rotulação perigosa dos indivíduos. Deixa-se de analisar o fato criminoso
objetivamente para realizar uma apreciação subjetiva do agente. Como se pode
justificar que uma mesma conduta, se praticada por Pedro, já condenada por
tráfico, seja enquadrada no tipo penal do artigo 33, enquanto a Paulo, primário e
de bons antecedentes, imputa-se o delito associado ao uso? Cremos mesmo ser
de constitucionalidade duvidosa essa disposição. O legislador erigiu aí verdadeira
presunção de culpabilidade em nada compatível com o princípio inverso, da
presunção de inocência. Corroborando o entendimento de Salo de Carvalho, Samuel Miranda Arruda (2007,
p. 31-32) salienta que se cria, dessa forma, p. 31-32) salienta que se cria, dessa forma, uma rotulação perigosa dos indivíduos. Deixa-se de analisar o fato criminoso
objetivamente para realizar uma apreciação subjetiva do agente. Como se pode
justificar que uma mesma conduta, se praticada por Pedro, já condenada por
tráfico, seja enquadrada no tipo penal do artigo 33, enquanto a Paulo, primário e
de bons antecedentes, imputa-se o delito associado ao uso? Cremos mesmo ser
de constitucionalidade duvidosa essa disposição. 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO O legislador erigiu aí verdadeira
presunção de culpabilidade em nada compatível com o princípio inverso, da
presunção de inocência. uma rotulação perigosa dos indivíduos. Deixa-se de analisar o fato criminoso
objetivamente para realizar uma apreciação subjetiva do agente. Como se pode
justificar que uma mesma conduta, se praticada por Pedro, já condenada por
tráfico, seja enquadrada no tipo penal do artigo 33, enquanto a Paulo, primário e
de bons antecedentes, imputa-se o delito associado ao uso? Cremos mesmo ser
de constitucionalidade duvidosa essa disposição. O legislador erigiu aí verdadeira
presunção de culpabilidade em nada compatível com o princípio inverso, da
presunção de inocência. Verifica-se, portanto, a existência de uma grande margem de discricionariedade
reservada ao juiz, haja vista que ele cria o seu próprio conceito de traficante e usuário. Isso
viabiliza a construção de estereótipos de ordem social, transformando os pobres em
traficantes em potencial e os ricos meros usuários de drogas (SHECAIRA, 2014, p. 50). Essa discussão também coloca a questão sobre o que faz com que uma determinada
droga seja essencialmente lícita ou ilícita, já que o critério é a mera previsão legal. Por
conseguinte, nota-se que as drogas podem ou não ser prejudiciais ao ser humano, haja
vista que algumas são usadas em pequenas doses, como remédios ou distrativos, mas, se
mal utilizadas, podem causar danos. Aqui, também podem ser usados como exemplos o
álcool e o tabaco, uma vez que transitam livremente pela sociedade, mesmo que se tenha
conhecimento de seus efeitos nefastos sobre a saúde humana. Ademais, pode-se
mencionar os estudos acerca da Cannabis sativa5, associada a tratamentos alternativos Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes para as pessoas que sofrem de dor Neuropática, Esclerose Múltipla, Doença de Parkinson,
Autismo e Epilepsia (HONÓRIO; ARROIO; SILVA, 2006). Apesar de sofrer preconceito por
grande parte da população por causa do seu potencial alucinógeno, a planta está
constantemente em estudo, trazendo expectativas a pacientes que hoje sofrem com
doenças de difícil tratamento pela indústria farmacêutica. Em razão da prática de infração penal, os dependentes e usuários de drogas, que
estiverem submetidos à medida de segurança ou à pena privativa de liberdade têm, em
tese, assegurados os serviços de atenção à saúde, conforme estipulado no art. 266 da Lei
de Drogas. Contudo, conforme o art. 6 “Art. 26. O usuário e o dependente de drogas que, em razão da prática de infração penal, estiverem cumprindo
pena privativa de liberdade ou submetidos a medida de segurança, têm garantidos os serviços de atenção à sua
saúde, definidos pelo respectivo sistema penitenciário” (BRASIL, 2006). 7 “Art. 47. Na sentença condenatória, o juiz, com base em avaliação que ateste a necessidade de encaminhamento do
agente para tratamento, realizada por profissional de saúde com competência específica na forma da lei, determinará
que a tal se proceda, observado o disposto no art. 26 desta Lei” (BRASIL, 2006). o Delta-9-tetrahidrocanabinol (Δ9-THC), que se atribui os efeitos alucinógenos, e o Canabidiol ao qual se atribui as
propriedades terapêuticas (CDB) (NASCIMENTO; DALCIN, 2019). 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO 477 da mencionada Lei, quem avalia a necessidade do
tratamento, com base nos laudos, é o juiz, sem a participação do destinatário da medida. Verifica-se, assim, que a intenção da referida Lei é, efetivamente, combater o tráfico
de drogas, bem como submeter ao tratamento os dependentes e aplicar medidas
socioeducativas aos usuários, como se fosse uma prevenção ao futuro delito. Neste
prisma, pode-se dizer que o Estado usa uma ideia de profilaxia social, sendo ela uma
estratégia pública de intervenção estatal direcionada ao controle de determinadas
pessoas. Com efeito, quando o Estado passa a adotar a estratégia da internação
compulsória enquanto forma de “tratamento” do usuário, os limites entre cerceamento e
liberdade do indivíduo tornam-se bastante obscuros. Isso porque a internação compulsória
materializa uma espécie de “medida de segurança atípica”, não resolvendo problema
algum e causando, isto sim, novos transtornos. Como assevera Hartmann (1999, p. 41), no lugar de se “transpor barreiras
profundamente arraigadas no preconceito social, no tocante à toxicomania”, permanece
um olhar de corte lombrosiano sobre o tema, revelando traços remanescentes de
darwinismo social na operacionalidade das instituições jurídico-penais, que seguem
operando de modo seletivo. O Estado brasileiro segue pautando a política de drogas pelo 133 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 clamor popular, insuflado pela abordagem seletiva da mídia, conduzindo uma política
criminal facilmente identificada com a ideia de “lei e ordem”, que supõe a necessidade de
uma verdadeira “guerra contra as drogas”, a qual se faz cada vez mais presente nos órgãos
legislativos do país. Sobre o tema, com a ideia da internação compulsória surgiu, em 20 de abril de
2010, o Projeto de Lei nº 7.663/2010 do deputado Osmar Terra, que propõe que o
dependente químico seja internado para tratamento sem que ele autorize. O autor da
medida tão polêmica, salienta que a mens legis é que se estabeleça a quantidade de
drogas como critério para diferenciação dos usuários e traficantes, mudando a forma
como os últimos são punidos e estabelecendo penas que variam conforme o entorpecente. 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO Em 5 de junho de 2019, referido Projeto foi transformado na Lei nº 13.840/2019, que
alterou trechos da Lei nº 11.343/2006, aumentando as hipóteses nas quais o dependente
de drogas possa ser internado involuntariamente – a pedido de familiar ou do responsável
legal ou, na absoluta falta deste, de servidor público da área de saúde, da assistência social
ou dos órgãos públicos integrantes do SISNAD (com exceção de servidores da área de
segurança pública) que constate a existência de motivos que justifiquem a medida (BRASIL,
2019). No tocante ao tratamento do usuário ou dependente de drogas, a Lei nº
13.840/2019 estabelece que deverá ocorrer em uma rede de atenção à saúde,
com prioridade para as modalidades de tratamento ambulatorial. A internação em
unidades de saúde e hospitais deve ser medida excepcional. Importante registrar que o
tratamento oferecido aos usuários e dependentes deverá ser orientado por protocolos
técnicos predefinidos, baseados em evidências científicas, devendo ser oferecido
atendimento individualizado com abordagem preventiva. Caberá à União dispor sobre os
protocolos técnicos de tratamento, em âmbito nacional (BRASIL, 2019). A referida lei estabelece, em seu art. 23-A, §3º, dois tipos de internação: a primeira
é a internação voluntária que é aquela que se dá com o consentimento do dependente de
drogas, mas que não será explorada no presente artigo em virtude do tempo; a segunda é
a internação involuntária, sendo aquela que se dá sem o consentimento do dependente de
tóxicos. Conforme a Lei, após o pedido realizado, a internação involuntária dependerá da 134 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes formalização da decisão por médico responsável, bem como será indicada depois da
avaliação sobre o tipo de droga utilizada, o padrão de uso e na hipótese comprovada da
impossibilidade de utilização de outras alternativas terapêuticas previstas na rede de
atenção à saúde. A internação involuntária perdurará apenas pelo tempo necessário à
desintoxicação, no prazo máximo de 90 dias, tendo seu término determinado pelo médico
responsável. A família ou o representante legal poderá, a qualquer tempo, requerer ao
médico a interrupção do tratamento (BRASIL, 2019). Fazendo uma análise do teor da Lei nº 13.840/2019, portanto, conclui-se que não
foi ofertado nada concreto em prol dos usuários e dependentes de drogas, mas sim a
segregação e a retirada dessas pessoas vulneráveis do seio da sociedade. 8 A partir de denúncias de violações aos direitos humanos de pessoas internadas, segundo Paulo Amarante (2006),
nasceu, em 1978, o movimento de trabalhadores da saúde mental e posteriormente, a luta antimanicomial, um
importante movimento social pela reforma psiquiátrica e pela extinção dos manicômios. Esse movimento denunciou
os maus-tratos aos pacientes psiquiátricos, desnudando o universo de milhares de pessoas abandonadas em
instituições financiadas pelo poder público. 2 UM PANORAMA HISTÓRICO DA LEGISLAÇÃO SOBRE DROGAS NO BRASIL SOB A
PERSPECTIVA DO TRATAMENTO DISPENSADO AO USUÁRIO Embora a lei fale
na priorização do tratamento ambulatorial, não há nenhuma menção de como o governo
pretende reforçar essa modalidade de tratamento. Não há profissionais multidisciplinares
que visem à reinserção dessas pessoas ao convívio em sociedade e, pior, em nenhum
momento a Lei estabelece a necessidade de laudo produzido por psiquiatra. Ademais, é
sabido que o sistema de saúde do Brasil é deficitário, pois clínicas e unidades para
tratamento de dependentes químicos praticam maus tratos e violência contra os internos. A lei somente trata das modalidades de internação e de inclusão em comunidades
terapêuticas, deixando nítido que o tratamento de usuário e dependentes de drogas deve
ocorrer primordialmente na rede substitutiva ao hospital psiquiátrico tradicional, ou seja,
em serviços de base comunitária. O usuário e dependentes de drogas não podem ser negligenciados e nem
segregados. Devem ser respeitadas as suas particularidades e o projeto terapêutico deve
atender às necessidades de cada paciente. A internação, pura e simples, sobremaneira a
involuntária, serve apenas para “condenar” o comportamento, o estilo de vida que não é
moralmente aceito na sociedade. Ao caracterizar o sujeito com a conduta desviante, a
internação articula uma esfera pública de julgamento criminal, criando um novo regime de
visibilidade: os comportamentos desviantes como doença e propensão ao crime. Os
comportamentos tornam-se alvo de discursividade de uma visibilidade própria, criando
meios de condenações moral e jurídica, que violam, dentre outros Direitos Humanos, o
devido processo legal, conforme análise a seguir empreendida. 135 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 9 Dispõe o parágrafo único do art. 2º da Lei nº 10.216/2001: “Parágrafo único. São direitos da pessoa portadora de
transtorno mental: 3 DIREITOS HUMANOS E A INTERNAÇÃO COMPULSÓRIA/INVOLUNTÁRIA DO USUÁRIO
DE DROGAS: A QUEBRA DO DEVIDO PROCESSO LEGAL No Brasil, na passagem dos anos 1970 para os anos 19808, assistiu-se ao surgimento
do movimento que propugnava pela chamada “Reforma Psiquiátrica”. Este movimento
culminou na edição da Lei nº 10.216/2001, que tinha como objetivo a criação de
mecanismos que possibilitassem o tratamento digno dos pacientes psiquiátricos. Foram
criadas diversas instituições auxiliares, como as Unidades Básicas de Saúde (UBS) e os
Centros de Atendimento Psicossocial (CAPS). Outrossim, a Lei passou a exigir locais
diferentes para tratamento psiquiátrico e para a internação dos pacientes que cumprem
medida de segurança (BRASIL, 2001). A referida Lei busca evitar a internação dos pacientes em instituições com
características semelhantes ao sistema prisional, justamente com o objetivo de evitar
confusões conceituais entre o cumprimento de pena e de medida de segurança – mesmo
que as finalidades de ambas, como se sabe, sejam idênticas –, e também para uma maior
garantia da dignidade humana dos portadores de transtorno mental, considerando os
efeitos deletérios do cárcere. Nos termos do art. 3º, §4º da Lei nº 10.216/2001, “é vedada
a internação de pacientes portadores de transtornos mentais em instituições com
características asilares, ou seja, aquelas desprovidas dos recursos mencionados no § 2o
[incluindo serviços médicos, de assistência social, psicológicos, ocupacionais, de lazer, e
outros] e que não assegurem aos pacientes os direitos enumerados no parágrafo único do
art. 2º9 (BRASIL, 2001). 9 Dispõe o parágrafo único do art. 2º da Lei nº 10.216/2001: “Parágrafo único. São direitos da pessoa portadora de
transtorno mental: I - ter acesso ao melhor tratamento do sistema de saúde, consentâneo às suas necessidades; II - ser tratada com humanidade e respeito e no interesse exclusivo de beneficiar sua saúde, visando alcançar sua
recuperação pela inserção na família, no trabalho e na comunidade; III - ser protegida contra qualquer forma de abuso e exploração; IV - ter garantia de sigilo nas informações prestadas; Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes Segundo Paulo Amarante (2010, p. 31), V - ter direito à presença médica, em qualquer tempo, para esclarecer a necessidade ou não de sua hospitalização
involuntária; Segundo Paulo Amarante (2010, p. 31), o processo de desinstitucionalização demonstra tornar-se possível construir e
inventar novas perspectivas de vida e subjetividade para aqueles mesmos
sujeitos, e não apenas ‘evoluções’ e ‘prognósticos’ da doença mental. *...+ No
contexto da desinstitucionalização, na medida em que esta coloca em discussão
os conceitos da loucura, periculosidade, anormalidade, desvio e outros tantos,
sobre os quais a psiquiatria construiu seu mandato terapêutico, legitimou e
autorizou determinada ação institucional violenta e tutelar sobre os sujeitos assim
classificados, e, na medida, ainda, em renúncia a esse mesmo mandato, abre-se
uma nova perspectiva de relação ética entre os homens e as instituições. No Brasil, em se tratando de internação de paciente psiquiátrico, existem três
possibilidades disciplinadas pela legislação quando se fala em doença mental, à luz do
parágrafo único do art. 6º da Lei nº 10.216/2001: internação voluntária, involuntária e
compulsória (BRASIL, 2001). No presente trabalho não se analisará a internação voluntária,
que é aquela na qual existe o consentimento do paciente. Analisar-se-á, sim, a internação
involuntária e a compulsória, no âmbito das quais se evidencia com maior contundência a
possibilidade de violação de direitos humanos do internado. Primeiramente, cumpre salientar que os usuários de drogas em estágio avançado de
drogadição nem sempre possuem condições de se autodeterminar livremente. Assim
sendo, compete à família ou ao ente público interná-los, mesmo contra a sua vontade
(internação involuntária). Conforme o art. 8º da Lei 10.216/2001, quem tem legitimidade
para provocar a internação involuntária ou compulsória é o Ministério Público ou terceiros,
in casu, os parentes do internando ou seus responsáveis legais (BRASIL, 2001). A
internação compulsória, por seu turno, é aquela determinada pelo Poder Judiciário, tendo
como diferencial a avaliação de um juiz. Nesses casos, a justiça toma para si a tutela do
dependente e determina a sua internação, nos termos do art. 9º da Lei nº 10.216/2001
(BRASIL, 2001). 137
V - ter direito à presença médica, em qualquer tempo, para esclarecer a necessidade ou não de sua hospitalização
involuntária;
VI - ter livre acesso aos meios de comunicação disponíveis;
VII - receber o maior número de informações a respeito de sua doença e de seu tratamento;
VIII - ser tratada em ambiente terapêutico pelos meios menos invasivos possíveis;
IX - ser tratada, preferencialmente, em serviços comunitários de saúde mental” (BRASIL, 2001). Segundo Paulo Amarante (2010, p. 31), VI - ter livre acesso aos meios de comunicação disponíveis; VII - receber o maior número de informações a respeito de sua doença e de seu tratamento; I - ser tratada em ambiente terapêutico pelos meios menos invasivos possíveis; IX - ser tratada, preferencialmente, em serviços comunitários de saúde mental” (BRASIL, 2001). 137 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 A internação involuntária deverá ser aplicada em caráter excepcional, razão pela
qual é imprescindível a apresentação de laudo médico, confirmando a necessidade, nos
termos do já referido art. 8º da Lei nº 10.261/2001. O §1º do dispositivo legal mencionado
preconiza que “a internação psiquiátrica involuntária deverá, no prazo de setenta e duas
horas, ser comunicada ao Ministério Público Estadual pelo responsável técnico do
estabelecimento no qual tenha ocorrido, devendo esse mesmo procedimento ser adotado
quando da respectiva alta” (BRASIL, 2001). Isso porque, nesse caso, existe um nítido
conflito com o direito à liberdade do internado. Da mesma forma, o §2º do dispositivo em
comento preconiza que “o término da internação involuntária dar-se-á por solicitação
escrita do familiar, ou responsável legal, ou quando estabelecido pelo especialista
responsável pelo tratamento” (BRASIL, 2001). Não há dúvida, portanto, de que tanto a internação involuntária quanto a
compulsória representam intervenções na vida do cidadão que podem ser consideradas
uma afronta aos direitos humanos. Nesse sentido, a Lei nº 13.840/2019 configura-se como
um retrocesso no que tange à proteção dos direitos humanos dos usuários de drogas. Isso
porque a internação, por si só, contraria os princípios da reforma psiquiátrica cristalizada
na Lei nº 10.216/2001, a qual prioriza justamente o atendimento não institucionalizado
(extra-hospitalar), a partir de uma consideração do usuário/paciente enquanto sujeito e
não como mero objeto de uma política pública, resguardando-se, portanto, a sua
autonomia como cidadão e sua dignidade humana. Sabe-se que a pena é a consequência jurídica de um crime e que há vários tipos de
sanções previstos pelo ordenamento jurídico-penal brasileiro: penas privativas de
liberdade, penas restritivas de direito e penas de multa, nos termos do art. 32 do Código
Penal (BRASIL, 1940). Ao lado das penas, estão as medidas de segurança, as quais são
aplicadas aos agentes que cometeram crimes e que, em decorrência de sua
inimputabilidade, não receberam nenhuma pena no sentido estrito. A partir deste panorama, impõe-se a pergunta: a internação involuntária e a
compulsória também seriam consequências jurídicas de um crime? Segundo Paulo Amarante (2010, p. 31), À primeira vista, a
resposta seria negativa, já que os pacientes submetidos à internação nestas modalidades
não cometeram crime algum. Isto é assim porque, por exemplo, a liberdade de locomoção, 138 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes constitucionalmente protegida, permite que qualquer pessoa vá, volte ou permaneça em
qualquer lugar público. Assim, o simples fato de permanecer, mesmo que durante dias, em
um espaço como uma “cracolândia”, por exemplo, representa, em princípio, o exercício de
um direito fundamental e não a prática de um delito. Percebe-se, assim, que a internação
involuntária/compulsória pode ser enquadrada como uma medida de segurança de
internação (ASENSI, 2011), razão pela qual deve durar enquanto a noção de periculosidade
estiver presente, conforme preconiza o art. 97 do Código Penal (BRASIL, 1940). Deve-se entender que, na verdade, a medida de segurança constitui-se
obrigatoriamente como tratamento ou internação, tendo em vista a prática de fato típico e
ilícito por aqueles que a lei penal considera inimputáveis. Sendo a internação compulsória
e involuntária um tratamento, constata-se uma contradição entre dispositivos penais e a
Lei nº 10.216/2001. Esta Lei estabelece, no seu art. 5º, que o paciente há longo tempo hospitalizado ou para o qual se caracterize situação de
grave dependência institucional, decorre de seu quadro clínico ou de ausência de
suporte social, será objeto de política específica de alta planejada e reabilitação
psicossocial assistida, sob responsabilidade da autoridade sanitária competente e
supervisão de instância a ser definida pelo Poder Executivo, assegurada a
continuidade do tratamento, quando necessário (BRASIL, 2001). Significa assim, conforme a mencionada Lei, que é vedada a internação perpétua,
sendo obrigatória a determinação de prazo. Ora, os efeitos de uma internação, seja ela
compulsória ou involuntária, são em tudo análogos aos efeitos de uma medida de
segurança: o paciente é recolhido a uma instituição psiquiátrica controlada pelo Estado,
para o fim oficial de receber tratamento; entretanto, sem saber ao certo o tempo mínimo
e máximo da internação. Levando em conta a magnitude da intervenção estatal na esfera
de liberdade do indivíduo, é evidente que as internações só se legitimariam, em um Estado
Democrático de Direito, precedidas de um devido processo legal. Com efeito, em um Estado Democrático de Direito, o processo penal está
circundado por limites e garantias. Segundo Paulo Amarante (2010, p. 31), Isso porque o Direito, em um Estado Democrático de
Direito, não cumpre mais com uma função de ordenação (como na fase liberal), ou apenas
de promoção (como na fase do Estado de Bem-estar Social), sendo “mais do que um plus
normativo em relação às fases anteriores, constituindo-se em um elemento qualificativo 139 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 para a sua própria legitimidade, uma vez que impulsiona o processo de transformação da
realidade” (STRECK, 2008, p. 279). Ou seja, o Estado Democrático de Direito pretende
superar os modelos de Estado Liberal e Social, adotando, do primeiro, a ideia de Estado de
Direito, isto é, de Estado governado pelo Direito emanado da vontade geral (art. 1º,
parágrafo único, da Constituição Federal), em contraposição a um Estado Absolutista, a fim
de cumprir com a exigência de defesa da sociedade em face do arbítrio estatal, o que se
pretende conseguir por meio da técnica formal da divisão dos poderes e do princípio da
legalidade. Já em relação ao Estado Social, adota-se a perspectiva segundo a qual devem
ser quebradas as barreiras que separam Estado e sociedade, quer dizer, o Estado é erigido
à condição de “motor ativo” da vida social, sendo chamado a modificar efetivamente as
relações sociais. Portanto, o Estado Democrático de Direito incorpora os núcleos liberal e social,
juntamente
com
um
projeto
de
sociedade
e
de
democracia
positivado
constitucionalmente. Visa, assim, a atender princípios como os da constitucionalidade,
democracia, sistema de direitos fundamentais, justiça social, divisão de poderes,
legalidade, segurança e certeza jurídica, para que se possa buscar a menor desigualdade
possível entre a coletividade (BOLZAN DE MORAIS, 1996). Como salienta Mir Puig (1994, p. 33-34), la fórmula ‘Estado social y democrático de Derecho’ supone no solo la tentativa
de someter la actuación del Estado social – a la que no se quiere renunciar – a los
límites formales del Estado de Derecho, sino también su orientación material
hacia la democracia real. Se pretende, por esta vía, acoger una modalidad de
Estado social – esto es, que tome partido efectivo en la vida social – al servicio de
todos los ciudadanos. En cuanto social y democrático, tal Estado deberá crear
condiciones sociales reales que favorezcan la vida del individuo, pero para
garantizar el control por el mismo ciudadano de tales condiciones deberá ser,
además, un Estado democrático de Derecho. Segundo Paulo Amarante (2010, p. 31), El carácter democrático de esse
Estado aparece vinculado, pues, a la síntesis del Estado social y del de Derecho, y
expresa tanto la necesidad de libertad ‘real’ – oponiéndose a que el ‘Estado social’
dirija sólo su intervención en beneficio de ciertos grupos – como ‘formal’ –
cerrando el paso a la posibilidad de un ‘Estado de Derecho’ no controlado por
todo el pueblo – para los ciudadanos. Como síntese do exposto, portanto, cumpre salientar a lição de Zaffaroni (2007, p. 169), no sentido de que “os Estados de direito não são nada além da contenção dos
Estados de polícia, penosamente conseguida como resultado da experiência acumulada ao 140 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes longo das lutas contra o poder absoluto.” Quer dizer, o pacto social da modernidade, o
Direito moderno e suas Constituições, estão umbilicalmente ligados ao intento de conter a
guerra, de civilizar e submeter a regras institucionais os conflitos políticos e sociais
(BARATTA, 2000). Com efeito, de acordo com Zúñiga Rodríguez (2001, p. 27), o reconhecimento dos
direitos fundamentais e a construção do conceito de Estado Democrático de Direito que o
acompanha, como paradigmas do Direito legítimo, podem ser tidos como a mais
importante invenção do Ocidente e a mais louvável conquista do ser humano, uma vez que
os direitos fundamentais e o conceito de Estado de Direito “constituyen ideales con una
legitimación axiológica capaz de oponerse a cualquier forma de opresión social e individual
en el mundo.” Nesse contexto, destaca-se, inicialmente, a garantia da jurisdição (art. 5º, XXXV, da
Constituição Federal), que significa ter um juízo imparcial, natural e que representa
exclusivamente o poder de jurisdição. No âmbito do processo penal, vige o princípio da
jurisdicionalidade, segundo o qual o processo é um pressuposto em relação à pena. Em
outras palavras, não há pena e não há culpa sem processo – nulla poena, nulla culpa sine
iudicio. Neste contexto, coloca-se a questão sobre a necessidade de um processo prévio
para a imposição da internação involuntária e compulsória. Na medida em que a
internação possui um caráter verdadeiramente sancionatório-penal, como já visto acima,
sua aplicação pelo Estado pressupõe um processo prévio e regrado, sob pena de violação
do princípio da jurisdicionalidade, bem como do princípio do devido processo legal. Assim, o mencionado princípio determina que algumas matérias fiquem submetidas
ao Poder Judiciário. Esta “reserva de jurisdição” aplica-se, também, em outras searas do
sistema jurídico. Segundo Paulo Amarante (2010, p. 31), Nesse sentido, impõe-se prestigiar os princípios e valores fundamentais inscritos na
Constituição da República, os quais demandam a observância do devido processo legal, do
contraditório, da ampla defesa, bem como a presunção geral de liberdade, e a necessidade
de observância da proporcionalidade de qualquer intervenção estatal. Relativamente à 141 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 tutela das garantias no âmbito jurídico-penal, Zaffaroni (2007, p. 173) destaca que essa
tutela é inerente ao Estado de direito porque as garantias processuais penais e as
garantias penais não são mais do que resultado da experiência de contenção
acumulada secularmente e constituem a essência da cápsula que encerra o Estado
de polícia, ou seja, são o próprio Estado de direito. O direito penal de um Estado
de direito, por conseguinte, não pode deixar de esforçar-se em manter e
aperfeiçoar as garantias dos cidadãos como limites redutores das pulsões do
Estado de polícia, sob pena de perder sua essência e seu conteúdo. Sob qualquer ângulo que se analise o tema, então, é forçoso concluir pela
inconstitucionalidade da internação involuntária/compulsória, sobretudo quando pensada
como instrumento de uma política pública para a abordagem da questão das drogas. Neste cenário, é notória a exacerbada intervenção do Estado em relação ao
indivíduo, haja vista que não se pode abandonar, em nome de uma propalada “defesa da
sociedade”, o princípio do devido processo legal. Sobremaneira, em um Estado
Democrático de Direito, que tem na dignidade da pessoa humana um de seus
sustentáculos. O art. 5º, LIV, da Constituição Federal, expressa que “ninguém será privado da
liberdade ou de seus bens sem o devido processo legal (BRASIL, 1988). Com isso, visa-se a
limitar a atividade penal do Estado, no sentido de proteger o direito à liberdade a favor do
justo processo e dos direitos humanos. De fato, compreender a Constituição como norma
fundamental que constitui o valor de referência de um programa de política criminal supõe
redescobrir seu significado como conjunto de normas substanciais dirigidas à garantia da
divisão dos poderes e dos direitos fundamentais de todos, quer dizer, os dois princípios
que são negados em um ambiente de Estado absoluto. Segundo Paulo Amarante (2010, p. 31), Assim, a legitimidade do Estado,
tanto no que diz respeito à sua política social quanto no que se refere à sua política
criminal, não se produz somente pela mera legalidade, mas também está condicionada
pela “estrita legalidade”, a qual, por sua vez, é motivada pelo conteúdo respeitoso aos
direitos fundamentais (ZÚÑIGA RODRÍGUEZ, 2001, p. 52). Sendo assim, o princípio do devido processo penal é, em essência, garantia de
liberdade. Trata-se de um direito consagrado na Declaração Universal dos Direitos
Humanos e na Convenção de São José da Costa Rica (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 142 142 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes 1948; ORGANIZAÇÃO DOS ESTADOS AMERICANOS, 1969), exercendo a função de um
importante “freio” para que sejam respeitados os direitos do ser humano e para que o
Estado não abuse da liberdade e não se rebele contra aqueles que tem a obrigação de
proteger e respeitar. Contudo, para a manutenção do Estado Democrático de Direito, é necessário o
respeito à Constituição, porque é ela que define o devido processo legal como uma
garantia do sistema penal e daqueles que estão sujeitos ao poder coercitivo do Estado. Caso contrário, corre-se o risco de se ter decisões voluntaristas e arbitrárias. Outrossim, o Estado Democrático de Direito deve garantir que as partes
interessadas estejam cientes sobre as informações contidas no processo. Tal princípio é
imperativo no processo penal. Portanto, é uma garantia constitucional que garante a
ampla defesa ao acusado, possibilitando às partes um meio de provar/convencer o juiz da
prova apresentada, já que o juízo só pode decidir a partir das provas contraditadas. Em outras palavras, o princípio do contraditório é o direito de informação,
conhecimento; de saber o que está acontecendo e do direito de acesso a tudo que está no
processo. É a possibilidade de reação da parte. O princípio da ampla defesa, por sua vez, trata da obrigação do Estado em
proporcionar a todo acusado condições para exercer seu direito de defesa, possibilitando-
lhe trazer ao processo os elementos que achar necessários para o esclarecimento da
verdade. Processualmente, tal princípio garante ao acusado o conhecimento inequívoco da
imputação que lhe é feita nos termos da acusação e fundamentos de fato e de direito. Posteriormente, fornece todos os meios possíveis para contrariá-la. Segundo Paulo Amarante (2010, p. 31), As garantias analisadas ligam-se à indisponibilidade dos interesses em litígio –
direito de punir versus direito de liberdade – motivo pelo qual os revestem de cautelas-
garantias. Enquanto a ampla defesa assegura a possibilidade de informação do acusado da
imputação que lhe é feita, são assegurados também os meios jurídicos para atacá-la; o
contraditório liga-se à efetividade e plenitude do exercício defensivo, frente à pretensão
punitiva estatal. A teor do art. 23-A da Lei 13.840/2019, a internação involuntária é um tipo de
internação psiquiátrica caracterizada por ser realizada sem o consentimento do 143 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 dependente de tóxicos. Conforme a Lei, após o pedido realizado, a internação involuntária
acontecerá a partir da formalização da decisão por médico responsável, bem como será
indicada depois da avaliação sobre o tipo de droga utilizada, o padrão de uso e na hipótese
comprovada da impossibilidade de utilização de outras alternativas terapêuticas previstas
na rede de atenção à saúde. A internação involuntária perdurará apenas pelo tempo
necessário à desintoxicação, no prazo máximo de 90 dias, tendo seu término determinado
pelo médico responsável. A família ou o representante legal poderá, a qualquer tempo,
requerer ao médico a interrupção do tratamento (BRASIL, 2019). Veja-se que o indivíduo internado não tem direito de expressar a sua
voluntariedade ao tratamento, bem como vê aplicada em seu desfavor uma medida de
segurança autoritária para o “bem da sociedade” e para a prevenção de futuros delitos. Assim, nota-se a supressão da ampla defesa e do contraditório, ou seja, o sujeito
sequer cometeu um crime e já é encaminhado a “cumprir uma pena” que deveria ser
aplicada a quem praticou delitos. Segundo Nereu Giacomolli (2014 p. 94), o sujeito “não
pode ser tratado como se já fosse ou nascesse culpado e nem como um objeto do
processo, mas como um ser humano e sujeito processual”. Em outras palavras, pode-se sugerir que a internação compulsória e a involuntária
são uma espécie de medida de segurança “atípica”, pois, como já mencionado, não estão
presentes na sua execução os princípios do devido processo legal: contraditório e ampla
defesa que são assegurados pelo art. 5º, LV10, da Constituição Federal. Verifica-se, portanto, que o Estado brasileiro abandonou a ideia de voluntariedade
do tratamento do dependente de drogas, colocando em seu lugar o instituto da internação
involuntária/compulsória como solução. 10 “Art. 5º. *...+ LV - aos litigantes, em processo judicial ou administrativo, e aos acusados em geral são assegurados o
contraditório e ampla defesa, com os meios e recursos a ela inerentes” (BRASIL, 1988). Segundo Paulo Amarante (2010, p. 31), Ora, consumir o tóxico ou não é um ato de
voluntariedade do cidadão, bem como o de se tratar ou não se tratar, e precisa ser, por
esta razão, respeitado pelo Estado. Nesta linha de pensamento, somente haverá crime
quando algo ou alguém for lesionado (princípio da lesividade); ao contrário, não há uma
justificativa para uma medida de segurança atípica apenas pelo uso de drogas. 144 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes No dizer de José Carlos Sousa Silva (2006, p. 41), “é dever de todos respeitar e
proteger a vida humana e cabe ao poder estadual punir quem causar-lhe ameaça ou
dano”. Assim, a Constituição Federal não autoriza expressamente o poder de internação
ao Estado. A isto devem ser somadas as observações de Ela Wiecko V. Castilho (2012, p. 36) sobre o instituto jurídico ora em exame: O tratamento compulsório é violência; segregação sem o devido processo legal. Significa negação da liberdade individual ou, antes, da própria condição para o
exercício dessa liberdade, isto é, negação da própria capacidade de discernimento
e tomada de decisões por parte do usuário-dependente – como decorrência do
uso de droga em si mesmo. A partir desta lógica, a exceção se torna regra, o
recolhimento se converte em medida preferencial – sobretudo quando se trata de
combater a “epidemia do crack. É preciso ter em conta que as internações involuntárias e compulsórias não
incidem de forma igual para todos os dependentes de drogas. Da mesma maneira
como ocorre com a pena privativa de liberdade e com a medida de segurança,
recai preferencialmente sobre pessoas e grupos sociais vulneráveis, em razão de
idade, de sexo ou identidade de gênero, de cor, de procedência, de deficiência, de
classe social. Assim é que funciona o que chamamos de sistema de justiça. A
seletividade, característica estruturante do sistema penal, existe em todos os
países independentemente do seu nível de desenvolvimento. Ademais, fornecendo valiosos dados empíricos a consubstanciar as vozes da
doutrina anteriormente referida, impõem-se de maneira contundente relatório elaborado
pelo Conselho Federal de Psicologia (2011, p. 191): A Lei nº 10.216 prevê a internação compulsória como medida a ser adotada por
juiz competente. Disto se depreende que ela deve ser parte de um processo
judicial, ou seja, decorrência da adoção de uma medida de segurança, tendo em
vista o cometimento de ato infracional por parte do usuário. Segundo Paulo Amarante (2010, p. 31), O que se vê na
prática, com os usuários de álcool e outras drogas, contraria o disposto na lei, pois
introduz a aplicação de medida jurídica fora de um processo judicial. É o recurso à
lei, o uso do aparato jurídico para segregar e não para mediar as relações do
sujeito com a Justiça e com a sociedade. A internação compulsória e involuntária serão, portanto, uma forma de punir o
usuário, sendo ele viciado ou não. Sobre o tema, Arlete Hartmann (1999, p. 42), já em
1999, fez a seguinte indagação: “pode o Estado forçar um doente a submeter-se a
tratamento médico? Como conciliar com esta norma o direito à liberdade (elencado como
direito fundamental na nossa Constituição e de aplicabilidade imediata)?” E conclui: 145 evista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021
Todo o nosso sistema penal estabelece que cada pessoa pode fazer com sua
saúde o que melhor lhe convém – não há penalização para a tentativa de suicídio,
nem para autolesão, tampouco pode o Estado invadir a vida privada, reprimindo o
uso de entorpecente enquanto esta atitude não invadir a esfera do bem comum,
de terceiros (HARTMANN, 1999, p. 55). Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 Todo o nosso sistema penal estabelece que cada pessoa pode fazer com sua
saúde o que melhor lhe convém – não há penalização para a tentativa de suicídio,
nem para autolesão, tampouco pode o Estado invadir a vida privada, reprimindo o
uso de entorpecente enquanto esta atitude não invadir a esfera do bem comum,
de terceiros (HARTMANN, 1999, p. 55). O Estado não deve incentivar, consentir e nem instituir a internação do usuário de
drogas, uma vez que ele precisa querer se tratar, e não ser tratado autoritariamente. É
necessário ter em mente que o Estado está ludibriando a população com seu discurso, já
que o tratamento não voluntário é ineficaz, porque não conta com o convencimento
pessoal do sujeito pela interrupção do hábito. É provável a recaída, o que na lógica da
internação, só pode gerar novos e sucessivos recolhimentos forçados. 4 CONSIDERAÇÕES FINAIS De acordo com o que foi afirmado na introdução deste trabalho, a temática da
internação involuntária/compulsória mostra-se cada vez mais atual e complexa. O
problema com as drogas sempre foi uma realidade no Brasil e no mundo. Leis são criadas
para contê-las, porém ainda não conseguiram extingui-las da sociedade e o consumo
continua crescente, ocasionando efeitos cada vez mais devastadores. No ano de 2006, surge, no Brasil, uma nova Lei de Drogas (Lei nº 11.343/06), que
não mais pune o uso de drogas, de modo que o indivíduo que for pego com certa
quantidade de drogas que caracterize o consumo pessoal não mais é submetido a uma
pena privativa de liberdade, mas simplesmente cumpre uma medida educativa elencada
na lei. Recentemente, no entanto, foi sancionada a Lei nº 13.840/2019, que altera trechos
da Lei nº 11.343/2006, aumentando as hipóteses nas quais dependentes e usuários podem
ser internados involuntariamente. A referida Lei vem na contramão da Reforma Psiquiátrica promovida no Brasil pela
Lei nº 10.216/2001, uma vez que não foi ofertado nada concreto em prol dos usuários e
dependentes de drogas, mas sim a segregação e a retirada dessas pessoas vulneráveis do
seio da sociedade. Embora a Lei nº 13.840/2019 fale na priorização do tratamento
ambulatorial, não há nenhuma menção de como o governo pretende reforçar essa
modalidade de tratamento. Não há profissionais multidisciplinares que visem a reinserção 146 Maiquel Ângelo Dezordi Wermuth e Luíza Hrynyszyn Menezes dessas pessoas ao convívio em sociedade e pior, em nenhum momento a Lei estabelece a
necessidade de laudo produzido por psiquiatra. A lei somente trata das modalidades de
internação e de inclusão em comunidades terapêuticas, deixa nítido que o tratamento de
usuário e dependentes de drogas devem ocorrer primordialmente na rede substitutiva ao
hospital psiquiátrico tradicional, ou seja, em serviços de base comunitária. Portanto, parece que essa alteração na orientação da Política Nacional de Drogas
representa um retrocesso para Brasil, uma vez que a lei pretende ampliar e obrigar a
internação psiquiátrica de usuários de drogas. Não é propondo a internação de usuários e
dependentes de drogas que o governo vai resolver um problema tão complexo, como é o
caso do abuso e dependência de substâncias psicoativas. Como um problema de saúde
pública, deve-se optar por cuidar das pessoas no seu território, evitando, assim,
enclausuramento e as longas internações, para que possam continuar convivendo em
sociedade. REFERÊNCIAS AMARANTE, Paulo. Rumo ao Fim dos Manicômios. Mente & Cérebro. 2006. AMARANTE, Paulo. O Homem e a serpente: outras histórias para a loucura e a psiquiatria. Rio de Janeiro: Fiocruz, 2010. ARRUDA, Samuel Miranda. Drogas: Aspectos Penais e Processuais Penais: Lei 11.343/06. São Paulo: Método, 2007. ASENSI. Felipe Dutra. Direito sanitário. Rio de Janeiro: Elsevier, 2011. 4 CONSIDERAÇÕES FINAIS A Lei não trata de matéria penal ou processual penal, mas está ligada a questões
envolvendo
políticas
públicas
para
de
usuários
e
dependentes
de
drogas. Independentemente do mérito de tais políticas, espera-se uma melhora no tratamento de
usuários e dependentes de drogas, e que o Supremo Tribunal Federal coloque em pauta o
Recurso Extraordinário proporcionando a discussão sobre a constitucionalidade do crime
de posse de droga para consumo pessoal. Tanto a internação compulsória quanto a internação involuntária, baseadas apenas
em laudo médico circunstanciado, violam o direito fundamental de liberdade, bem como
os princípios do devido processo legal, do contraditório e da ampla defesa, sendo,
portanto, inconstitucionais. A supressão do devido processo legal impulsiona consequências determinantes na
sociedade, uma vez que gera o incômodo da população em relação ao Estado que é
Democrático de Direito e não autoritário. Nesse sentido, é preciso enfatizar que a solução
não é somente a internação dos usuários e dependentes de drogas deixando-os afastados
da sociedade e esquecidos, mas sim tratá-los de forma digna. Estas ações seriam realizadas
por profissionais especializados da área de saúde física e mental. O Poder Judiciário é o
guardião de direitos humanos, por essa razão, não pode violar a liberdade e obrigar os 147 Revista Brasileira de Sociologia do Direito, v. 8, n. 3, set./dez. 2021 usuários e dependentes de drogas a se submeterem ao tratamento de maneira
compulsória ou involuntária. Levando em consideração o que foi exposto neste trabalho, conclui-se que o
tratamento de usuário e dependentes de drogas, no Brasil, requer mudanças, devendo ser
respeitadas as escolhas dos cidadãos. O Estado brasileiro, ao invés de honrar o seu dever
de amparar e assistir os usuários e dependentes de drogas, está impulsionando estes seres
vulneráveis para exclusão da sociedade, mediante a aplicação de medidas flagrantemente
violadoras de direitos humanos. ASENSI. Felipe Dutra. Direito sanitário. Rio de Janeiro: Elsevier, 2011. Dispõe sobre a prevenção, o tratamento,
a fiscalização, o controle e a repressão à produção, ao uso e ao tráfico ilícitos de produtos,
substâncias ou drogas ilícitas que causem dependência física ou psíquica, assim elencados
pelo
Ministério
da
Saúde,
e
dá
outras
providências. Disponível
em:
http://www.planalto.gov.br/ccivil_03/LEIS/2002/L10409.htm. Acesso em: 25 maio 2021. BRASIL. Lei nº 10.409, de 11 de janeiro de 2002. Dispõe sobre a prevenção, o tratamento,
a fiscalização, o controle e a repressão à produção, ao uso e ao tráfico ilícitos de produtos,
substâncias ou drogas ilícitas que causem dependência física ou psíquica, assim elencados
pelo
Ministério
da
Saúde,
e
dá
outras
providências. Disponível
em:
http://www.planalto.gov.br/ccivil_03/LEIS/2002/L10409.htm. Acesso em: 25 maio 2021. BRASIL. Lei nº 11.343, de 23 de agosto de 2006. Institui o Sistema Nacional de Políticas
Públicas sobre Drogas - Sisnad; prescreve medidas para prevenção do uso indevido,
atenção e reinserção social de usuários e dependentes de drogas; estabelece normas para
repressão à produção não autorizada e ao tráfico ilícito de drogas; define crimes e dá
outras providências. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2004-
2006/2006/lei/l11343.htm. Acesso em: 25 maio 2021. BRASIL. Lei nº 11.343, de 23 de agosto de 2006. Institui o Sistema Nacional de Políticas
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Alegre: Livraria do Advogado, 1996. BRASIL. Decreto-Lei nº 2.848, de 7 de dezembro de 1940. Institui o Código Penal
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Aprovado em: 31/05/2021 151
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Carbon Resistances
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Physical review
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public-domain
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No. 6.]
CARBON
RESISTANCES.
355
CARBON RESISTANCES.
BY A. C. LONGDEN.
A
L T H O U G H our dependence upon carbon as a high resistance
material is not as nearly absolute as it was a few years ago, yet
I suppose it is true to-day that the majority of electrical resistances
ranging from 1,000,000 to 100,000,000 ohms and used for general
experimental purposes are made of carbon. Perhaps the most
common form is the graphite line on a ground-glass surface.
Of course carbon resistances at best are very much inferior to
wire resistances, but when a number of resistances of several m e g ohms each are required, the majority of us have to look for something cheaper than wire. T h e use of a less expensive substitute is
not a matter of choice, but a necessity.
A few years ago, while looking for a satisfactory form of carbon
for a resistance material, I hit upon the idea of carbonizing sugar in
clay sticks. In April, 1897, I mixed 10 grams of pipe clay and 2
grams of flake graphite with 10 cubic centimeters of a 20-per-cent.
solution of ordinary granulated sugar. T h e moist clay was then
pressed into cylindrical sticks about 5 millimeters in diameter.
These sticks were thoroughly dried in an oven at 150 degrees and
then carbonized by keeping them at a red heat 8 hours in a covered
crucible, packed with graphite.
I then prepared another series of carbon sticks in every way similar to those just described, except that the graphite was omitted, so
that the resulting sticks contained no carbon except that which was
furnished by the sugar. In both cases the results were tolerably
satisfactory, although there was no great uniformity in either. T h e
resistance of the carbon sticks in the first series was from 1,000 to
5,000 ohms per centimeter, while that of the second series was several megohms per centimeter.
Another lot, similar to the first (with graphite), but more thoroughly carbonized (20 hours) varied in resistance from 25 to 200
356
A.
C. LONG DEN.
[VOL.
XV.
ohms per centimeter; and still another lot, carbonized 3 hours only,
varied from 5,000 to 40,000 ohms per centimeter. Of course, even
if the carbonization,were thorough in every case, the resistance of
these sticks may be varied indefinitely by adjusting the amounts of
g r a p h i t e and sugar in the mixture before carbonization.
O n e of the objections to these resistances is the rapid increase of
resistance with time. This increase is represented graphically by
the curves in Fig. 1, where time is plotted on the X-axis and resistance on the Y-axis. These curves are very fair representatives of
100
200
Time in Days
300
400
Fig. 1.
the class of resistances described above. It is to be noted that while
the resistance increases very rapidly at first, it is also true that after
a sufficient length of time the change is so slow as to be quite negligible during a brief interval, such as a few days, or even a few weeks.
If no other objection could be urged against these resistances, they
might be used in any short series of experiments, and, if standardized
immediately before and after the series of experiments, their values
during the experiments might be known to a degree of accuracy
which would satisfy the requirements of all ordinary work. A n -
No. 6.]
CARBON
RESISTANCES.
357
other, and a more serious objection, however, demands our consideration. It is the unreliable character of the junctions between the
carbon sticks and their terminal wires. T h e resistance of the j u n c tion should be small and constant. A s a matter of fact, if the wires
are fastened on with clamps, the resistance of the joint is usually
high and quite variable. T h e variations are sometimes so great as
to indicate that there is more resistance in the joints than anywhere
else. F o r example, on one occasion a resistance fell from 1,840,0 0 0 to 746,000 ohms upon tightening the clamps, and, upon still
further increasing the pressure, the resistance fell to 357,000 o h m s .
Doubtless the variations which occur after the clamps are permanently set are largely due to the difference in coefficient of e x pansion between the carbon and the clamp. A t any rate, temperature changes frequently produce changes in resistance which can
hardly be accounted for on any other hypothesis.
In support of this view I cite another example. In this case the
terminal wires were twisted around the ends of the carbon stick and
then a mass of melted lead was cast around the joint. T h e lead of
course answers the purpose of a closely fitting clamp, binding upon
all sides of the carbon stick ; but as the lead and the carbon have
different coefficients of expansion, the pressure of the clamp is regulated by the temperature. T h e effect upon the resistance of the joint
is shown in Table I., which displays the results of a number of resistance measurements made upon the same carbon stick at different
temperatures.
TABLE
I.
Resistance in Ohms.
Dif.
Temperature.
Dif.
Change in Resistance
per Degree.
43495
42030
41120
40621
42810
1465
910
499
2189
14.1°
16.2
17.5
18.2
15.1
2.1
' 1.3
.7
3.1
698
700
713
706
F r o m the results in the last column it will be seen that the resistance changes followed the temperature changes within two per
cent., which is quite as close agreement as the thermometer readings
would warrant. Of course the experiment suggests temperature
358
A.
C.
LONGDEV.
[VOL.
XV.
coefficient; but the magnitude of the change in resistance per
degree is very much greater than the temperature coefficient of the
material would explain. I am inclined to believe that the contact
resistance between a metal clamp and a piece of high resistance
material is in general very much higher and very much less constant than manufacturers of high resistances have supposed. Of
course, conducting wires are fastened to carbon battery plates by
means of clamps, but the carbon used in battery plates is a low resistance material. A s the specific resistance of the material increases, the contact resistance increases also. Professor Rood, in a
recent article, 1 " O n the Electrical Resistance of Glass, Quartz, Mica,
Ebonite and Gutta-Percha," says : " Mere mechanical contact is
not sufficient to abolish contact resistance, which is apt to be quite
large.'' In making this statement Professor Rood was discussing
millions of megohms.
T h e point to the quotation is that contact resistances may be quite large, even in comparison with very large
resistances, and that contact resistance increases with the specific
resistance of the material used.
A b o u t three years ago I had occasion to test a graphite megohm
which had been prepared by rubbing a pencil point upon a strip of
ground glass. The terminals were strips of tin foil, fastened on with
clamps. T h e manufacturer had tried to eliminate the inconstancy
so common in graphite resistances by making the line of graphite
very broad, and, of course, proportionally long. I subjected this
m e g o h m to a large number of tests, comparing it with platinum film
resistances, 2 and also with a wire resistance made by Elliott Bros.
T h e only conclusion at which I could arrive was that the resistance
of the graphite megohm varied as much as ten per cent.—sometimes
as much as three or four per cent, within a few minutes.
In a paper read before the British Association in September,
1896, Mr. W. M. M o r d e y 3 offers an improvement upon the ordinary
graphite line, in the form of a strip of cartridge paper, coated with
graphite and burnished. T h e advantage of this form is said to be
its increased cross-section, t h o u g h I can see no reason why a
1
Am. Jour, of Sci., XIV., 161.
PHYS. REV., XI., 40-55 and 84-94.
3 Phil. Mag., S. 5, XLIL, 450.
2
No. 6.]
CARBON
RESISTANCES.
359
graphite line on ground glass might not have as large a cross-section
as the same material on cartridge paper, A n objection to the paper
is that it is flexible, and the resistance of the conducting layer of
graphite changes when the paper bends. Of course the paper may
be mounted in such a way as to minimize the probability of bending,
but the untrustworthy character of the clamped contacts is still a
serious objection. In June, 1901, I measured the resistance of a
film of graphite on cartridge paper, prepared by one of the best
manufacturing firms in the world, and bearing their name. On the
25th of the month its resistance was 93,850 ohms. Only one measurement was made on that day. On the 26th, a number of measurements were made, with results varying from 94,000 to 97,000.
O n the 27th the resistance had fallen to 92,090. All the measurements were made with the same apparatus and under the same conditions of temperature, electromotive force, etc. It is m y belief
that much of the erratic behavior of carbon resistances which has
been attributed to small cross-section could justly be charged to
poor contacts.
Being thus thoroughly at war with clamp contacts for high-resistance materials, I tried the plan of electroplating copper terminals to
the clay-carbon sticks. Of course
this could not be done by passing the plating current t h r o u g h
the entire stick, on occount of its
high resistance, but it is very successfully and easily done by passing the current from the anode,
t h r o u g h the solution, to a fine
copper wire twisted several times
around the end of the carbon stick, as in Fig. 2. In this figure,
A is the a n o d e ; B, the clay-carbon stick; C, the conducting
wire; D, the surface of the plating bath, and Ey the glass dish
in which the process is conducted. T h e copper is at first deposited only upon the copper wire, but in a short time it begins to creep out over the surface of the clay-carbon stick and soon
forms a perfect coating over as m u c h of the surface as is immersed
in the bath. T h e contact between the copper and the carbon is so
close as to fully satisfy the requirements of the case.
360
A.
C.
LONGDEN.
[VOL.
XV.
I have described these clay-carbon resistances because they have
been very serviceable to me and to a number of others who have
used them, but I wish also to describe another form of carbon resistance which I find even more satisfactory. It consists of a film
of smoke deposited upon a strip of glass. It makes very little diference what kind of smoke or how deposited, though I think perhaps camphor smoke gives results which are a little more nearly
uniform than candle smoke or the smoke from a gas jet. T h e
resistance of a film having a given length and breadth, depends,
of course, upon its thickness. A film 5 cm. long by 1.5 cm.
wide may have any resistance from about 50,000 ohms to about
100 megohms.
A s in the work already described, the most serious difficulty to
be overcome was the matter of making satisfactory contacts between
the carbon and the terminal wires. It was found possible to deposit
copper electrolytically upon the ends of the smoke films, but the
resistance of the films almost invariably increased abnormally afterwards, many of them rising to infinity. A microscopic examination
revealed the fact that the connections between the films and the
terminal wires had been weakened or severed by minute cracks in
the films, just at the edge of the copper deposit.
Having had an experience similar to this in dealing with platinum
films,1 I resorted to the method which had been so successfully applied to them. It consists in first coating the ends of the glass strip
with metallic silver, chemically deposited, and then depositing the
resistance material in a uniform layer over the entire surface of glass
and silver. T h e silver tips may be thick enough to stand electrolytic depositions of copper at the extreme ends, or they may even be
made so thick that fine copper wires may be soldered directly to the
silver.
Tin-foil terminals attached to the glass with shellac and thoroughly baked were tried, but in this case the smoke films which
were afterwards deposited were always thinner at the edge of the
tin-foil than elsewhere, and in the case of very thin films there was
lack of continuity at this point. Even in films of average thickness
the resistance at the joint was very high.
X
PHYS. REV., XL,
53.
No. 6.]
CARBON
RESISTANCES.
36l
Efforts were made to correct this defect in three ways: First, by
rubbing down the edge of the tin-foil with very fine emery paper.
Second, by overlaying the joint with gold leaf. Third, by brushing
smoke into the joint with a camel's-hair brush. In each case the
treatment of the joint was previous to the final deposition of smoke.
All of these methods gave fairly good results, but none of them
equal to coating the ends of the glass with silver by chemical
means. The perfection of this method lies in the fact that the portion of the glass which is below the plane of the surface of the silvering bath is heavily coated with silver, while a small portion just
above this plane receives silver from only the small amount of solution which rises above the plane of the surface by capillary action.
For this reason the silver film, however thick, always terminates in
an indefinitely thin edge, so that the layer of smoke which is afterwards deposited, is perfectly continuous and uniform over the entire
surface of glass and silver.
A cross-section of one of the tin-foil joints, without preliminary
treatment, considerably magnified, is represented in Fig. 3. Fig. 4
is a similar representation of a silver-tip joint.
Notwithstanding the perfect smoothness of the silver-tip joint, I
was not without apprehension in regard to the possibility of the
smoke lying so loosely upon the surface of the silver as to render a
high contact resistance possible. I have, therefore, tested these
joints in a variety of ways and in almost every case I find their resistance quite negligible.
One.of the methods used in testing joints is as follows :
QA film is split lengthwise by completely removing the smoke and
silver from the central portion of the glass, except at one end, and
two terminal wires are provided for the split end. The film is then
in the form of a U, with a terminal at each extremity and one at
362
4. C
LONGDEN.
[VOL.
XV.
the bend, as in Fig. 5. T h e smoke film is represented at EEy in
the figure, and the silver tips at DDD.
T h e resistance is measured
DD
Fig. 5.
between A and B, and also between B and C. If the resistance
between the smoke and the silver is negligible, the resistance AC
will be equal to the sum of AB and BC.
On account of the extreme delicacy of a film of smoke upon
glass, it is difficult to handle and use the unprotected films without
danger of injuring them. Fortunately, a simple bath in alcohol
vapor is sufficient to harden a smoke film to such an extent that it
may be further protected by flowing upon its surface a coat of shellac
varnish, india-rubber varnish, 1 or melted paraffin ; or it is even possible to imbed a film in paraffin without injuring it. This lastmentioned process, however, is not recommended because of the
change in volume which the paraffin subsequently undergoes when
changes in temperature occur. A thin layer of the material is perfectly safe and quite sufficient to protect the films if ordinary care
is used in handling them. If shellac varnish is used as a protecting
material, the varnished film should be pretty thoroughly baked at
about 110 degrees, in order to remove all the alcohol and water
from the varnish. Of course, a film can not be said to be perfectly
protected until it is enclosed in a dry wood or hard rubber case and
provided with binding posts.
It has not seemed worth while to attempt to adjust these resistances to particular values, on account of the changes in value which
would occur afterwards. It really is not very important that they
should be adjusted, for it is so easy to make them in large quantities
that one may have almost an unlimited variety from which to select.
However, it ought to be mentioned here that putting a coat of insulating varnish upon a film increases its resistance considerably.
1
A solution of india-rubber in carbon bisulphide.
No. 6.]
CARBON
RESISTANCES.
363
If the film is dipped into the varnish or melted paraffin and then
removed immediately and allowed to harden quickly, the protecting
layer of insulating material is mainly upon the surface, and the resistance is not very greatly increased ; but if the film is allowed to
remain immersed in the varnish or melted paraffin for a considerable
length of time, the insulating material will soak into the film, separating the particles of carbon from each other and thus greatly increasing the resistance of the film. T h e preliminary treatment with
alcohol vapor reduces the resistance, probably because it simply
packs the particles of carbon closer together.
A n o t h e r difference between the tin-foil and silver terminals o u g h t
to be pointed out in this connection. W h e n tin-foil terminals are
used, even if the joints are successfully made, many of the films
which give good results for a time, ultimately increase in resistance,
usually to infinity; probably on account of a rupture at the joint,
d u e to the unequal expansion of the different materials.
Especially
is this true of films which have been coated with shellac or paraffin
or even hardened with alcohol. T h e silver-tipped films are free
from this defect. Films which have not been coated with varnish
or paraffin, but in which the smoke lies just as it was deposited,
are not so likely to suffer in this way, even if they have tin-foil
terminals, provided the joints are originally good. I have a n u m ber of such films in splendid condition, now over four years o l d ;
but of course these unprotected films are always in great danger of
being injured by handling.
It has already been stated that the alcohol vapor treatment reduces the resistance of a smoke film. This is always the case, if the
joints are good. A n increase of resistance under this treatment is
always an indication of faulty joints.
T h e constancy of the silver-tipped smoke films as compared with
the clay-carbon sticks with copper-plated terminals is shown b y
comparing Figs. 1 and 6. T h e curves in Fig. 1 are for claycarbon sticks. Those in Fig. 6 are for smoke films. T h e two
sets of curves are drawn on the same scale and are for resistances
of about the same range, from one to three or four m e g o h m s .
It
will be seen at a glance that the increase in resistance is very m u c h
less rapid in the smoke films.
A
364
c
-
> LONG DEN.
[VOL. XV.
These curves evidently become practically straight lines after a
1
-
•
1
F
1
1
i
i ...__
[l
100
200
T i m a in Days
j
3
|
300
Fig. 6.
sufficient length of time, but they probably never become quite
parallel to the X-axis.
In some cases, notably films with tin-foil terminals, it was noted
that the resistance would fall when the electromotive force was increased. This fall of resistance amounted in some cases to as much
as 4 or 5 per cent, upon increasing the E.M.F. from 1 to 5 volts.
It is believed that the cause of the change in resistance is the
closing of the joints at the edges of the tin-foil terminals, by heating, and therefore lengthening the film between the joints. Of
course it is true that the amount of heat developed in a film carrying such a small current as these high-resistance films carry, is
very small; but, that a small amount of heat will produce a marked
effect upon the resistance of one of these joints is made evident by
the fact that a poor joint, when used as a bolometer, is capable of
detecting temperature changes as small as one ten-thousandth of a
degree or less.
No. 6.]
CARBON
RESISTANCES.
365
The change of resistance with voltage just considered is a change
which invariably occurs when the joints are poor.
In comparison with the changes just noted, Table II. exhibits the
very small changes which occur under similiar conditions when the
silver tips are used instead of tin-foil.
TABLE
Film Number.
53
Resistance in Ohms
\
f
I
f
54
L
55
II.
\
r
I
107030
107010
106750
2848900
2848100
2836200
63391
63390
63310
E M F in Volts.
4.2
28
140
4.2
28
140
4.2
28
140
These changes, amounting to only a few tenths of one per cent.
for 140 volts, are so small as to be fully explained by the temperature coefficient of the carbon ; and they leave no room for doubt
in regard to the perfection of the contacts.
I have in a number of cases subjected resistances of this kind to
a potential difference of 104 volts, alternating, for ten days at a time,
without producing any perceptible effect upon them, except to
hasten the aging process a little.
K N O X COLLEGE PHYSICAL LABORATORY,
GALESBURG, I I I . , September 8, 1902.
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English
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Optimization of selection for growth in Menz Sheep while minimizing inbreeding depression in fitness traits
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Genetics selection evolution
| 2,013
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cc-by
| 2,941
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Open Access Open Access Open Access Abstract The genetic trends in fitness (inbreeding, fertility and survival) of a closed nucleus flock of Menz sheep under
selection during ten years for increased body weight were investigated to evaluate the consequences of selection
for body weight on fitness. A mate selection tool was used to optimize in retrospect the actual selection and
matings conducted over the project period to assess if the observed genetic gains in body weight could have
been achieved with a reduced level of inbreeding. In the actual selection, the genetic trends for yearling weight,
fertility of ewes and survival of lambs were 0.81 kg, –0.00026% and 0.016% per generation. The average inbreeding
coefficient remained zero for the first few generations and then tended to increase over generations. The genetic
gains achieved with the optimized retrospective selection and matings were highly comparable with the observed
values, the correlation between the average breeding values of lambs born from the actual and optimized matings
over the years being 0.99. However, the level of inbreeding with the optimized mate selections remained zero until
late in the years of selection. Our results suggest that an optimal selection strategy that considers both genetic
merits and coancestry of mates should be adopted to sustain the Menz sheep breeding program. Research problem and hypothesis Selective breeding is expected to reduce fitness of animal
populations through its negative effect on genetic diver-
sity, particularly in small closed populations. The reduc-
tion in fitness caused by intensive selection for production
traits is more severe under random mating systems due
to more frequent mating between close relatives [1]. Optimum genetic gains while minimizing inbreeding can
be achieved by adopting appropriate selection and mating
designs. Such designs include the family mating design
[2], where mating permissions are restricted within mating
groups, and minimum coancestry mating, which involves
individual mate selection that limits the contribution of
individuals to future generations [3-5]. © 2013 Gizaw 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. Findings paper were to evaluate the current Menz sheep breeding
program in terms of genetic trends in fitness (fertility
and survival), as related to inbreeding, and to investigate
whether the observed genetic gains in body weight could
have been achieved with a reduced level of inbreeding
by optimizing the actual selection and matings con-
ducted over the project years by adopting the individual
mate selection approach. Optimization of selection for growth in Menz
Sheep while minimizing inbreeding depression in
fitness traits Solomon Gizaw1*, Tesfaye Getachew1, Aynalem Haile2, Barbara Rischkowsky2, Johann Sölkner3 Solomon Gizaw1*, Tesfaye Getachew1, Aynalem Haile2, Barbara Rischkowsky2, Johann Sölkner3 and Markos Tibbo4 * Correspondence: s_gizaw@yahoo.com
1Debre Birhan Agricultural Research Centre, P.O. Box 112, Debre Birhan,
Ethiopia
Full list of author information is available at the end of the article Ge n e t i c s
Selection
Evolution Ge n e t i c s
Selection
Evolution Gizaw et al. Genetics Selection Evolution 2013, 45:20
http://www.gsejournal.org/content/45/1/20 Ge n e t i c s
Selection
Evolution Ge n e t i c s
Selection
Evolution Estimation of breeding values A closed nucleus population of Menz sheep was established
in 1998 in the subalpine highlands at the Debre Birhan
Agricultural Research Center in Ethiopia. Selection of
breeding stocks was based on estimated breeding values
(EBV) for yearling weight which were estimated using the
best linear unbiased prediction (BLUP) procedure fitting a
univariate model. Mating was carried out according to a
family mating design, in which random mating of indivi-
duals was allowed within mating groups [6,7]. The mating
groups were created by dividing the foundation flock into
five groups at the beginning of the breeding program. The A selective breeding program for the Menz sheep
breed in Ethiopia has been ongoing in a closed nucleus
flock since 1998. Remarkable improvements in body
weight have been recorded [6]. The objectives of this Gizaw et al. Genetics Selection Evolution 2013, 45:20
http://www.gsejournal.org/content/45/1/20 Page 2 of 5 Figure 1 Genetic trends in yearling weight in a closed nucleus population of Menz sheep selected for yearling weight. mating groups remained closed with female replacements
coming from within the group, but rams were not permit-
ted to mate in their groups of origin. nature of the binary traits fertility and survival. However,
several studies have shown that linear models that as-
sume underlying continuous distributions can be applied
to estimate genetic parameters [8,9] and linear and
threshold models have given very similar heritability esti-
mates for survival in another Menz sheep population
[10]. Breeding values and inbreeding coefficients (F)
for each animal were estimated simultaneously using
WOMBAT [11] by fitting the following univariate
model: Data obtained from the Menz sheep nucleus flock
from 1998 to 2008 was used to estimate genetic trends
in yearling weight, ewe fertility, pre-weaning lamb sur-
vival and level of inbreeding. Ewe fertility and pre-
weaning lamb survival are binary traits and are defined
respectively, as number of ewes lambing combined as a
proxy measure to pregnancy, and as survival of a lamb
from birth to weaning at three months. Ewes lambing
and lambs surviving were assigned a value of 1, whereas
ewes not lambing and lambs not surviving a value of 0. The pedigree data used to estimate breeding values
consisted of 2194 animals born from 709 dams and 80
sires, spanning over seven generations of pedigree for
yearling weight and survival and six generations for
fertility. Estimation of breeding values A total of 1142, 1842 and 2152 records were
available for yearling weight, survival and fertility, res-
pectively. Breeding values were estimated for all traits by
fitting mixed linear models. This ignores the categorical Yi ¼ Xibi þ Ziai þ ei where Yi is a vector of observations for trait i, bi a
vector of fixed effects for trait i (sex, season, year and
dam age for yearling weight and lamb survival; sea-
son, year and age for ewe fertility), ai a vector of ran-
dom animal effects for trait i, ei a vector of random
residual effects for trait i and Xi and Zi are incidence
matrices relating records for trait i to fixed and ran-
dom animal effects, respectively. where Yi is a vector of observations for trait i, bi a
vector of fixed effects for trait i (sex, season, year and
dam age for yearling weight and lamb survival; sea-
son, year and age for ewe fertility), ai a vector of ran-
dom animal effects for trait i, ei a vector of random
residual effects for trait i and Xi and Zi are incidence
matrices relating records for trait i to fixed and ran-
dom animal effects, respectively. Figure 2 Inbreeding trends in a closed nucleus population of Menz sheep selected for yearling weight. Figure 2 Inbreeding trends in a closed nucleus population of Menz sheep selected for yearling weight. Gizaw et al. Genetics Selection Evolution 2013, 45:20
http://www.gsejournal.org/content/45/1/20 Page 3 of 5 Figure 3 Genetic trends in ewe fertility in a closed nucleus population of Menz sheep selected for yearling weight. Retrospective optimization trend per year, calculated by regressing average EBV on
year of birth, was 0.81 kg. The average inbreeding coeffi-
cient remained zero for the first few generations and
below the acceptable level of 1% in later generations
(Figure 2). However, the trend increased over genera-
tions. The rate of inbreeding (ΔF) per generation calcu-
lated by regressing F on generation number was 0.17%. These results show that a high rate of genetic progress
in growth can be achieved with an acceptable increase of
less than 1% F per generation, at least in the short term. The actual matings carried out in the Menz sheep breed-
ing program were revised in retrospect. For the opti-
mized retrospective selection and mating, we used the
actual candidates and parameters in each mating year,
with selection on yearling weight and individual mate se-
lection with minimum coancestry using the mate selec-
tion tool MateSel version 1.5 [3], based on the principles
of optimal contributions of parents to future generations
[4,5]. Optimization was attempted by placing a hard
constraint on genetic gain and constraining the thresh-
old of progeny inbreeding to 1%. A maximum of five
and a minimum of three sires (mating groups) were con-
sidered in each mating year. The average EBV and in-
breeding coefficients of lambs born in each year of the
optimized matings were compared with the actual values
obtained in the breeding program. The level of fertility was inconsistent over the period
of selection (Figure 3). However, the overall genetic
trend was negative, with an average decline in EBV of
0.00026% per generation. Average EBV for survival
showed an average increase of 0.016% per generation
(Figure 4). Correlations of individual coefficients of in-
breeding with EBV for ewe fertility (r = −0.023) and lamb
survival (r = 0.074), taken as indicators of the effect of
inbreeding on fitness, were small and statistically not
significant (p > 0.05). These findings indicate that genetic
progress in growth can be achieved without a significant
decline in fitness, at least in the short term, provided Conclusion Family mating has proved to be an efficient mating tool
to achieve maximum gains in productive traits with min-
imal losses in fitness traits in the short term. However, a
strategy involving optimal mate selection that considers
both genetic merits and coancestry of mates should be
adopted to sustain the Menz sheep breeding program in
the long run. 5. Meuwissen THE: Maximizing the response of selection with a predefined
rate of inbreeding. J Anim Sci 1997, 75:934–940. 5. Meuwissen THE: Maximizing the response of selection with a predefined
rate of inbreeding. J Anim Sci 1997, 75:934–940. 6. Gizaw S, Lemma S, Komen H, van Arendonk JAM: Estimates of genetic
trends and genetic parameters for live weight and fleece traits in Menz
sheep. Small Ruminant Res 2007, 70:145–153. 7. Gizaw S, Getachew T, Tibbo M, Haile A, Dessie T: Congruence
between selection of breeding rams based on breeding values for
production traits and farmers ram choice criteria. Animal 2011,
7:995–1001. 8. Phocas F, Sapa J: Genetic parameters for growth, reproductive
performance, calving ease and suckling performance in beef cattle
heifers. Anim Sci 2004, 79:41–48. Authors’ contributions
SG
d TG
d
d h Authors’ contributions SG and TG conducted the breeding program and collected the data. MT
suggested the research question for this paper. SG analyzed the data; SG, BR,
MT, AH, TG and JS wrote the paper. All authors read and approved the final
manuscript. References 1. Moreno A, Salgado C, Piqueras P, Gutiérrez JP, Toro MA, Ibanez-Escriche N,
Nieto B: Restricting inbreeding while maintaining selection
response for weight gain in Mus musculus. J Anim Breed Genet 2011,
128:276–283. 1. Moreno A, Salgado C, Piqueras P, Gutiérrez JP, Toro MA, Ibanez-Escriche N,
Nieto B: Restricting inbreeding while maintaining selection
response for weight gain in Mus musculus. J Anim Breed Genet 2011,
128:276–283. 1. Moreno A, Salgado C, Piqueras P, Gutiérrez JP, Toro MA, Ibanez-Escriche N,
Nieto B: Restricting inbreeding while maintaining selection
f
i h
i
i
M
l
J A i
B
d G
2011 2. Croston D, Pollot G: Planned sheep production. Oxford: Blackwell Scientific
Publications; 1994. 3. Kinghorn BP: An algorithm for efficient constrained mate selection. Genet
Sel Evol 2011, 43:4. 3. Kinghorn BP: An algorithm for efficient constrained mate selection. Genet
Sel Evol 2011, 43:4. 4. Meuwissen THE, Sonesson AK: Maximizing the response of selection with
predefined rate of inbreeding: overlapping generations. J Anim Sci 1998,
76:2575–2583. Acknowledgements g
The Menz sheep breeding program is financed by the Amhara Regional
Agricultural Research Institute, Ethiopia. Contributions of the animal
breeding staff of Debre Birhan Agricultural Research Centre are greatly
acknowledged. The genetic gains in yearling weight that were achieved
in the optimized retrospective selection and matings
were compatible with the observed values (Table 1), the
correlation between the average EBV of lambs born from
the actual and optimized matings over the years being
0.99. However, the level of inbreeding with optimized
mate selections remained zero until late in the years of
selection, with only minimal inbreeding in the last two
generations (F < 1%). This was in contrast with the ear-
lier buildup of inbreeding in the actual selection pro-
gram. Similarly, the percentage of individuals with
inbreeding coefficients greater than 1% reached 25 to
50% in the actual breeding program, compared to only 3
to 5% in the retrospectively optimized mating scheme. Furthermore, among the animals born in 2008, all were
inbred in the actual selection program, with F ranging
from 0.098 to 6.25%, whereas only 45.0% were inbred
in the simulated population, with F ranging from 0.1
to 1.56%. Author details
1 1Debre Birhan Agricultural Research Centre, P.O. Box 112, Debre Birhan,
Ethiopia. 2International Centre for Agricultural Research in the Dry Areas, P.O. Box 5466, Aleppo, Syria. 3BOKU, University of Natural Resources and Life
Sciences, 1180, Vienna, Austria. 4Food and Agriculture Organization of the
United Nations, Regional Office for the Near East, P.O. Box 2223, Dokki, Cairo,
Egypt. Received: 3 December 2012 Accepted: 23 May 2013
Published: 19 June 2013 Observed genetic trends Average EBV for yearling weight of lambs increased over
the years of selection (Figure 1). The average genetic Figure 4 Genetic trends in preweaning survival in a closed nucleus population of Menz sheep selected for yearling weight. Figure 4 Genetic trends in preweaning survival in a closed nucleus population of Menz sheep selected for yearling weight. Gizaw et al. Genetics Selection Evolution 2013, 45:20
http://www.gsejournal.org/content/45/1/20 Page 4 of 5 Page 4 of 5 Table 1 Optimized genetic progress in yearling weight (EBV) and inbreeding (F) compared with values observed in a
closed nucleus population of Menz sheep selected for yearling weight
Birth year
Average EBV (kg)
Average F (%)
Percent progeny with F > 1%
Optimized
Observed
Optimized
Observed
Optimized
Observed
1998
0.31
0.87
0
0
0
0
2000
1.33
1.83
0
0
0
0
2001
1.87
1.49
0
0
0
0
2002
3.36
2.88
0
0.28
0
4.7
2003
4.05
3.97
0
0.19
0
6.0
2004
5.36
4.96
0
0.23
0
6.1
2005
5.94
5.30
0
0.47
0
13.9
2006
6.58
6.44
0.14
0.86
3.1
24.7
2008
7.58
7.16
0.32
1.67
5.1
50.0 Table 1 Optimized genetic progress in yearling weight (EBV) and inbreeding (F) compared with values observed in a
closed nucleus population of Menz sheep selected for yearling weight Table 1 Optimized genetic progress in yearling weight (EBV) and inbreeding (F) compa
closed nucleus population of Menz sheep selected for yearling weight that a family mating scheme, rather than a random mat-
ing scheme is adopted. Random mating has been experi-
mentally shown to result in a higher level of inbreeding
and reduced fitness [1]. p
g
The authors declare they have no competing interests. Competing interests
h
h
d
l
h p
g
The authors declare they have no competing interests. Page 5 of 5 Page 5 of 5 Gizaw et al. Genetics Selection Evolution 2013, 45:20
http://www.gsejournal.org/content/45/1/20 9. Kadarmideen HN, Thompson R, Coffey MP, Kossaibati MA: Genetic
parameters and evaluations from single and multiple trait analysis
of dairy cow fertility and milk production. Livest Prod Sci 2003,
81:183–195. 10. Gizaw S, Joshi BK: Genetic and non-genetic factors affecting survivability
of Menz and Awassi x Menz crossbred sheep in Ethiopia. Indian J Anim
Sci 2004, 74:887–889. 11. Meyer K: WOMBAT - a tool for mixed model analyses in quantitative
genetics by REML. J Zhejiang Univ Sci B 2007, 8:815–821. doi:10.1186/1297-9686-45-20
Cite this article as: Gizaw et al.: Optimization of selection for growth in
Menz Sheep while minimizing inbreeding depression in fitness traits. Genetics Selection Evolution 2013 45:20. 9. Kadarmideen HN, Thompson R, Coffey MP, Kossaibati MA: Genetic
parameters and evaluations from single and multiple trait analysis
of dairy cow fertility and milk production. Livest Prod Sci 2003,
81:183–195. 9. Kadarmideen HN, Thompson R, Coffey MP, Kossaibati MA: Genetic
parameters and evaluations from single and multiple trait analysis
of dairy cow fertility and milk production. Livest Prod Sci 2003,
81:183–195. 10. Gizaw S, Joshi BK: Genetic and non-genetic factors affecting survivability
of Menz and Awassi x Menz crossbred sheep in Ethiopia. Indian J Anim
Sci 2004, 74:887–889. 10. Gizaw S, Joshi BK: Genetic and non-genetic factors affecting survivability
of Menz and Awassi x Menz crossbred sheep in Ethiopia. Indian J Anim
Sci 2004, 74:887–889. 11. Meyer K: WOMBAT - a tool for mixed model analyses in quantitative
genetics by REML. J Zhejiang Univ Sci B 2007, 8:815–821. 11. Meyer K: WOMBAT - a tool for mixed model analyses in quantitative
genetics by REML. J Zhejiang Univ Sci B 2007, 8:815–821. doi:10.1186/1297-9686-45-20
Cite this article as: Gizaw et al.: Optimization of selection for growth in
Menz Sheep while minimizing inbreeding depression in fitness traits. Genetics Selection Evolution 2013 45:20. Gizaw et al. Genetics Selection Evolution 2013, 45:20
http://www.gsejournal.org/content/45/1/20 Submit your next manuscript to BioMed Central
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and take full advantage of:
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• Immediate publication on acceptance
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English
| null |
Stat3 and CCAAT/enhancer binding protein beta (C/EBP-beta) regulate Jab1/CSN5 expression in mammary carcinoma cells
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Breast cancer research
| 2,011
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cc-by
| 12,494
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Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Open Access Stat3 and CCAAT/enhancer binding protein beta
(C/EBP-beta) regulate Jab1/CSN5 expression in
mammary carcinoma cells Terry J Shackleford1, Qingxiu Zhang1, Ling Tian1, Thuy T Vu1, Anita L Korapati1, Angela M Baumgartner1,
Xiao-Feng Le2, Warren S Liao2 and Francois X Claret1* © 2011 Shackleford 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: fxclaret@mdanderson.org
1Department of Systems Biology, Unit 950, University of Texas - MD
Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030,
USA
Full list of author information is available at the end of the article Abstract Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 2 of 16 overexpression occurs would be of great interest. The
extent to which Jab1 amplification on chromosome 8q is
responsible for its frequent overexpression in cancer has
not yet been investigated. However, additional mechan-
isms of regulation through transcriptional control are
likely to also be of importance and may link its regulation
to upstream signaling pathways. We hypothesize that
overexpression of Jab1 in breast cancer can be attributed
to an increase in transcriptional activity over that seen in
normal tissue. We therefore studied the transcriptional
regulation of Jab1 in breast cancer cells. In this present
study, we describe the cloning and characterization of the
human Jab1 promoter. We also identify a region whereby
CCAAT/enhancer binding protein-beta (C/EBP-b), signal
transducer and activator of transcription-3 (Stat3), and
GATA1 induce Jab1 transcription and identify a potential
upstream oncogenic signaling molecule that may be key
to the regulation of Jab1 expression in cancer. The region
we describe here has also recently been identified by
another group to contain a T-cell transcription factor
(TCF)-4 and Sp1 binding site that was found to be
important for transcription and activated by human epi-
dermal growth factor receptor (HER)-2 activation of the
AKT/b-catenin pathway [21], which points to the impor-
tance of this region in driving the transcription of Jab1
and possibly linking its expression to potent oncogenic
signaling pathways. Abstract Introduction: The c-Jun coactivator, Jun activation-domain binding protein 1 (Jab1) also known as the fifth
component of the COP9 signalosome complex (CSN5), is a novel candidate oncogene whose aberrant expression
contributes to the progression of breast carcinoma and other human cancers. The mechanism of Jab1 gene
expression and its deregulation in cancer cells remains to be identified. We therefore investigated the
transcriptional regulatory mechanisms of Jab1 expression in human breast carcinoma cells. Methods: To identify potential regulators of Jab1 transcription, we cloned the 5’ upstream region of the human
Jab1 gene and mapped its transcriptional start site. We identified binding sequences for the CCAAT/enhancer
binding protein (C/EBP) and GATA, as well as a signal transducer and activator of transcription-3 (Stat3) consensus
sequence overlapping the C/EBP site, using 5’- deletion analysis and a gene reporter assay. Mutational analysis of
these binding sites was performed to confirm their roles in promoting Jab1 transcription in breast cancer cells. We
further confirmed these binding sites using electrophoretic mobility shift assays (EMSAs) and chromatin
immunoprecipitation (ChIP) assays. We also analyzed whether the siRNA-mediated inactivation of Stat3 and Src
could reduce Jab1-promoter activity and whether interleukine-6 (IL-6) could mediate increased Jab1 expression
through Stat3 signaling. Results: We identified binding sequences for C/EBP, GATA, as well as a Stat3 consensus sequence overlapping the
C/EBP site in the promoter region of Jab1. C/EBP-beta2 is a potential transcriptional activator of Jab1 and mutation
of the C/EBP/Stat3 binding site significantly reduced Jab1-promoter activity. In addition, inhibiting Stat3 significantly
reduced Jab1-promoter activation. EMSA and ChIP assays confirmed that C/EBP, GATA1 and Stat3 bind to Jab1
promoter in breast carcinoma cells. We also found that Src, an activator of Stat3, is involved in Jab1-promoter
activation. siRNA knockdown of Src reduced the Jab1-promoter activity, similar to the results seen when Stat3 was
inhibited in breast carcinoma cells. Interestingly, reactivation of Stat3 in normal mammary epithelial cells (MCF-10A,
MCF-10F) is sufficient to reactivate Jab1 expression. Treatment with the cytokine IL-6 resulted in increased Jab1
expression that was blocked by inhibition of Stat3. Conclusions: These findings reveal a novel mechanism of Jab1 gene regulation and provide functional and
mechanistic links between the Src/Stat3 and IL-6/Stat3 signaling axes that are involved in the activation of Jab1
transcription and regulation of this novel oncogenic protein. Full list of author information is available at the end of the article Shackleford et al. Introduction c-Jun activation domain-binding protein-1 (Jab1) is a
multifunctional protein that regulates cell proliferation
and oncogenesis. Since its identification as a c-Jun coac-
tivator [1], Jab1 has been found to be an integral com-
ponent of the COP9 signalosome (CSN) complex, a
multifunctional protein complex involved in modulating
signal transduction, gene transcription, and protein sta-
bility [2-4]. Jab1 is the fifth subunit of the CSN and is
also referred to as CSN5. One of the most recognized
functions of the CSN is the deneddylation of the cullin-
RING-ubiquitin ligase (CRL) and this function is reliant
on the Jab1 MPN domain metalloenzyme (JAMM) motif
that serves as the catalytic center [5,6]. Jab1 exists not
only as a member of the CSN holocomplex and smaller
CSN complexes, but also as a monomer with a number
of different unique protein interactions and functions
outside of the CSN. Jab1 functionally inactivates several key negative regu-
latory proteins by affecting their subcellular localization,
degradation, phosphorylation, and deneddylation,
thereby acting as a positive regulator of cellular prolif-
eration. Through these interactions, Jab1 plays a crucial
role in the inactivation of several key tumor suppressors,
including cyclin-dependent kinase inhibitor p27Kip1, p53,
and Smad4/7 [7-10]. It can also interact with several
important intracellular signaling molecules including
hypoxia inducible factor-1 alpha (HIF-1a), macrophage
migration inhibitory factor (MIF), E2F1, and cullin 1
(CUL-1) [11,12]. Mutagenesis of the Jab1 promoter Site-directed mutagenesis of CEBP and GATA1 was per-
formed according to the QuickChange II method (Stra-
tagene, La Jolla, CA, USA). The following mutagenic
primers were used: CEBP-Mut, GATA1-Mut, and
GATA1-Del (see Table 1 for sequences). All mutants
were verified by sequencing. Nuclear extract preparation and electrophoretic mobility
shift assay Nuclear extracts were prepared as previously described
[1]. Briefly, MCF7 and MDA-MB-468 cells were lysed in
10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM DTT, and protease inhi-
bitor cocktail (Roche Applied Science, Indianapolis, IN,
USA). After incubation for 10 minute on ice, nuclei
were recovered by centrifugation at 3,000 × g at 4°C for
one minute and resuspended in 20 mM HEPES-KOH
(pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1
mM DTT, and protease inhibitor cocktail. Protein con-
centrations were determined using the DC Protein
Assay (Bio-Rad Laboratories, Hercules, CA, USA). The
following double-stranded DNA oligonucleotides were
used in the electrophoretic mobility shift assays
(EMSAs): -462/-436 CEBP-WT, -462/-436 CEBP-M1, Cell lines, reagents and antibodies Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 3 of 16 Page 3 of 16 Transient transfection with reporter constructs and
luciferase assay MCF7, MDA-MB-231, and MDA-MB-468 cells were
plated into 24-well tissue culture dishes at 4 × 104 cells/
well 24 hours before transfection. Transfections were
performed in triplicate according to the manufacturer’s
protocol using Lipofectamine PLUS reagent (Invitrogen,
Carlsbad, CA, USA). Briefly, 0.4 μg reporter plasmid
Jab1-Luc (Firefly luciferase) together with 10 ng of pRL
(Renilla luciferase) were cotransfected. Luciferase assays
were performed 36 hours after transfection using a
Dual-Luciferase Reporter Assay System (Promega, Madi-
son, W, USA). Firefly and Renilla luciferase activities
were read on a Monolight 3010 luminometer (BD
Bioscience, Rockville, MD, USA). Firefly luciferase activ-
ity was normalized to Renilla luciferase readings in each
well. Each experiment was conducted at least twice in
triplicate. Primer extension 5 minutes, followed by 30 cycles of 30 seconds at 94°C,
30 seconds at 60°C, and 30 seconds at 68°C. After a
final extension at 72°C for 10 minutes, PCR products
were resolved on 1.2% agarose gels and visualized by
ethidium bromide transillumination under UV light. Pri-
mers used were: Jab1 F296-314, Jab1 R1094-1076, Jab1
R883-864, GAPDH F, and GAPDH R. For these and all
following primer sequences please refer to Table 1. An antisense primer, P1 was designed 5’ of the ATG
translation site of the Jab1 gene and end-labeled with T4
polynucleotide kinase and 32P-g-ATP, followed by purifi-
cation using Nu-Clean D25 columns (Shelton Scientific-
IBI, Peosta, IA, USA). Total RNA was isolated from
MDA-MD-231 cells using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA), and 20 μg of RNA was hybridized
with 1 × 106 c.p.m. of 32P-labeled P1 oligonucleotide. The protocol for the Primer Extension System-AMV
Reverse Transcriptase (Promega, Madison, WI, USA) was
followed. The size of the extended product was deter-
mined by electrophoresis on a 6% polyacrylamide gel
containing 6 M urea. To determine the size of the primer
extended fragment, the labeled Promega marker and a
sequencing reaction with Jab1 cDNA as template and
primer P1 using the SequiTherm Excel II DNA Sequen-
cing Kit (Epicentre, Madison, WI, USA) were run simul-
taneously on the gel. Computer analysis Transcription factor binding sites were predicted using
Genomatix software (Munich, Germany); the MatIn-
spector program, which uses TRANSFAC matrices; and
the WebGene HCtata program’s Hamming clustering
method for TATA signal prediction in eukaryotic genes. Cloning and analysis of the human Jab1 promoter To clone the 5’-flanking region of the human Jab1 gene,
a bacterial artificial chromosome clone containing the
region corresponding to Jab1 was used as a template for
PCR. The amplified DNA fragments were subcloned
into the luciferase reporter vector pGL3 (Promega,
Madison, W, USA). Progressive deletion mutants of the
pGL3-Jab1 promoter were created by PCR. The integrity
of constructs was confirmed by DNA sequencing. The
following primers were used: +83R, -2006F, -2958F,
-946F, -658F, -472F, -344F, -127F, and -59F (Table 1). Cell lines, reagents and antibodies The human breast cancer cell lines, MCF7, MDA-MB-
468, MDA-MB-231, BT-474, ZR-75-1, BT-549, MDA-
MB-453, T47D and non-tumorigenic human breast
epithelial MCF-10A, MCF-10F, HMEC, and 184A, were
purchased from the American Type Culture Collection
(Manassas, VA, USA). None were derived directly from
tumor tissue for the purposes of this study. MCF7 cells
were grown in DMEM. Breast cancer cells were grown
in RPMI medium supplemented with 10% FBS. MCF-
10A and MCF-10F cells were grown in 50% DMEM,
50% F-12 medium supplemented with 5% horse serum,
100 units/ml penicillin, 100 μg/ml streptomycin, 10 μg/
ml insulin, 100 ng/ml cholera toxin, 0.5 μg/ml hydrocor-
tisone, 20 ng/ml recombinant human epidermal growth
factor, and 1 mM CaCl2. The following antibodies were
used in the study: C/EBP-a (N-19), C/EBP-b (C-19),
GATA-1 (H-200), and Stat3 (C-20) (Santa Cruz Biotech-
nology, Santa Cruz, CA, USA). The following antibodies
were obtained from Cell Signaling (Danvers, MA, USA):
Src, Phospho-Stat3(Y705), b-Tubulin, and b-actin. Anti-
Flag was obtained from Sigma-Aldrich (St. Louis, MO,
USA). IL-6 was obtained from Invitrogen (Carlsbad, CA,
USA) and used at 40 ng/mL. The Stattic inhibitor
(Sigma, St. Louis, MO, USA) was used at 20 nM. Abnormal overexpression of Jab1 has been detected in
several types of cancer in humans and in some cases
correlates with poor prognosis and low-level expression
of p27 [13-18]. However, the molecular mechanism for
up-regulation of Jab1 in cancer cells is still unclear. Our
studies have shown that Jab1 and p27 protein levels are
inversely correlated in invasive breast carcinoma speci-
mens and that Jab1 is highly expressed in breast tumor
samples relative to paired normal-tissue samples [14]. Jab1, along with the oncogene Myc, reside on the fre-
quently amplified region on chromosome 8 and were
identified to induce a wound signature in human breast
cancer cells [19]. Further investigations identified the
isopeptidase activity of Jab1 to be critical for its ability
to promote transformation and progression in breast
epithelial cells and inhibition of this activity is sufficient
to block breast cancer progression driven by MYC and
RAS [20]. These findings suggest that Jab1 is an impor-
tant regulator in cancer development and preclinical
studies suggest that inhibition of Jab1 delays tumor
growth [14]. Given the frequency of Jab1 overexpression in human
cancers and its potential role in the development of can-
cer,
identifying
the
mechanism
by
which
Jab1 Shackleford et al. Chromatin immunoprecipitation (ChIP) assay -435/-417 GATA1-WT, and -435/-417 GATA1-M1 (see
Table 1 for sequences). -435/-417 GATA1-WT, and -435/-417 GATA1-M1 (see
Table 1 for sequences). PCR and RT-PCR The PCR reaction contained 100 ng of DNA template,
1.5 mM MgCl2, 0.2 mM dNTP (Roche Applied Science,
Indianapolis, IN, USA), 1 uM of primers, and Taq High
Fidelity DNA polymerase (Invitrogen, Carlsbad, CA,
USA). The reverse transcriptase (RT) assay was performed
from 2 μg of total RNA using Superscript II RT (Invitro-
gen, Carlsbad, CA, USA) according to the manufac-
turer’s procedure. A reaction without RT was performed
in parallel to ensure the absence of genomic DNA con-
tamination. PCR amplification was carried out as
described previously [22]. Conditions for the PCR reac-
tion consisted of an initial denaturation step at 94°C for Page 4 of 16 Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Table 1 Sequences of oligonucleotides used in RT-PCR, primer extension, cloning site- directed mutagenesis, EMSA
and ChIP
Oligonucleotide
Sequence (5’ à 3’)
Purpose
Jab1 F296-314
ATCTCAGCATTGGCTCTGC
RT-PCR
Jab1 R1094-1076
TAACCTGAGACATCAATC
RT-PCR
Jab1 R883-864
AGAACTCAACGTATTCACCC
RT-PCR
GAPDH F
CCACCCATGGCAAATTCCATGGCA
RT-PCR
GAPDH R
TCTAGACGGCAGGTCAGGTCCACC
RT-PCR
P1
CGCTCCCGGACGCCGCC
Primer extension
+83R
CTGAAAGCTTCGCTCCCGGACGCCGCC
Cloning
-2006F
CTGACTCGAGGTGGTAGTGAGAGG
Cloning
-2958F
CTGACTCGAGCGCATGGGAACCAA
Cloning
-946F
TGAACTCGAGCCTGCTCCCTGTGTC
Cloning
-658F
CTGACTCGAGCCCACTGCCTCCTCG
Cloning
-472F
CTGAGCTAGCCAACAGACAGCCTT
Cloning
-344F
CTGACTCGAGGAGGCCGAGCCTGC
Cloning
-127F
TGAGCTAGCGTCCCGGAAAGGTCCCC
Cloning
-59F
TGACTCGAGCTGCCCCAAGAGTC
Cloning
-31F
CTGAGCTAGCGTTCCCGTGGTGCGG
Cloning
CEBP-Mut
GCCTTACCTTTTAGTCTTTCctgAAACTTATCTC
Site-directed mutagenesis
GATA1-Mut
CAACAAACTTgagTCATTTAAGGTACCTATACCC
Site-directed mutagenesis
GATA1-Del
GTCTTTCAACAAACTT....CATTTAAGGTACC
Site-directed mutagenesis
-462/-445 CEBP WT
CCTTACCTTTTAGTCTTTCAACAAACT
EMSA
-462/-445 CEBP Mut
CCTTACCTTTTAGTCTTTCctgcAAACT
EMSA
-444/-417 GATA1 WT
AATCATTTATCTCATTTAAGGTACC
EMSA
-444/-417 GATA1 M1
GGAGTCATTTAAGGTACCTATACCC
EMSA
-472F
CTGACTCGAGCAACAGACAGC
ChIP
-343R
CTGAAAGCTTCGCAGGCT
ChIP
Lower-case letters represent mutated oligonucleotides. ChIP, chromatin immunoprecipitation; Del, deletion; EMSA, electrophoretic mobility shift assay; F, forward; Mut, mutant; M1, mutant-1; P, primer; R, reverse; RT-
PCR, reverse-transcription polymerase chain reaction; WT, wild-type. Table 1 Sequences of oligonucleotides used in RT-PCR, primer extension, cloning site- directed mutagenesis, EMSA ucleotides used in RT-PCR, primer extension, cloning site- directed mutagenesis, EMSA Lower-case letters represent mutated oligonucleotides. ChIP, chromatin immunoprecipitation; Del, deletion; EMSA, electrophoretic mobility shift assay; F, forward; Mut, mutant; M1, mutant-1; P, primer; R, reverse; RT-
PCR, reverse-transcription polymerase chain reaction; WT, wild-type. Lower case letters represent mutated oligonucleotides. ChIP, chromatin immunoprecipitation; Del, deletion; EMSA, electrophoretic mobility shift assay; F, forward; Mut, mutant; M1, mutant-1; P, primer; R, reverse; RT-
PCR, reverse-transcription polymerase chain reaction; WT, wild-type. Chromatin immunoprecipitation (ChIP) assay Mapping the transcription initiation start site of the
human Jab1 gene To determine the transcription start site of the Jab1
gene, primer extension analysis was performed. An anti-
sense oligonucleotide (P1 primer) was synthesized corre-
sponding
to
the
sequence
located at the
ATG
translational start site according to the published
sequence [GenBank accession no. NT008183]. The P1
primer was radio-labeled and extended using avian mye-
loblastosis virus reverse transcriptase and analyzed on a
polyacrylamide-urea gel along with a DNA cycle sequen-
cing reaction using the same primer. The primer exten-
sion experiment revealed an extension product of 71
nucleotides using the P1 primer (Figure 1a). The nucleo-
tide 71 bp upstream of the ATG translation start site
was designated the +1 transcription initiation site in the
numbering of the nucleotide sequence throughout this
study. Identification of the transcription elements located in the
Jab1 promoter Based on the determined location of the transcription
start site, we analyzed the Jab1 5’ flanking region for a
functional promoter. Using the MatInspector program,
which uses TRANSFAC transcription factor binding site
matrices, we identified a number of putative transcrip-
tion factor binding sites upstream of the Jab1 transcrip-
tion start site (Figure 1b). A typical TATA box [23] was
found 40 bp upstream of the transcription start site,
along with a CAAT box at -99 bp. A number of putative
transcription factor-binding elements were present in
the promoter sequence, including STAT, ELK, p53, E2F,
C/EBP, GATA, c-Myb, and AP-1. Figure 1 Identification of the transcriptional start site and
promoter region of the human Jab1 gene. (a) Primer extension Figure 1 Identification of the transcriptional start site and
promoter region of the human Jab1 gene. (a) Primer extension
(PE) analysis was performed using an oligonucleotide corresponding
to the antisense sequence at the translational start site (+1). A
sequencing reaction using the same primer labeled with [g -32P]
ATP was included as a size marker and is shown on the left. An
extension product of 71 bp was observed. (b) Sequence of the
promoter region of Jab1, with potential regulatory elements
(underlined) identified according to the TRANSFAC databases. bp,
base pairs; Jab1, c-Jun activation domain-binding protein-1. -435/-417 GATA1-WT, and -435/-417 GATA1-M1 (see
Table 1 for sequences). An
extension product of 71 bp was observed. (b) Sequence of the
promoter region of Jab1, with potential regulatory elements
(underlined) identified according to the TRANSFAC databases. bp,
base pairs; Jab1, c-Jun activation domain-binding protein-1. nmol and 50 nmol) were transfected in complete med-
ium without antibiotics. The -472 Jab1-Luc construct
and pRL were cotransfected 24 hours later using the
manufacturer’s protocol for Lipofectamine PLUS trans-
fection reagent. Luciferase assays were performed after
48 hours. -435/-417 GATA1-WT, and -435/-417 GATA1-M1 (see
Table 1 for sequences). The manufacturer’s protocol for the chromatin immu-
noprecipitation (ChIP) Assay Kit (Upstate Biotechnol-
ogy, Temecula, CA, USA) was followed. Briefly, MCF7
cells or MDA-MB-231 cells transfected with control
pcDNA vector or C/EBP-b2 and were incubated with
1% formaldehyde for 20 minutes at 37°C. Cells were col-
lected, lysed, sonicated, and incubated with 4 μg of anti-
bodies to C/EBP-a, C/EBP-b, GATA-1, Stat3, or b-actin
overnight. PCR was used to amplify DNA bound to the
immunoprecipitated histones after reversing the histone-
DNA cross-links. The following primers were used for
PCR: -472F and -344R (see Table 1 for sequences). q
)
Oligonucleotides were end-labeled with [g-32P] ATP
(MP Biomedicals, Solon, OH, USA) by T4 polynucleo-
tide kinase (New England Biolabs, Ipswich, MA, USA)
and purified with Quick Spin G-50 Columns (Roche
Applied Science, Indianapolis, IN, USA). Pre-binding of
5 μg of nuclear extract to 0.05 mg/mL poly(dI:dC)
(Roche, Applied Science, Indianapolis, IN, USA) was
performed in a buffer containing 20 mM HEPES (pH
7.4), 0.1 mM EDTA (pH 8.0), 75 mM KCl, 2.5 mM
MgCl2, 1 mM DTT, and 5% glycerol for 20 minutes at
room temperature (22 to 24°C) before addition of
60,000 c.p.m. of labeled probe. For competition assays,
25- and 100-fold excess cold competitor oligonucleotide
duplex was added to the reaction buffer 10 minutes
before addition of the labeled probes. For supershift
assays, antibodies were added for 20 minutes at 4°C
prior to addition of the labeled probe. Reactions were
resolved by electrophoresis on a 4.5% nondenaturing
polyacrylamide gel run in 0.5 × TBE, vacuum-dried with
heat, and exposed to film at -80°C. Transfection of small interfering RNA oligonucleotides
Small interfering RNA (siRNA) for Stat3, Src, and Con-
trol (LUC) were obtained from Dharmacon (Lafayette,
CO, USA). Oligonucleotides were transfected using Oli-
gofectamine (Invitrogen, Carlsbad, CA, USA) following
the manufacturer’s protocol. For luciferase assay experi-
ments, MDA-MB-468 cells were plated at 4 × 104 cells
per well in a 24-well tissue culture dish. siRNA (20 Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 5 of 16 Figure 1 Identification of the transcriptional start site and
promoter region of the human Jab1 gene. (a) Primer extension
(PE) analysis was performed using an oligonucleotide corresponding
to the antisense sequence at the translational start site (+1). A
sequencing reaction using the same primer labeled with [g -32P]
ATP was included as a size marker and is shown on the left. Identification of Cis-Acting elements within the Jab1 gene
promoter MCF7 breast cancer cells (Figure 2a). Serial 5’-deletion
mutations of the full-length promoter revealed a pattern
of functional activity in transfected cells. The results
suggest that the core promoter elements that drive max-
imal promoter activity are within the sequence spanning
-472 and -345 upstream of the transcription initiation
site, as the -344 Jab1-Luc construct displayed a marked
loss of promoter activity (Figure 2a). Similar patterns of
activity were seen in other breast cancer cell lines
including BT-549, MDA-MB-231, SKBR3, and BT-474
(data not shown). Additionally, we evaluated the promo-
ter strength in mammary tumor cells versus non-tumor To analyze the mechanisms responsible for transcrip-
tional regulation of Jab1, luciferase constructs were gen-
erated to evaluate Jab1 promoter activity. A fragment of
the human Jab1 promoter region from -2958 to +68
was amplified, sequenced, and subcloned into the luci-
ferase reporter vector pGL3. To identify regulatory
sequences that are important for transcriptional control
of the Jab1 gene, we created a series of 5’-deletion con-
structs of the full length Jab1 promoter and analyzed for
their ability to drive the expression of luciferase in Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 6 of 16 Figure 2 5’ deletion of the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. (a)
Identification of the minimal promoter region of Jab1 using progressive deletion of the 5’ region in luciferase reporter constructs. The plasmids Figure 2 5’ deletion of the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. (a)
Identification of the minimal promoter region of Jab1 using progressive deletion of the 5’ region in luciferase reporter constructs. The plasmids
were cotransfected with Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Values
represent mean ± standard error of triplicate experiments. Luciferase assays shown in subsequent figures were performed similarly. (b) Jab1
promoter 5’ deletion constructs were transfected into MCF7 and MCF-10A cells and subjected to luciferase reporter assays. Promoter activity was
higher in MCF7 epithelial breast cancer cells than in non-tumorigenic epithelial MCF-10A cells. Deletion of the region -472 to -344 resulted in a
loss of promoter activity in both cell lines. (c) A panel of breast cancer cells and normal mammary cells were transfected with the -472 Jab1-Luc
construct. Identification of Cis-Acting elements within the Jab1 gene
promoter Significance is noted as * P < 0.05 and ** P < 0.01 and was determined by t-test. (d) Jab1 protein levels for the cell lines in (c) were
detected by western blotting, b-actin is shown as a control. Jab1, c-Jun activation domain-binding protein-1. Figure 2 5’ deletion of the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. (a)
Identification of the minimal promoter region of Jab1 using progressive deletion of the 5’ region in luciferase reporter constructs. The plasmids
were cotransfected with Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Values
represent mean ± standard error of triplicate experiments. Luciferase assays shown in subsequent figures were performed similarly. (b) Jab1
promoter 5’ deletion constructs were transfected into MCF7 and MCF-10A cells and subjected to luciferase reporter assays. Promoter activity was
higher in MCF7 epithelial breast cancer cells than in non-tumorigenic epithelial MCF-10A cells. Deletion of the region -472 to -344 resulted in a
loss of promoter activity in both cell lines. (c) A panel of breast cancer cells and normal mammary cells were transfected with the -472 Jab1-Luc
construct. Significance is noted as * P < 0.05 and ** P < 0.01 and was determined by t-test. (d) Jab1 protein levels for the cell lines in (c) were Figure 2 5’ deletion of the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. (a)
Identification of the minimal promoter region of Jab1 using progressive deletion of the 5’ region in luciferase reporter constructs. The plasmids
were cotransfected with Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Values
represent mean ± standard error of triplicate experiments. Luciferase assays shown in subsequent figures were performed similarly. (b) Jab1
promoter 5’ deletion constructs were transfected into MCF7 and MCF-10A cells and subjected to luciferase reporter assays. Promoter activity was Figure 2 5’ deletion of the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. (a)
Identification of the minimal promoter region of Jab1 using progressive deletion of the 5’ region in luciferase reporter constructs. The plasmids
were cotransfected with Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Values the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. Identification of Cis-Acting elements within the Jab1 gene
promoter (a)
l
f
b
d l
f h
l
f
h
l
d Figure 2 5 deletion of the Jab1 promoter analysis reveals increased activity in breast cancer cells compared with normal cells. (a)
Identification of the minimal promoter region of Jab1 using progressive deletion of the 5’ region in luciferase reporter constructs. The plasmids
were cotransfected with Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Values
represent mean ± standard error of triplicate experiments. Luciferase assays shown in subsequent figures were performed similarly. (b) Jab1
promoter 5’ deletion constructs were transfected into MCF7 and MCF-10A cells and subjected to luciferase reporter assays. Promoter activity was
higher in MCF7 epithelial breast cancer cells than in non-tumorigenic epithelial MCF-10A cells. Deletion of the region -472 to -344 resulted in a
loss of promoter activity in both cell lines. (c) A panel of breast cancer cells and normal mammary cells were transfected with the -472 Jab1-Luc
construct. Significance is noted as * P < 0.05 and ** P < 0.01 and was determined by t-test. (d) Jab1 protein levels for the cell lines in (c) were
detected by western blotting, b-actin is shown as a control. Jab1, c-Jun activation domain-binding protein-1. Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 7 of 16 cells. Jab1 promoter activity was three times higher in
MCF7 breast cancer cells when observing the -472 con-
struct than in MCF-10A normal mammary epithelial
cells (Figure 2b). Further analysis of a panel of breast
cancer cell lines including BT-474, ZR-75-1, MCF7, BT-
549, and MDA-MB-453 revealed higher Jab1 promoter
activity compared to normal mammary epithelial cells
MCF-10A, HMEC and 184A (Figure 2c). Jab1 protein
levels were also found to be higher in the panel of
breast cancer cells compared with the normal mammary
epithelial cells HMEC and MCF-10A (Figure 2d). There-
fore, the transcription factors responsible for promoting
Jab1 transcription are either not present in normal cells
or not as active as in tumor cells. Another possibility is
that normal cells may express transcription suppressors
acting on this region. In Figure 2b, the activity of the
deletion constructs demonstrated differing ratios of luci-
ferase activity between MCF7 and MCF-10A suggesting
that different dominant regulatory factors may exist in
each of these cell lines. Identification of C/EBP and GATA transcription elements
located in the -472/-345 region of the Jab1 promoter Identification of C/EBP and GATA transcription elements
located in the -472/-345 region of the Jab1 promoter
Our data suggest that the -472 to -345 (-472/-345)
region is key to driving the transcription of Jab1. We
therefore sought to identify the transcription factors that
regulated Jab1 through this region. Using the TRANS-
FAC database, we identified a number of putative tran-
scription factor binding sites in the -472/-345 region,
including sites for C/EBP (-444/CAAC/-441) and
GATA-1 (-435/TATCT/-431) (Figure 1b). Because these
transcription factors are activated in some cases of
tumorigenesis [24,25], we speculated that they contribu-
ted to the increased transcription of Jab1. C/EBP-a, C/EBP-b, and GATA-1 transactivate the Jab1
promoter Identification of Cis-Acting elements within the Jab1 gene
promoter However, interestingly, the same
differential in promoter activity was seen between the
-472 and -344 constructs in both cell lines. are capable of binding transcription factors, we per-
formed a series of EMSA experiments with nuclear
extracts from MCF7 breast cancer cells. The oligonu-
cleotides and mutants for the C/EBP and GATA-1 bind-
ing sites are shown in Figure 3b. The -462/436 and
-435/-417 probes showed transcription factor binding
activity to the DNA containing the C/EBP and GATA-1
binding sites, respectively (Figure 3c). The cold specific
oligonucleotides competed efficiently for binding,
whereas a cold mutant competitor containing a muta-
tion in the C/EBP or GATA-1 binding site did not. Further, a supershift was observed when the oligonu-
cleotides were incubated with antibodies to C/EBP-b
and GATA-1 (Figure 3c). To assess whether any of these sites is important for
expression of Jab1, each was mutated individually in
luciferase reporter plasmids. We introduced mutations
in the -472/-345 region of the Jab1 promoter to disrupt
C/EBP and GATA-1 binding and compared their activ-
ity with the -472 Jab1-Luc promoter construct in transi-
ent transfection assays. Mutation of either C/EBP or
GATA-1 binding sequence reduced Jab1 promoter
activity by approximately 40% and 20%, respectively, and
mutation of both sites resulted in a reduction of
approximately 75% (Figure 3d). Interestingly, C/EBP and
GATA binding sites homology from the human and
mice promoter regions were found very well conserved
[see Figure S1 in Additional file 1]. C/EBP-a, C/EBP-b, and GATA-1 transactivate the Jab1
promoter Supershift of the complex was observed following incubation with C/EBPb or GATA-1 antibodies as shown in the right panels. (d) MCF7 cells were transfected with the -472 Jab1-Luc, -472 C/EBP mutant (Mut1 C/EBP), -472 GATA-1 mutant (Mut 2 GATA1), Mut1+2, or -344
Jab1-Luc plasmids. Mutation of the C/EBP(Mut1C/EBP) and GATA-1 (Mut2GATA1) binding sites reduced Jab1 promoter activity by about 60% and
20%, respectively, and by 75% when mutated together. The firefly luciferase activity of each sample was normalized to that of Renilla luciferase
(pRL). Data present the mean ± standard deviation of three independent experiments. All values were compared with the -472 Jab1-Luc reporter
by Student’s t test. C/EBP, CCAAT/enhancing binding protein; EMSA, electrophoretic mobility shift assay; Jab1, c-Jun activation domain-binding
protein-1; NS, nonspecific binding; SS, supershift. Figure 3 Functional analysis of the Jab1 promoter. (a) The -472/-312 region contains a regulatory sequence that is sufficient to activate
transcription of the Jab1 promoter when cloned in front of the TATA and CAAT boxes. MCF7 cells were transfected with either the -105/+83-
Luc or -472/-312, and -105/+83 Jab1-Luc constructs and subjected to luciferase reporter assays. Values were compared with the -105 Jab1-Luc
reporter by Student’s t test, * P < 0.01. (b) Sequence of the probes used in EMSA assays. The probes span the sequence from -440 to -414 or
from -412 to -389 of the Jab1 promoter and encompass the C/EBP or GATA sites. (c) Binding of C/EBP and GATA-1 to the Jab1 promoter. EMSAs
showing binding of nuclear proteins C/EBP and GATA-1 to the C/EBP and GATA-1 binding sites of the Jab1 promoter. End-labeled
oligonucleotide probes corresponding to the -440/-414 (C/EBP) or -413/-389 (GATA-1) regions of the WT Jab1 promoter sequence were
incubated with nuclear extract proteins from MCF7 cells and separated on a 4.5% polyacrylamide gel. Lane 1 shows binding between probe and
nuclear extract. Specificity was determined by addition of 50 and 100 molar excess of unlabeled specific cold probe, and mutant probe as
specific and nonspecific (NS) competitors as indicated. Lane 4 shows complexes observed in the presence of probes containing mutations in C/
EBP or GATA-1. Supershift of the complex was observed following incubation with C/EBPb or GATA-1 antibodies as shown in the right panels. (d) MCF7 cells were transfected with the -472 Jab1-Luc, -472 C/EBP mutant (Mut1 C/EBP), -472 GATA-1 mutant (Mut 2 GATA1), Mut1+2, or -344
Jab1-Luc plasmids. C/EBP-a, C/EBP-b, and GATA-1 transactivate the Jab1
promoter MCF7 cells were transfected with either the -105/+83- Figure 3 Functional analysis of the Jab1 promoter. (a) The -472/-312 region contains a regulatory sequence that is sufficient to activate
transcription of the Jab1 promoter when cloned in front of the TATA and CAAT boxes. MCF7 cells were transfected with either the -105/+83-
Luc or -472/-312, and -105/+83 Jab1-Luc constructs and subjected to luciferase reporter assays. Values were compared with the -105 Jab1-Luc
reporter by Student’s t test, * P < 0.01. (b) Sequence of the probes used in EMSA assays. The probes span the sequence from -440 to -414 or
from -412 to -389 of the Jab1 promoter and encompass the C/EBP or GATA sites. (c) Binding of C/EBP and GATA-1 to the Jab1 promoter. EMSAs
showing binding of nuclear proteins C/EBP and GATA-1 to the C/EBP and GATA-1 binding sites of the Jab1 promoter. End-labeled
oligonucleotide probes corresponding to the -440/-414 (C/EBP) or -413/-389 (GATA-1) regions of the WT Jab1 promoter sequence were Figure 3 Functional analysis of the Jab1 promoter. (a) The -472/-312 region contains a regulatory sequence that is sufficient to activate
transcription of the Jab1 promoter when cloned in front of the TATA and CAAT boxes. MCF7 cells were transfected with either the -105/+83-
Luc or -472/-312, and -105/+83 Jab1-Luc constructs and subjected to luciferase reporter assays. Values were compared with the -105 Jab1-Luc
reporter by Student’s t test, * P < 0.01. (b) Sequence of the probes used in EMSA assays. The probes span the sequence from -440 to -414 or
from -412 to -389 of the Jab1 promoter and encompass the C/EBP or GATA sites. (c) Binding of C/EBP and GATA-1 to the Jab1 promoter. EMSAs
showing binding of nuclear proteins C/EBP and GATA-1 to the C/EBP and GATA-1 binding sites of the Jab1 promoter. End-labeled
oligonucleotide probes corresponding to the -440/-414 (C/EBP) or -413/-389 (GATA-1) regions of the WT Jab1 promoter sequence were
incubated with nuclear extract proteins from MCF7 cells and separated on a 4.5% polyacrylamide gel. Lane 1 shows binding between probe and
nuclear extract. Specificity was determined by addition of 50 and 100 molar excess of unlabeled specific cold probe, and mutant probe as
specific and nonspecific (NS) competitors as indicated. Lane 4 shows complexes observed in the presence of probes containing mutations in C/
EBP or GATA-1. C/EBP-a, C/EBP-b, and GATA-1 transactivate the Jab1
promoter Next, we examined which of the C/EBP and GATA
family members are important for Jab1 promoter activ-
ity. We cotransfected different members of the C/EBP
and GATA family, C/EBP-a, C/EBP-b, or C/EBP-δ, or
GATA1-6, into MCF7 cells, along with the -472 Jab1-
Luc plasmid (Figure 4a). Compared with control cells
transfected with vector alone, cells transfected with C/
EBP-a, C/EBP-b, or GATA-1 showed the greatest
increase in -472 Jab1-Luc reporter activity (2.2-, 3.0-,
and 2.6-fold, respectively). GATA-2 and GATA-3 also
showed a significant increase in activity but for the pur-
pose of this study were not studied in detail. C/EBPs are a family of basic leucine-zipper transcrip-
tion factors, including C/EBP-a, C/EBP-b, -∂, -ε, -g, and
-ζ [26]. Of these, C/EBP-a, -b, and -∂are expressed in
the mammary gland, with C/EBP-b proposed to play a
role in breast cancer [27,28]. The GATA family of tran-
scription factors is required for erythroid and megakar-
yocytic differentiation [29]. Unlike other GATA
members, GATA-1 has not been associated with any
solid tumors, but mutations in GATA-1 are associated
with essentially all cases of acute megakaryoblastic leu-
kemia in children with Down syndrome [30]. Interestingly, the transcription factor C/EBP-b has
been associated with breast cancer [27,31]. It is trans-
lated into three different isoforms: C/EBP-b1, a 55-kDa
liver-enriched activating protein (LAP1); C/EBP-b2, a
42- to 46-kDa protein also called LAP2 and; C/EBP-b3
a 20-kDa liver-enriched inhibitory protein (LIP) [25,32]. Of the three C/EBP-b isoforms, LAP2 has been specifi-
cally observed in breast cancer, and overexpression of
this isoform can induce epithelial-mesenchymal transi-
tion [28]. LIP, however, is unable to activate gene tran-
scription, but is still able to bind to DNA and dimerize, To pinpoint the functional significance of the C/EBP
and GATA-1 binding sites detailed within the Jab1 pro-
moter we performed promoter and EMSA analysis of
these regions. Cloning of this region in front of the
-105/+83 sequence containing the TATA and CAAT
boxes was sufficient to drive Jab1 promoter activity (Fig-
ure 3a). To determine whether these putative elements Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 8 of 16 Figure 3 Functional analysis of the Jab1 promoter. (a) The -472/-312 region contains a regulatory sequence that is sufficient to activate
transcription of the Jab1 promoter when cloned in front of the TATA and CAAT boxes. C/EBP-a, C/EBP-b, and GATA-1 transactivate the Jab1
promoter Mutation of the C/EBP(Mut1C/EBP) and GATA-1 (Mut2GATA1) binding sites reduced Jab1 promoter activity by about 60% and
20%, respectively, and by 75% when mutated together. The firefly luciferase activity of each sample was normalized to that of Renilla luciferase
(pRL). Data present the mean ± standard deviation of three independent experiments. All values were compared with the -472 Jab1-Luc reporter
by Student’s t test. C/EBP, CCAAT/enhancing binding protein; EMSA, electrophoretic mobility shift assay; Jab1, c-Jun activation domain-binding
protein-1; NS, nonspecific binding; SS, supershift. Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 9 of 16 Figure 4 C/EBP-a, C/EBP-b and GATA-1 transactivate the Jab1 promoter. (a) -472/+83 Jab1-Luc reporter plasmid was cotransfected with
expression plasmids for C/EBP-a, C/EBP-b, C/EBP-δ, and GATA-1-6. The results are presented as fold activation compared with cells transfected
with pcDNA3.1 vector control. All values were compared with the vector control by Student’s t test. (b) Left, LAP1 (C/EBP-b1), LAP2 (C/EBP-b2),
or LIP (C/EBP-b3) plasmids, along with the -474 Jab1-Luc promoter construct, were cotransfected with the Renilla luciferase plasmid pRL-null
(transfection control) into MCF7 cells, and luciferase activity was assayed. Right, western blot analyses to demonstrate the protein expression of
LAP1, LAP2 and LIP. (c) Endogenous expression of the C/EBPb isoforms (LAP1, LAP2, and LIP) and GATA-1 were detected at higher levels in
breast cancer cell lines compared with normal mammary epithelial cells by western blot analysis. (d) Identification of proteins in complexes by
ChIP assays. Antibodies to b-actin, C/EBP-a, C/EBP-b, or GATA-1 were used to immunoprecipitate protein/DNA complexes from the sonicated
lysates of MCF7 cells. After reversing the cross-linking, DNA was precipitated and PCR was performed using primers to amplify the -472/-344
promoter DNA. As a negative control, primers for the upstream promoter region -642/-475 were used. Bottom panel represents quantification of
the data with ImageJ software. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun activation domain-
binding protein-1; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein; PCR, polymerase chain reaction; pRL, Renilla
luciferase reporter vector; RT-PCR, reverse transcription polymerase chain reaction. Figure 4 C/EBP-a, C/EBP-b and GATA-1 transactivate the Jab1 promoter. (a) -472/+83 Jab1-Luc reporter plasmid was cotransfected with
expression plasmids for C/EBP-a, C/EBP-b, C/EBP-δ, and GATA-1-6. The results are presented as fold activation compared with cells transfected
with pcDNA3.1 vector control. All values were compared with the vector control by Student’s t test. C/EBP-a, C/EBP-b, and GATA-1 transactivate the Jab1
promoter (b) Left, LAP1 (C/EBP-b1), LAP2 (C/EBP-b2),
or LIP (C/EBP-b3) plasmids, along with the -474 Jab1-Luc promoter construct, were cotransfected with the Renilla luciferase plasmid pRL-null
(transfection control) into MCF7 cells, and luciferase activity was assayed. Right, western blot analyses to demonstrate the protein expression of
LAP1, LAP2 and LIP. (c) Endogenous expression of the C/EBPb isoforms (LAP1, LAP2, and LIP) and GATA-1 were detected at higher levels in
breast cancer cell lines compared with normal mammary epithelial cells by western blot analysis. (d) Identification of proteins in complexes by
ChIP assays. Antibodies to b-actin, C/EBP-a, C/EBP-b, or GATA-1 were used to immunoprecipitate protein/DNA complexes from the sonicated
lysates of MCF7 cells. After reversing the cross-linking, DNA was precipitated and PCR was performed using primers to amplify the -472/-344
promoter DNA. As a negative control, primers for the upstream promoter region -642/-475 were used. Bottom panel represents quantification of
the data with ImageJ software. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun activation domain-
binding protein-1; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein; PCR, polymerase chain reaction; pRL, Renilla
luciferase reporter vector; RT-PCR, reverse transcription polymerase chain reaction. Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 10 of 16 and therefore acts as a dominant negative [27]. It has
been suggested that the LAP/LIP ratio may be an
important indicator of C/EBP-b transcription [25]. We
found that LAP2 activates the Jab1 promoter (over two-
fold), whereas LAP1 had little effect and LIP decreased
activity by about 16% (Figure 4b). Further, analysis of
the C/EBPb isoforms in breast cancer cells compared
with normal, revealed higher levels of the LAP-1 and -2
isoforms in MCF7, MDA-MB-468 and MDA-MB-231
breast cancer cells compared with normal mammary
epithelial cells HMEC and MCF-10A (Figure 4c). Inter-
estingly, the LIP expression was also expressed at higher
levels in the breast cancer cells compared with normal
(Figure 4c). This in line with the increased endogenous
Jab1 expression detected in these breast cancer cells
compared with the normal mammary epithelial cells as
shown in Figure 2d. We also detected higher GATA-1
expression in the breast cancer cells compared with nor-
mal mammary epithelial cells and taken together could
be a driving force in leading Jab1 expression in breast
cancer. sequence (-472 to -345) and was not identified by tran-
scription factor database searches (Figure 5b). Inhibition of Stat3 and Src decrease Jab1 promoter
activity and protein expression To further demonstrate the regulation of Jab1 by Stat3,
we examined the effect of inhibition of Stat3 and also
its upstream activator Src using siRNA [37,38]. Inhibi-
tion of Stat3 and Src by siRNA resulted in a dramatic
reduction of Jab1 promoter activity (Figure 6a), mRNA
levels (Figures 6b and 6c), and Jab1 protein expression
(Figure 6d). Similar results were obtained with MDA-
MB-231 cells (data not shown). Collectively, these
results demonstrate that the Src/Stat3 signaling pathway
plays an important role in the regulation of Jab1
transcription. Stat3 enhances the activity of C/EBP-a and C/EBP-b on
the Jab1 promoter The C/EBP transcription factors form heterodimers with
other C/EBP family members and other transcription
factors. We observed that Jab1-driven luciferase reporter
activity increased upon addition of conditioned medium
to MCF7 cells (data not shown). One of the pathways
activated by conditioned medium is the Stat3 pathway,
which has also been linked with breast tumorigenesis
[33]. Moreover, Stat3 has been shown to interact with
the C/EBP transcription factors and increase their activ-
ity [24]. We therefore investigated whether Stat3 can
transactivate the Jab1 promoter, alone or in combina-
tion with C/EBP-a or C/EBP-b. Stat3 alone increased
Jab1 promoter activity, but Stat3 along with C/EBP-a
and C/EBP-b synergistically increased Jab1 promoter
activity (Figure 5a), suggesting that Stat3 interacts with
C/EBP on the Jab1 promoter. To that end, we observed
a STAT general consensus site (TTN5AA) [34] that
overlaps the C/EBP site within the Jab1 promoter C/EBP-a, C/EBP-b, and GATA-1 transactivate the Jab1
promoter Stat1,
Stat3, Stat4, and Stat92E all bind to this generalized
consensus site [34,35]. We next performed a ChIP assay to determine
whether Stat3 could bind with C/EBP-b2 co-operatively
to the Jab1 promoter using MDA-MB-231 cells, which
have constitutively activated Stat3. Since C/EBP-b2 (or
LAP2) seems to be a major activator of the Jab1 promo-
ter (Figure 4), we next focused only on C/EBP-b2. Bind-
ing of Stat3 to the Jab1 promoter was increased greater
than seven-fold when C/EBP-b2 was transfected into the
cells (Figure 5c). Taken together, these data suggest that
C/EBP-b2 and Stat3 bind to the Jab1 promoter to
increase Jab1 promoter activities. We further investigated whether inhibition of Stat3
affects Jab1 promoter activity in MDA-MB-468 cells,
which have constitutively activated Stat3. Expression of
a dominant-negative mutant of Stat3 reduced over 80%
Jab1 promoter activity (Figure 5d). The EEE/VV muta-
tion renders the protein incapable of DNA binding [36]. Expression of exogenous wild type Stat3 increased Jab1
expression, whereas the dominant-negative EEE/VV
mutation reduced Jab1 protein levels (Figure 5e). C/EBP-a, C/EBP-b, and GATA-1 bind to the Jab1 promoter
To further determine whether C/EBP isoforms and
GATA-1 bind to the Jab1 promoter, we performed
ChIP assays on chromatin obtained from MCF7 cells
(Figure 4d). When chromatin was incubated with b-
actin, no product was observed. When chromatin was
incubated with antibodies to C/EBP-a, C/EBP-b, and
GATA-1 and the -472/-344 region was amplified by
PCR, a product was observed. These data suggest that
C/EBP-a, C/EBP-b, and GATA-1 bind specifically to the
-472/-344 region of the Jab1 promoter. Inhibition of Stat3 and Src decrease Jab1 promoter
activity and protein expression Stat3-induced Jab1 transcriptional activation and protein
expression (b) A potential STAT-binding element (TTN5AA consensus sequence) overlaps the C/EBP binding site
(CAAC). (c) Binding of Stat3 to the Jab1 promoter is enhanced by the addition of C/EBP. MDA-MB-231 cells were transfected with either control
of C/EBP-b2 and collected for ChIP assays using an antibodies against Stat3 and primers for the -472/-344 region of the Jab1 promoter. Right
panel represents quantification of the band intensities and normalized to the input with ImageJ software. (d) MDA-MB-468 cells were
cotransfected with either pcDNA control, a Flag-tagged Stat3 (Flag-Stat3), or Flag-tagged dominant negative (DN)-Stat3EEE/VV, along with the -472
Jab1-Luc construct, and subjected to luciferase reporter assays. (e) Overexpression of Flag-tagged Stat3 increased Jab1 protein levels while a
reduction was seen with Flag-DN- Stat3EEE/VV. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun
activation domain-binding protein-1. Figure 5 Stat3 can synergize with C/EBP-a and C/EBP-b to transactivate the Jab1 promoter. (a) The -472/+83 J Figure 5 Stat3 can synergize with C/EBP-a and C/EBP-b to transactivate the Jab1 promoter. (a) The -472/+83 Jab1-Luc reporter plasmid
was cotransfected with expression plasmids for C/EBP-a, or C/EBP-b. Plasmid amounts were normalized by the addition of empty expression
plasmid. Following transfection, MCF7 cells were incubated for another 36 hours. The results are presented as fold activation compared with cells
transfected with pcDNA vector control. (b) A potential STAT-binding element (TTN5AA consensus sequence) overlaps the C/EBP binding site
(CAAC). (c) Binding of Stat3 to the Jab1 promoter is enhanced by the addition of C/EBP. MDA-MB-231 cells were transfected with either control
of C/EBP-b2 and collected for ChIP assays using an antibodies against Stat3 and primers for the -472/-344 region of the Jab1 promoter. Right
panel represents quantification of the band intensities and normalized to the input with ImageJ software. (d) MDA-MB-468 cells were
cotransfected with either pcDNA control, a Flag-tagged Stat3 (Flag-Stat3), or Flag-tagged dominant negative (DN)-Stat3EEE/VV, along with the -472
Jab1-Luc construct, and subjected to luciferase reporter assays. (e) Overexpression of Flag-tagged Stat3 increased Jab1 protein levels while a
reduction was seen with Flag-DN- Stat3EEE/VV. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun
activation domain-binding protein-1. We further investigated whether an upstream activator
of Stat3, the cytokine IL-6, could be driving increased
Jab1 expression. Stat3-induced Jab1 transcriptional activation and protein
expression To determine the biological significance of Stat3-
mediated Jab1 expression, the role of Stat3 in normal
human breast epithelial cells was investigated. As Jab1
expression in normal mammary epithelial cells is low,
we asked whether overexpression of Stat3 could enhance
Jab1 transcription in these cells. Ectopic expression of
Stat3 in normal mammary epithelial cells (MCF-10A
and MCF-10F) resulted in increased Jab1 mRNA and
protein levels (Figures 7a and 7b). Therefore, the Stat3
transcription factor is in part responsible for promoting
Jab1 transcription in breast cancer cells and is either
not present or not as active in the MCF-10A and MCF-
10F cells. Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 11 of 16 Figure 5 Stat3 can synergize with C/EBP-a and C/EBP-b to transactivate the Jab1 promoter. (a) The -472/+83 Jab1-Luc reporter plasmid
was cotransfected with expression plasmids for C/EBP-a, or C/EBP-b. Plasmid amounts were normalized by the addition of empty expression
plasmid. Following transfection, MCF7 cells were incubated for another 36 hours. The results are presented as fold activation compared with cells
transfected with pcDNA vector control. (b) A potential STAT-binding element (TTN5AA consensus sequence) overlaps the C/EBP binding site
(CAAC). (c) Binding of Stat3 to the Jab1 promoter is enhanced by the addition of C/EBP. MDA-MB-231 cells were transfected with either control
of C/EBP-b2 and collected for ChIP assays using an antibodies against Stat3 and primers for the -472/-344 region of the Jab1 promoter. Right
panel represents quantification of the band intensities and normalized to the input with ImageJ software. (d) MDA-MB-468 cells were
cotransfected with either pcDNA control, a Flag-tagged Stat3 (Flag-Stat3), or Flag-tagged dominant negative (DN)-Stat3EEE/VV, along with the -472
Jab1-Luc construct, and subjected to luciferase reporter assays. (e) Overexpression of Flag-tagged Stat3 increased Jab1 protein levels while a
reduction was seen with Flag-DN- Stat3EEE/VV. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun
activation domain-binding protein-1. http://breast-cancer-research.com/content/13/3/R65 can synergize with C/EBP-a and C/EBP-b to transactivate the Jab1 promoter. (a) The -472/+83 Jab1-Luc reporter pla Figure 5 Stat3 can synergize with C/EBP-a and C/EBP-b to transactivate the Jab1 promoter. (a) The -472/+83 Jab1-Luc reporter plasmid
was cotransfected with expression plasmids for C/EBP-a, or C/EBP-b. Plasmid amounts were normalized by the addition of empty expression
plasmid. Following transfection, MCF7 cells were incubated for another 36 hours. The results are presented as fold activation compared with cells
transfected with pcDNA vector control. Stat3-induced Jab1 transcriptional activation and protein
expression Treatment with IL-6 for 30 minutes
increased phosphorylation of Stat3 on tyrosine 705 and
resulted in increased Jab1 mRNA (Figure 7c) and pro-
tein levels (Figure 7d) within the short time of 30 min-
utes, 1 hour, and 4 hours that was partially blocked by
the addition of the Stat3 inhibitor Stattic. The same
trends were observed in the breast cancer cell line T47D and the mammary epithelial cells, MCF-10A. IL-6 also
resulted in increased Jab1 promoter activity in MCF7
and T47D cells [See Figure S2 in Additional file 1]. Taken together, it is evident that both IL-6 and Src
signaling through Stat3 is contributing to Jab1 transcrip-
tion and increased expression in breast cancer. Further,
it is possible that Stat3 and C/EBPb could be binding to
the Jab1 promoter either separately or together to med-
iate increased Jab1 transcriptional activity. A proposed and the mammary epithelial cells, MCF-10A. IL-6 also
resulted in increased Jab1 promoter activity in MCF7
and T47D cells [See Figure S2 in Additional file 1]. Taken together, it is evident that both IL-6 and Src
signaling through Stat3 is contributing to Jab1 transcrip-
tion and increased expression in breast cancer. Further,
it is possible that Stat3 and C/EBPb could be binding to
the Jab1 promoter either separately or together to med-
iate increased Jab1 transcriptional activity. A proposed Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 12 of 16 Figure 6 Inhibition of Stat3 and Src by siRNA reduces Jab1 promoter activity in breast carcinoma cells. (a) MDA-MB-468 cells were
cotransfected with siRNA targeting Stat3 (siStat3) or Src (siSrc), along with either the -472 Jab1-Luc construct or -472-Mut1C/EBP Jab1-Luc (as a
control) and subjected to luciferase reporter assays, western blot analyses to demonstrate the efficient knockdown of Stat3 and Src protein levels
are shown in the right panel. (b) MDA-MD-468 cells were transfected with increasing doses of siStat3 and siSrc and Jab1 was detected by RT-
PCR, quantification of Jab1 RNA levels is shown in (c). (d) Western blot analysis demonstrates that Jab1 protein levels decreased following
inhibition of Stat3 and Src by siRNA. Jab1, c-Jun activation domain-binding protein-1; Mut, mutation; siRNA, small interfering RNA; RT-PCR, reverse
transcription polymerase chain reaction; STAT, signal transducer and activator of transcription. Figure 6 Inhibition of Stat3 and Src by siRNA reduces Jab1 promoter activity in breast carcinoma cells. Stat3-induced Jab1 transcriptional activation and protein
expression (a) MDA-MB-468 cells were
cotransfected with siRNA targeting Stat3 (siStat3) or Src (siSrc), along with either the -472 Jab1-Luc construct or -472-Mut1C/EBP Jab1-Luc (as a
control) and subjected to luciferase reporter assays, western blot analyses to demonstrate the efficient knockdown of Stat3 and Src protein levels
are shown in the right panel. (b) MDA-MD-468 cells were transfected with increasing doses of siStat3 and siSrc and Jab1 was detected by RT-
PCR, quantification of Jab1 RNA levels is shown in (c). (d) Western blot analysis demonstrates that Jab1 protein levels decreased following
inhibition of Stat3 and Src by siRNA. Jab1, c-Jun activation domain-binding protein-1; Mut, mutation; siRNA, small interfering RNA; RT-PCR, reverse
transcription polymerase chain reaction; STAT, signal transducer and activator of transcription. model for the activation of Jab1 transcription is shown
in Figure 7e. which Jab1 is regulated is currently not known and our
data suggest that this may occur at the transcriptional
level. In this study, the Jab1 promoter was analyzed to
identify the molecular basis of Jab1 gene expression and
to give insight into the mechanisms by which Jab1 is
overexpressed in cancer. Jab1 promoter analysis led to Discussion Jab1, c-Jun activation domain-binding
protein-1; RT-PCR, reverse transcription polymerase chain reaction; STAT, signal transducer and activator of transcription. Figure 7 Stat3 pathway contributes to Jab1 transcriptional activation and increased protein levels. (a) Increased expression of Stat3 in Figure 7 Stat3 pathway contributes to Jab1 transcriptional activation and increased protein levels. (a) Increased expression of Stat3 in
the normal mammary epithelial MCF-10A cells increased Jab1 RNA levels as detected by RTPCR, quantification of Jab1 RNA levels is shown in
the bottom panel. (b) Ectopic expression of Flag-Stat3 (0, 1, 2, and 3 μg) in normal mammary epithelial cells (MCF-10A and MCF-10F) resulted in
increased Jab1 protein levels. (c) Treatment with the cytokine IL-6 increased Jab1 mRNA levels - seen by RT-PCR - in both normal MCF-10A and
breast cancer T47D cells that were blocked by the Stat3 inhibitor Stattic. (d) Treatment with IL-6 increased Jab1 protein levels following
activation of Stat3 (P-Stat3 Y705) that was inhibited by the addition of Stattic. (e) Schematic representation of the major molecular mechanism of
Stat3-induced transcriptional activation of Jab1 through two signaling axes - Src/Stat3 and IL-6/Stat3. Jab1, c-Jun activation domain-binding
protein-1; RT-PCR, reverse transcription polymerase chain reaction; STAT, signal transducer and activator of transcription. the identification of C/EBP-b, GATA-1, and Stat3 as
positive regulators of Jab1 transcription in breast cancer
cells. Promoter deletion studies identified a region
between -472 and -345 that has significant transcrip-
tional activity, as evidenced by the dramatic reduction in
luciferase reporter activity when this region has been
deleted (Figure 2a). Mutation of both the C/EBP and
GATA-1 sites together resulted in decreased luciferase
reporter activity of approximately 75% when combined. We identified C/EBP, GATA-1, and Stat3 consensus
sequences located in this region and their binding was
confirmed by EMSA and ChIP assays. We established
that C/EBP, GATA-1, and Stat3 are positive regulators
of Jab1 promoter activity. As these transcription factors
are activated during tumorigenesis, and because Jab1 is
overexpressed in a number of tumors, we demonstrate the identification of C/EBP-b, GATA-1, and Stat3 as
positive regulators of Jab1 transcription in breast cancer
cells. Promoter deletion studies identified a region
between -472 and -345 that has significant transcrip-
tional activity, as evidenced by the dramatic reduction in
luciferase reporter activity when this region has been
deleted (Figure 2a). Mutation of both the C/EBP and
GATA-1 sites together resulted in decreased luciferase
reporter activity of approximately 75% when combined. Discussion Jab1 is commonly overexpressed in patients with breast
cancer as well as other tumor types. The mechanism by Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 13 of 16 Figure 7 Stat3 pathway contributes to Jab1 transcriptional activation and increased protein levels. (a) Increased expression of Stat3 in
the normal mammary epithelial MCF-10A cells increased Jab1 RNA levels as detected by RTPCR, quantification of Jab1 RNA levels is shown in
the bottom panel. (b) Ectopic expression of Flag-Stat3 (0, 1, 2, and 3 μg) in normal mammary epithelial cells (MCF-10A and MCF-10F) resulted in
increased Jab1 protein levels. (c) Treatment with the cytokine IL-6 increased Jab1 mRNA levels - seen by RT-PCR - in both normal MCF-10A and
breast cancer T47D cells that were blocked by the Stat3 inhibitor Stattic. (d) Treatment with IL-6 increased Jab1 protein levels following
activation of Stat3 (P-Stat3 Y705) that was inhibited by the addition of Stattic. (e) Schematic representation of the major molecular mechanism of
Stat3-induced transcriptional activation of Jab1 through two signaling axes - Src/Stat3 and IL-6/Stat3. Jab1, c-Jun activation domain-binding
protein-1; RT-PCR, reverse transcription polymerase chain reaction; STAT, signal transducer and activator of transcription. Figure 7 Stat3 pathway contributes to Jab1 transcriptional activation and increased protein levels. (a) Increased expression of Stat3 in
h
l
h l l MCF
A
ll
d J b
RNA l
l
d
d b RTPCR
f
f J b
RNA l
l
h Figure 7 Stat3 pathway contributes to Jab1 transcriptional activation and increased protein levels. (a) Increased expression of Stat3 in
the normal mammary epithelial MCF-10A cells increased Jab1 RNA levels as detected by RTPCR, quantification of Jab1 RNA levels is shown in
the bottom panel. (b) Ectopic expression of Flag-Stat3 (0, 1, 2, and 3 μg) in normal mammary epithelial cells (MCF-10A and MCF-10F) resulted in
increased Jab1 protein levels. (c) Treatment with the cytokine IL-6 increased Jab1 mRNA levels - seen by RT-PCR - in both normal MCF-10A and
breast cancer T47D cells that were blocked by the Stat3 inhibitor Stattic. (d) Treatment with IL-6 increased Jab1 protein levels following
activation of Stat3 (P-Stat3 Y705) that was inhibited by the addition of Stattic. (e) Schematic representation of the major molecular mechanism of
Stat3-induced transcriptional activation of Jab1 through two signaling axes - Src/Stat3 and IL-6/Stat3. Discussion We identified C/EBP, GATA-1, and Stat3 consensus
sequences located in this region and their binding was
confirmed by EMSA and ChIP assays. We established
that C/EBP, GATA-1, and Stat3 are positive regulators
of Jab1 promoter activity. As these transcription factors
are activated during tumorigenesis, and because Jab1 is
overexpressed in a number of tumors, we demonstrate that these transcription factors indeed increase tran-
scription of Jab1. In our study, we identified C/EBP as a potential tran-
scriptional activator for Jab1, specifically C/EBPa and
C/EBPb-2. C/EBPb-1 is expressed in normal breast cells
while expression of C/EBPb-2 is known to be expressed
specifically in invasive primary breast tumor samples or
cells lines [39]. Of the three isoforms of C/EBP-b, the
transactivating form of LAP2 resulted in a two-fold
increase in Jab1 transcriptional activity while the inhibi-
tor isoform LIP decreased activity. C/EBP-b appears to
play a critical role in the development of both the mam-
mary gland and cancers therein through its involvement
in development, differentiation, and proliferation of
mammary epithelial cells [25,27,31]. As Jab1 is fre-
quently upregulated in breast cancer, it is possible that Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 Page 14 of 16 However, trastuzumab did inhibit Jab1 protein levels in
BT-474 breast cancer cells as well as phosphorylation of
AKT and Stat3 (data not shown). The regulation of Jab1
expression by HER2 through the AKT pathway is of
great interest, and further studies could strengthen our
understanding on the role of Jab1 in the tumorigenic
process. However, trastuzumab did inhibit Jab1 protein levels in
BT-474 breast cancer cells as well as phosphorylation of
AKT and Stat3 (data not shown). The regulation of Jab1
expression by HER2 through the AKT pathway is of
great interest, and further studies could strengthen our
understanding on the role of Jab1 in the tumorigenic
process. LAP2 is a major factor in driving Jab1 transcription dur-
ing the tumorigenic process. Of note, our study detected
higher levels of all three C/EBP-b isoforms in a panel of
breast cancer cell lines compared with normal mam-
mary epithelial cells (Figure 4d), which is contrary to
previous studies that identified mainly higher expression
of LAP2 in breast cancer. Yet, LAP2 was the isoform
that resulted in the greatest increase in transcriptional
activity of Jab1. Discussion This increased expression could cer-
tainly be driving increased Jab1 activity in breast cancer
cells. As overexpression of Jab1 is frequently observed in
breast cancer, further investigation of the pathways that
modulate Jab1 transcription would provide insight into
the role Jab1 plays in the tumorigenic process therein. Activation of the Stat3 pathway in breast cancer can
occur through many pathways, including those of EGFR,
HER2, IL-6 receptors, IL-11 receptors, and progesterone
receptors [42]. Experimental activation of these path-
ways, followed by evaluation of Jab1 promoter activity
and mRNA levels, could provide insight into the
mechanisms by which Jab1 transcription is activated. Our data provide evidence of activation of Jab1 tran-
scription through IL-6 and Src mediated activation of
Stat3 as shown in Figure 7e. It is possible that other
activators upstream of Stat3 could be mediating this
downstream effect as well and warrants further
investigation. Further, we identified a co-existing Stat3 binding site
within the C/EBP binding site on the Jab1 promoter. Ectopic overexpression of Stat3 increased transcriptional
activity as well as mRNA and protein levels of Jab1. This was further increased with overexpression of the
activated form of Stat3. Constitutive activation of Stat3
occurs commonly in cancer, including breast cancer and
has been demonstrated to contribute to tumorigenic
processes [40]. Stat3 can mediate signaling through
upstream receptor tyrosine kinases such as epidermal
growth factor receptor (EGFR) and platelet-derived
growth factor receptor (PDGR) as well as upstream
non-receptor tyrosine kinases such as Abl and Src-
related kinases. These receptors are constitutively acti-
vated in cancer, typically through genetic alterations
[40]. The oncogenic Src protein kinase itself is overex-
pressed in a large number of tumor types and interacts
with multiple tyrosine kinase receptors, including EGFR
and HER2 [41] to mediate its oncogenic effects of pro-
moting growth and metastasis. We found that Src, an
activator of Stat3, is involved in Jab1 transcription. Overexpression of both Stat3 and Src in normal mam-
mary epithelial cells resulted in increased Jab1 mRNA
and protein levels. These data provide the first evidence
that Jab1 is a direct downstream target of Stat3 and Src. Additionally, inhibition of Src by siRNA reduced Jab1
promoter activity in a manner similar to inhibition of
Stat3. We further identified one upstream activator of
Stat3, IL-6, that mediated activation of Jab1 expression. Additional material Additional file 1: Figure S1. C/EBP and GATA binding sites homology
from the human and mice promoter regions.Figure S2. IL-6 mediated
activation of Jab1 promoter activity. Additional file 1: Figure S1. C/EBP and GATA binding sites homology
from the human and mice promoter regions.Figure S2. IL-6 mediated
activation of Jab1 promoter activity. Conclusions In summary, the present study demonstrates that the
Src/Stat3 and C/EBP signaling pathways positively regu-
late the expression of the Jab1 oncogene. Our results
show that Stat3 and LAP2 (C/EBP-b2) are the two
major transcription factors that contribute to Jab1 over-
expression that leads to increased proliferation of breast
cancer cells. Our findings reveal a novel mechanism of
Jab1 regulation and provide functional and mechanistic
links between two major signaling axes - Src/Stat3 and
IL-6/Stat3 - that are involved in controlling Jab1 onco-
genic protein activation. Understanding the mechanisms
by which Jab1 expression is deregulated may help in the
development of drugs that target additional key ele-
ments responsible for this important deregulation. Since the present study began, Jab1 expression has
been linked to the HER2 signaling pathway. HER2 has
been found to stimulate Jab1 transcriptional activity in
NIH3T3 cells stably expressing the HER2 receptor [21]. This stimulation took place through the AKT/b-catenin/
TCF-4 signaling pathway in breast cancer cells overex-
pressing the HER2 receptor. The TCF binding site is in
the same area as our region of interest, between -472
and -344. In our laboratory, we also found overexpres-
sion of Jab1 in NIH3T3 and MCF7 cells that stably
express the HER2 receptor (data not shown). However,
inhibition of this pathway by the anti-HER2 antibody
trastuzumab (Herceptin) or AKT inhibitors in MCF7
and SKBR3 cells did not reduce Jab1 promoter activity. Abbreviations
C/EBP: CCAAT/enhancing binding protein; ChIP: chromatin
immunoprecipitation; CRL: cullin-RING-ubiquitin ligase; CSN: COP9
signalosome; CUL-1: cullin 1; DMEM: Dulbecco’s modified Eagle’s medium;
EGFR: epidermal growth factor receptor; EMSA: electrophoretic mobility shift
assay; FBS: fetal bovine serum; Jab1: c-Jun activation domain-binding
protein-1; JAMM: Jab1 MPN domain metalloenzyme; HER: human epidermal References 1. Claret FX, Hibi M, Dhut S, Toda T, Karin M: A new group of conserved
coactivators that increase the specificity of AP-1 transcription factors. Nature 1996, 383:453-457. 26. Ramji DP, Foka P: CCAAT/enhancer-binding proteins: struct
and regulation. Biochem J 2002, 365:561-575. and regulation. Biochem J 2002, 365:561-575. 27. Grimm SL, Rosen JM: The role of C/EBPbeta in mammary gland
development and breast cancer. J Mammary Gland Biol Neoplasia 2003,
8:191-204. 2. Schwechheimer C, Deng XW: COP9 signalosome revisited: a novel
mediator of protein degradation. Trends Cell Biol 2001, 11:420-426. 2. Schwechheimer C, Deng XW: COP9 signalosome revisited: a novel
mediator of protein degradation. Trends Cell Biol 2001, 11:420-426. 3. Chamovitz DA, Segal D: JAB1/CSN5 and the COP9 signalosome. A
complex situation. EMBO Rep 2001, 2:96-101. 3. Chamovitz DA, Segal D: JAB1/CSN5 and the COP9 signalosome. A
complex situation. EMBO Rep 2001, 2:96-101. 28. Bundy LM, Sealy L: CCAAT/enhancer binding protein beta (C/EBPbeta)-2
transforms normal mammary epithelial cells and induces epithelial to
mesenchymal transition in culture. Oncogene 2003, 22:869-883. 4. Shackleford TJ, Claret FX: JAB1/CSN5: a new player in cell cycle control
and cancer. Cell Div 2010, 5:26. 29. Lowry JA, Mackay JP: GATA-1: one protein, many partners. Int J Biochem
Cell Biol 2006, 38:6-11. 5. Cope G, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ:
Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of
Nedd8 from Cul1. Science 2002, 298:608-611. 5. Cope G, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ
Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of
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protease. Trends Biochem Sci 2008, 33:592-600. 7. Tomoda K, Kubota Y, Kato J: Degradation of the cyclin-dependent-kinase 30. Crispino JD: GATA1 in normal and malignant hematopoiesis. Semin Cell
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cancer and associations with receptor tyrosine kinases. Expert Rev Mol
Med 2009, 11:e12. p
7. Tomoda K, Kubota Y, Kato J: Degradation of the cyclin-dependent-kinase
inhibitor p27Kip1 is instigated by Jab1. Nature 1999, 398:160-165. 32. Author details
1 1Department of Systems Biology, Unit 950, University of Texas - MD
Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030,
USA. 2Department of Experimental Therapeutics, University of Texas - MD
Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030,
USA. 18. Richardson KS, Zundel W: The emerging role of the COP9 signalosome in
cancer. Mol Cancer Res 2005, 3:645-653. 19. Adler AS, Lin M, Horlings H, Nuyten DS, van de Vijver MJ, Chang HY:
Genetic regulators of large-scale transcriptional signatures in cancer. Nat
Genet 2006, 38:421-430. Authors’ contributions 20. Adler AS, Littlepage LE, Lin M, Kawahara TL, Wong DJ, Werb Z, Chang HY:
CSN5 isopeptidase activity links COP9 signalosome activation to breast
cancer progression. Cancer Res 2008, 68:506-515. TJS carried out all of the studies and drafted the manuscript. QZ assisted in
many aspects of the study including the execution of deletion analysis and
siRNA analysis. LT contributed to the cloning of Jab1 promoter and its
analysis. VTT and AMB participated in cell culture, siRNA, and western
blotting experiments. ALK provided plasmid constructs, assisted in the
primer extension analysis and participated in study design. XFL and WSL,
participated in the study design, and provided important intellectual
support. FXC conceived the study, participated in its design, coordination
and interpretation of the results and finalized the manuscript. All authors
read and approved the final manuscript. 21. Hsu MC, Chang HC, Hung WC: HER-2/neu transcriptionally activates Jab1
expression via the AKT/beta-catenin pathway in breast cancer cells. Endocr Relat Cancer 2007, 14:655-667. 22. Mansouri A, Ridgway LD, Korapati AL, Zhang Q, Tian L, Wang Y, Siddik ZH,
Mills GB, Claret FX: Sustained activation of JNK/p38 MAPK pathways in
response to cisplatin leads to Fas ligand induction and cell death in
ovarian carcinoma cells. J Biol Chem 2003, 278:19245-19256. 23. Nikolov DB, Burley SK: RNA polymerase II transcription initiation: a
structural view. Proc Natl Acad Sci USA 1997, 94:15-22. Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 12. Wei N, Deng XW: The COP9 signalosome. Annu Rev Cell Dev Biol 2003,
19:261-286. growth factor receptor; HIF-1α: hypoxia inducible factor-1 alpha; LAP: liver-
enriched activating protein; LIP: liver-enriched inhibitory protein; MIF:
macrophage migration inhibitory factor; PCR: polymerase chain reaction;
PDGR: platelet-derived growth factor receptor; RT-PCR: reverse-transcription
polymerase chain reaction; siRNA: small interfering RNA; STAT: signal
transducer and activator of transcription; TCF: T-cell transcription factor. growth factor receptor; HIF-1α: hypoxia inducible factor-1 alpha; LAP: liver-
enriched activating protein; LIP: liver-enriched inhibitory protein; MIF:
macrophage migration inhibitory factor; PCR: polymerase chain reaction;
PDGR: platelet-derived growth factor receptor; RT-PCR: reverse-transcription
polymerase chain reaction; siRNA: small interfering RNA; STAT: signal
transducer and activator of transcription; TCF: T-cell transcription factor. 13. Sui L, Dong Y, Ohno M, Watanabe Y, Sugimoto K, Tai Y, Tokuda M: Jab1
expression is associated with inverse expression of p27(kip1) and poor
prognosis in epithelial overian tumors. Clin Cancer Res 2001, 7:4130-4135. 14. Kouvaraki MA, Tian L, Mansouri A, Zhang Q, Kumar R, Kittas C, Claret FX:
Jun activation domain-binding protein 1 expression in breast cancer
inversely correlates with the cell cycle inhibitor p27(Kip1). Cancer Res
2003, 63:2977-2981. Acknowledgements TJS is a recipient of fellowships from Susan G. Komen Foundation and the
Department of Defense Breast Cancer Research Program; TTV is a recipient
of fellowships from Vietnam Education Foundation. This study was
supported by grants from the National Cancer Institute (RO1-CA90853) and
the Susan G. Komen Breast Cancer Foundation to FXC. The authors thank Dr
Prahlad Ram for the Stat3 DNA constructs and Dr. Linda Sealy for the C/
EBPb1-3 (LAP1, LAP2, and LIP) plasmids. They appreciate the technical
assistance of Sarah May and editing assistance of Pierrette Lo. 15. Rassidakis GZ, Claret FX, Lai R, Zhang Q, Sarris AH, McDonnell TJ,
Medeiros LJ: Expression of p27(Kip1) and c-Jun activation binding
protein 1 are inversely correlated in systemic anaplastic large cell
lymphoma. Clin Cancer Res 2003, 9:1121-1128. 16. Shintani SLC, Mihara M, Hino S, Nakashiro K, Hamakawa H: Skp2 and Jab1
expression are associated with inverse expression of p27(KIP1) and poor
prognosis in oral squamous cell carcinomas. Oncology 2003, 65:355-362. 17. Dong Y, Sui L, Watanabe Y, Yamaguchi F, Hatano N, Tokuda M: Prognostic
significance of Jab1 expression in laryngeal squamous cell carcinomas. Clin Cancer Res 2005, 11:259-266. Competing interests
Th
h
d
l
h 24. Numata A, Shimoda K, Kamezaki K, Haro T, Kakumitsu H, Shide K, Kato K,
Miyamoto T, Yamashita Y, Oshima Y, Nakajima H, Iwama A, Aoki K, Takase K,
Gondo H, Mano H, Harada M: Signal transducers and activators of
transcription 3 augments the transcriptional activity of CCAAT/enhancer-
binding protein alpha in granulocyte colony-stimulating factor signaling
pathway. J Biol Chem 2005, 280:12621-12629. The authors declare that they have no competing interests. Received: 8 October 2010 Revised: 21 March 2011
Accepted: 20 June 2011 Published: 20 June 2011 Received: 8 October 2010 Revised: 21 March 2011 Accepted: 20 June 2011 Published: 20 June 2011 Accepted: 20 June 2011 Published: 20 June 2011 25. Zahnow CA: CCAAT/enhancer binding proteins in normal mammary
development and breast cancer. Breast Cancer Res 2002, 4:113-121. Abbreviations C/EBP: CCAAT/enhancing binding protein; ChIP: chromatin
immunoprecipitation; CRL: cullin-RING-ubiquitin ligase; CSN: COP9
signalosome; CUL-1: cullin 1; DMEM: Dulbecco’s modified Eagle’s medium;
EGFR: epidermal growth factor receptor; EMSA: electrophoretic mobility shift
assay; FBS: fetal bovine serum; Jab1: c-Jun activation domain-binding
protein-1; JAMM: Jab1 MPN domain metalloenzyme; HER: human epidermal Page 15 of 16 Page 15 of 16 Page 15 of 16 Shackleford et al. Breast Cancer Research 2011, 13:R65
http://breast-cancer-research.com/content/13/3/R65 References Singh SP, Lipman J, Goldman H, Ellis FH, Aizenman L, Cangi MG,
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the cytoplasmic localization and degradation of p53 in coordination
with Hdm2. J Biol Chem 2006, 281:17457-17465. 33. Clevenger CV: Roles and regulation of stat family transcription factors in
human breast cancer. Am J Pathol 2004, 165:1449-1460. 34. Horvath CM, Wen Z, Darnell JE Jr: A STAT protein domain that determines
DNA sequence recognition suggests a novel DNA-binding domain. Genes Dev 1995, 9:984-994. 10. Wan M, Cao X, Wu Y, Bai S, Wu L, Shi X, Wang N: Jab1 antagonizes TGF-
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recognition conferred by the STAT amino-terminal domain. Science 1996,
273:794-797. 11. Bae MK, Ahn MY, Jeong JW, Bae MH, Lee YM, Bae SK, Park JW, Kim KR,
Kim KW: Jab1 interacts directly with HIF-1alpha and regulates its
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http://breast-cancer-research.com/content/13/3/R65 36. Kaptein A, Paillard V, Saunders M: Dominant negative stat3 mutant
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Cite this article as: Shackleford et al.: Stat3 and CCAAT/enhancer
binding protein beta (C/EBP-beta) regulate Jab1/CSN5 expression in
mammary carcinoma cells. Breast Cancer Research 2011 13:R65. Submit your next manuscript to BioMed Central
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https://www.cambridge.org/core/services/aop-cambridge-core/content/view/2D5267359172C286C2659CDE7AC80A88/S0955603600103071a.pdf/div-class-title-span-class-italic-an-introduction-to-therapeutic-communities-span-by-david-kennard-london-jessica-kingsley-1998-166-pp-14-95-isbn-1853026034-div.pdf
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English
| null |
An Introduction to Therapeutic Communities. By David Kennard. London: Jessica Kingsley. 1998. 166 pp. £14.95. ISBN 1853026034
|
Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin
| 1,999
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cc-by
| 534
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Book reviews An Introduction
to Therapeutic
Communities. By David
Kennard. London:
Jessica
Kingsley. 1998. 166 pp. £14.95. ISBN 1853026034 play was written) would today have been labelled
as suffering from borderline
personality
disorder
rather
than
schizophrenia. The book was first
published
in 1983 and although
extensively
re
written,
one or two passages
seem dated. The
imaginary
first day for three new staff members
echoed
my experience
as a junior
psychiatrist
but
showed
little
awareness
of the
interim
changes:
more clarity of structure
and attempts
to create
a reassuring
secure
base
for staff as
well as residents. An Introduction
to Therapeutic
Communities
is
written
in
a
jargon-free,
questioning,
non-
dogmatic,
conversational
style. This accessibility
makes
it an
appropriate
choice
to start
this
exciting
new series. David Kennard's
gift is to
make
therapeutic
community
ideas
seem
ob
vious,
and
to show
how they
grow out
of a
common sense approach
to caring for each other,
based on ideas of collective responsibility,
agape
(fellowship, brotherly
love) and citizenship
which
are part of our heritage
and shared
humanity. Chapters
on moral treatment,
planned
envir
onmental
therapy,
the
therapeutic
community
approach
and
anti-psychiatry
raise
important,
perhaps
unfashionable
questions,
about
how
society
approaches
mental
ill health
and what
psychiatry's
role
should
be. The
relationship
between
wider changes
in society
and
how we
care for people
with mental
illnesses
and
dis
turbances
is a fascinating
one. Why, for example,
should
therapeutic
communities
have
been
popular
in Holland in the 1980s and be on the
demise
now, whereas
in Britain
the reverse
is
happening? How have
Christianity,
Marxism,
two
World
Wars
and
the
swinging
sixties
influenced
the practice
of psychiatry? In a time
of rapid and government-led
change
it is surely
important
that
psychiatrists
think
about
these
questions
if we are to affect the organisation
of
care
rather
than
passively
reflect
prevailing
ideologies. This
book
is a good starting
point
and I would encourage
all consultants
to read it
and recommend
it to their trainees. p
g
y
Kennard
brings
a
broad
and
international
perspective
to
the
subject. The
chapter
on
behavioural
or concept therapeutic
communities
for drug
addicts,
which
are widespread
in the
USA,
describes
them
sympathetically,
and
although
some of the religious
and hierarchical
ideas
will not appeal
to those
involved
in the
British
therapeutic
community
movement,
the
attempt
to
bridge
this
divide
is
welcome. Penelope
Campling
Constatant
Psychotherapist.
Francis Dixon Lodge, Gipsy Lane, Leicester LE5
OTD Psychiatric Bulletin (1999), 23, 511 Book reviews The
influence
of the
therapeutic
community
approach
on psychiatry
generally
is described
and the book makes
it clear that
the future
of
therapeutic
community
ideas has relevance
for
the community
and the voluntary
sector as much
as for specific NHS units. p
The
chapter
on personality
disorder
is the
weakest
in the
book
with
no mention
of the
changes
in this service user group or advances
in
our
understandings. The
definitions
are
also
misleading
and
do not tie up with ICD-10
or
DSM-IV. Similarly,
the
chapter
on
anti-
psychiatry
would have been more understand
able to today's readers
if it had pointed
out that
Mary Barnes
(about whom a book and
then
a 511 Psychiatric Bulletin (1999), 23, 511 https://doi.org/10.1192/pb.23.8.511 Published online by Cambridge University Press
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https://openalex.org/W4292058578
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https://scipost.org/10.21468/SciPostPhys.13.2.022/pdf
|
English
| null |
Fingerprints of freeze-in dark matter in an early matter-dominated era
|
SciPost physics
| 2,022
|
cc-by
| 9,401
|
Abstract We study the impact of an alternate cosmological history with an early matter-dominated
epoch on the freeze-in production of dark matter. Such early matter domination is trig-
gered by a meta-stable matter field dissipating into radiation. In general, the dissipation
rate has a non-trivial temperature and scale factor dependence. Compared to the usual
case of dark matter production via the freeze-in mechanism in a radiation-dominated
universe, in this scenario, orders of magnitude larger coupling between the visible and
the dark sector can be accommodated. Finally, as a proof of principle, we consider a spe-
cific model where the dark matter is produced by a sub-GeV dark photon having a kinetic
mixing with the Standard Model photon. We point out that the parameter space of this
model can be probed by the experiments in the presence of an early matter-dominated
era. Copyright A. Banerjee and D. Chowdhury. This work is licensed under the Creative Commons
Attribution 4.0 International License. Published by the SciPost Foundation. Received 06-05-2022
Accepted 21-07-2022
Published 16-08-2022
Check for
updates
doi:10.21468/SciPostPhys.13.2.022 Copyright A. Banerjee and D. Chowdhury. This work is licensed under the Creative Commons
Attribution 4.0 International License. Published by the SciPost Foundation. Received 06-05-2022
Accepted 21-07-2022
Published 16-08-2022
Check for
updates
doi:10.21468/SciPostPhys.13.2.022
Contents
1
Introduction
2
2
Early matter domination with generalized dissipation
3
3
Dark matter freeze-in during matter domination
6
4
Case study: dark photon portal to dark matter
8
5
Conclusions
11
References
11
1 Received 06-05-2022
Accepted 21-07-2022
Published 16-08-2022
Check for
updates
doi:10.21468/SciPostPhys.13.2.022 Copyright A. Banerjee and D. Chowdhury. This work is licensed under the Creative Commons
Attribution 4.0 International License. Published by the SciPost Foundation. Fingerprints of freeze-in dark matter
in an early matter-dominated era 1 Department of Physics, Chalmers University of Technology,
Fysikgården, 41296 Göteborg, Sweden
2 Department of Physics, Indian Institute of Technology Kanpur,
Kanpur 208016, India ⋆avik@chalmers.se † debtoshc@iitk.ac.in SciPost Phys. 13, 022 (2022) 1
Introduction Despite the overwhelming evidences in favor of the ‘dark matter’ (DM) having a major share in
the total energy budget of the Universe [1–4], barely anything is known about its nature and
the origin. Most of the speculations regarding the particle identity of the DM revolve around
the Weakly interacting massive particle (WIMP) framework, where the DM freezes-out from
the thermal bath after a period of equilibrium with the visible matter. Despite strong theoretical
motivations, dearth of any pointers from experiments [5–8] drive us to look for alternatives of
WIMP paradigm. One of the notable alternative to the WIMP paradigm is the Feebly interacting
massive particle (FIMP) framework [9, 10]. In this scenario, DM is produced out of thermal
equilibrium via the so-called ‘freeze-in’ production mechanism. The interaction of the DM with
the Standard Model (SM) particles in the freeze-in scenario needs to be extremely small such
that they never attain a thermal equilibrium [11–29]. Such a tiny interaction strength makes
freeze-in dark matter models challenging to detect at the experiments compared to the usual
WIMP scenarios. For recent attempts to detect FIMP, see for example [30–33]. The present day relic abundance of DM produced by freeze-in mechanism significantly
depends on the cosmological history as well as the initial DM abundance at the end of inflation. Any modification in the standard picture of the radiation domination after the inflation till
the big bang nucleosynthesis (BBN) alters the production of the dark matter [34–55]. In
this paper, we investigate the impact of a pre-BBN matter-dominated epoch on freeze-in DM
production. A number of motivated scenarios beyond the Standard Model (BSM) of particle
physics predict some meta-stable matter fields, for instance, string theory inspired moduli
fields [56–58], supersymmetric condensates and gravitino [59,60], scenarios with primordial
black hole formation [61–64], inflaton fields [65,66], curvaton [67], dilaton [68], Q-balls [69]
etc. This kind of late-decaying species can give rise to an early matter-dominated (EMD) epoch
after the inflation. Subsequently the decay of these long-lived particles into the radiation
leads to a second phase of reheating at the end of which the universe once again enters into a
radiation-dominated phase. The temperature evolution of the universe depends on the detailed dynamics of how the
matter field leading to the EMD dissipates into the radiation. Contents 1
Introduction
2
2
Early matter domination with generalized dissipation
3
3
Dark matter freeze-in during matter domination
6
4
Case study: dark photon portal to dark matter
8
5
Conclusions
11
References
11
1 1
Introduction
2
2
Early matter domination with generalized dissipation
3
3
Dark matter freeze-in during matter domination
6
4
Case study: dark photon portal to dark matter
8
5
Conclusions
11
References
11 1 SciPost Phys. 13, 022 (2022) 1
Introduction In general, the dissipation rate
depends on the instantaneous temperature of the thermal bath and the expansion rate of the
universe [70]. In such a scenario, end of the EMD era is preceded by an epoch of entropy
production which has a direct impact of diluting the present day relic abundance of the DM. Existing works illustrating such dilution of DM relic density generally consider a constant dis-
sipation rate of the meta-stable field into the radiation [34–44]. Here instead, we consider a
more general parametrization of the dissipation rate which depends on the temperature and
the scale factor [70]. We show that the freeze-in production of DM is significantly affected by the presence of
an EMD epoch with such generalized dissipation rate. In this scenario, orders of magnitude
larger couplings between the DM and the visible matter are required for the freeze-in mech-
anism, compared to the usual case of freeze-in in a radiation-dominated universe. Moreover,
we present some specific benchmark cases where the dissipation rates have non-trivial temper-
ature and scale factor dependence and show that even larger couplings can be accommodated
compared to an EMD epoch with constant dissipation rate. As a consequence, in this case
even a freeze-in dark matter model can come under the microscope of experimental facilities,
in stark contrast with freeze-in in a radiation-dominated universe. As a proof of principle, we finally consider a motivated dark matter scenario where the
freeze-in mechanism is facilitated through a dark photon portal having an extremely small
kinetic mixing with the SM photon [31, 71–78]. In the absence of any matter-dominated
epoch, the parameter space of this model is far beyond the reach of any current observations 2 SciPost Phys. 13, 022 (2022) or future experiments. We point out that for a sub-GeV dark photon, the kinetic mixing re-
quired to satisfy the observed relic density in the presence of an EMD epoch is accessible to
the experiments. In particular, we show that the bounds from the supernova observations and
beam-dump experiments can rule out a considerable part of the relevant parameter space. The paper is organized as follows. In Sec. 2, we discuss the cosmological history with an
early matter-dominated epoch. The impact of such matter-dominated epoch on the freeze-in
DM production is shown in Sec. 3. In Sec. 1
Introduction 4, we study the parameter space of a concrete
model with a dark photon acting as a portal between the visible and the dark sector before
concluding in Sec. 5. φ
3Note that we use different normalizations suitable for our context in Eq. (1) as compared to [54,70]. 1We assume that the post-inflationary reheating is instantaneous.
2 2
Early matter domination with generalized dissipation In this section, we explain the details of the alternate cosmological history with an early matter-
dominated epoch. We consider that the total energy of the universe is dominated by a thermal
plasma at the end of the inflation (characterized by a temperature Tinf)1, leading to an early
radiation domination (ERD). However, below some temperature Teq the total energy budget is
assumed to be dominated by a new meta-stable species (denoted generically by φ) red-shifting
slower than the radiation2. This marks the beginning of an early matter-dominated epoch. The meta-stable field φ with equation of state ω ∈(−1,1/3) oscillates around its potential
minima and would eventually decay into the SM radiation through its coupling to the SM
particles. This would eventually lead to entropy production and a second phase of reheating
towards a radiation-dominated (RD) universe below the temperature TRH, compatible with
the constraints from BBN [79–81]. We assume that the standard cosmological history prevails
below TRH. The dissipation rate (Γφ) of such a species φ mainly depends on the (i) temperature of
the surrounding environment – in this case the temperature of the background radiation bath
and (ii) on the expansion rate of the universe through its field dependent decay width to SM
particles [70, 82–84]. For instance, it was shown that decay of scalar particle in a thermal
environment leads to a temperature dependent decay width due to the back reaction of the
plasma of decay products [85–92]. As an example, the dissipation rate of a moduli field in
the early universe is given by Γφ ∝T 3 for temperatures larger than its mass [88]. On the
other hand, the dissipation rate of a meta-stable species with a potential V(φ) ∝φp (which
corresponds to ω = p −2/p + 2) can be written as Γφ→f ¯f ∝mφ(t) ∝a−3(p−2)/(p+2) , (for fermionic decay),
Γφ→ηη ∝m−1
φ (t) ∝a3(p−2)/(p+2) , (for bosonic decay), where mφ(t) ∝〈φ(t)〉(p−2)/2. We remain agnostic about the detailed dynamics of φ and
express the envelop of φ averaged over larger time scales as 〈φ(t)〉∼a−6/p+2 [93–95]. where mφ(t) ∝〈φ(t)〉(p−2)/2. We remain agnostic about the detailed dynamics of φ and
express the envelop of φ averaged over larger time scales as 〈φ(t)〉∼a−6/p+2 [93–95]. 2In what follows we refer to φ as ‘matter’ because it is assumed to have a slower red-shift than the radiation
3N t th t
diff
t
li
ti
it bl f
t
t i
E
(1)
d t
[54 70] 2In what follows we refer to φ as ‘matter’ because it is assumed to have a slower red-shift than the radiation.
3Note that we use different normalizations suitable for our context in Eq. (1) as compared to [54,70]. 1We assume that the post-inflationary reheating is instantaneous.
2 2
Early matter domination with generalized dissipation Motivated by these effects we parametrize the dissipation rate of the matter field into th
radiation, as follows [70]
k Γφ = ˆΓ
T
Teq
n
a
aeq
k
,
(1) (1) where aeq is the scale factor at Teq and ˆΓ is a mass dimensional parameter dependent on the
zero-temperature decay width3. The values of the exponents n and k depend on the detailed where aeq is the scale factor at Teq and ˆΓ is a mass dimensional parameter dependent on the
zero-temperature decay width3. The values of the exponents n and k depend on the detailed 3 SciPost Phys. 13, 022 (2022) SciPost Phys. 13, 022 (2022
Table 1: Representative cases illustrating the dependence of Γφ on the temperature
and scale factor. The last column depicts its consequence on the temperature evolu-
tion during entropy production era. Table 1: Representative cases illustrating the dependence of Γφ on the temperature
and scale factor. The last column depicts its consequence on the temperature evolu-
tion during entropy production era. Table 1: Representative cases illustrating the dependence of Γφ on the temperature
and scale factor. The last column depicts its consequence on the temperature evolu-
tion during entropy production era. Dynamics of φ / V(φ)
Γφ
(n, k,ω)
T(z) during EMDNA
V(φ) ∼φ2 [70,96]
const., mφ ≫T
(0,0,0)
decreases with z
T, mφ ≪T
(1,0,0)
decreases with z
V(φ) ∼φp
〈φ〉
p−2
2 , mφ ≫T
(0,−3(p−2)
p+2 , p−2
p+2)
decreases with z if p ≥1
remains constant if p = 1
(fermionic decay of φ) [82–84]
V(φ) ∼φp
〈φ〉−p−2
2 , mφ ≫T
(0, 3(p−2)
p+2 , p−2
p+2)
decreases with z if p ≥0
(bosonic decay of φ) [82–84]
Rotating scalar, V(φ) ∼φ†φ
〈φ〉−2, mφ ≫T
(0,3,0)
increases with z
log
φ
T
FµνF µν interaction [70,89,90]
T 3
〈φ〉2 , mφ ≪T
(3,3,0)
increases with z
Oscillating scalar, V(φ) ∼φp
〈φ〉−1, mφ ≫T
(0,
6
p+2, p−2
p+2)
remains constant if p = 2
decreases with z if p ≥2
log
φ
T
FµνF µν interaction [70,89,90]
T 2
〈φ〉, mφ ≪T
(2,
6
p+2, p−2
p+2) dynamics of the oscillating matter field and its interactions with the thermal plasma. Different
possibilities for n and k in specific scenarios are discussed in [54, 70, 89, 90]. In Tab. 2
Early matter domination with generalized dissipation 1, we
list some representative cases where the dissipation rate depends on the temperature of the
thermal bath and 〈φ(t)〉. The evolution of energy densities of the late-decaying matter ρφ
and radiation ργ are given by the following equations dynamics of the oscillating matter field and its interactions with the thermal plasma. Different
possibilities for n and k in specific scenarios are discussed in [54, 70, 89, 90]. In Tab. 1, we
list some representative cases where the dissipation rate depends on the temperature of the
thermal bath and 〈φ(t)〉. The evolution of energy densities of the late-decaying matter ρφ
and radiation ργ are given by the following equations ˙ρφ + 3(1 + ω)Hρφ = −(1 + ω)Γφρφ ,
(2)
˙ργ + 4Hργ = (1 + ω)Γφρφ ,
(3) (2)
(3) (2) (2)
(3) (3) where the Hubble expansion rate is H = pρφ + ργ/
p
3Mp with Mp being the reduced Planck
mass. In order to solve Eq. (3), we make the crucial assumption that the radiation produced
from φ thermalizes instantaneously. This allows us to relate ργ with an instantaneous bath
temperature as ργ = (π2/30)g∗(T) T 4. For simplicity we assume that the effective energetic
and entropic degrees of freedom are constant, g∗(T) = g∗S(T) = 106.75. It is convenient to
express the solutions for the energy densities in terms of a dimensionless variable z ≡a/aeq
(with dz = zHdt). In Fig. 1 we present the variation of the temperature of radiation bath as a function of z for
different values of (n, k, w), keeping Teq and TRH fixed. For 0 ≤n < 4 and δ ≡5−2n+2k−3ω > 0,
the EMD epoch can be further divided into two regions: an epoch of adiabatic evolution of
the temperature (EMDA), followed by an entropy production phase where the thermal plasma
becomes non-adiabatic (EMDNA). The approximate solutions for the Eq. (2) and Eq. (3) in the
EMD epoch are given by ρφ(z) ≃ργ(Teq)z−3(1+ω) ,
ργ(z) = z−4
ργ(Teq)
4−n
4 +
p
3MpˆΓ(4 −n)(1 + ω)
2δργ(Teq)
(n−2)
4
zδ/2 −1
4
4−n
. (4) (4) SciPost Phys. 13, 022 (2022) Figure 1: Evolution of the thermal plasma temperature as a function of z, obtained by
numerically solving Eq. (2) and Eq. (3) for different values of (n, k,ω) as given in the
legend. 2
Early matter domination with generalized dissipation The colored vertical lines show the approximate values of zNA for different
cases with specific (n, k,ω). For the purpose of illustration, we assume Tinf = 104
GeV, Teq = 103 GeV and, TRH = 1 GeV. Note that the value of zRH depends on ω. For
(2,6/5,1/5) case, the value of zRH is shown by the vertical dashed line. Figure 1: Evolution of the thermal plasma temperature as a function of z, obtained by Figure 1: Evolution of the thermal plasma temperature as a function of z, obtained by
numerically solving Eq. (2) and Eq. (3) for different values of (n, k,ω) as given in the
legend. The colored vertical lines show the approximate values of zNA for different
cases with specific (n, k,ω). For the purpose of illustration, we assume Tinf = 104
GeV, Teq = 103 GeV and, TRH = 1 GeV. Note that the value of zRH depends on ω. For
(2,6/5,1/5) case, the value of zRH is shown by the vertical dashed line. Figure 1: Evolution of the thermal plasma temperature as a function of z, obtained by
numerically solving Eq. (2) and Eq. (3) for different values of (n, k,ω) as given in the
legend. The colored vertical lines show the approximate values of zNA for different
cases with specific (n, k,ω). For the purpose of illustration, we assume Tinf = 104
GeV, Teq = 103 GeV and, TRH = 1 GeV. Note that the value of zRH depends on ω. For
(2,6/5,1/5) case, the value of zRH is shown by the vertical dashed line. Table 2: Approximate expressions for the evolution of T(z) and H(z) in different
cosmological epochs. Epoch
z
T(z)
H(z)
ERD
zinf < z < 1
Teq
z
s
ργ(Teq)
3M2
p z−2
EMDA
1 < z < zNA
Teq
z
s
ργ(Teq)
3M2
p z−3
2 (1+ω)
EMDNA
zNA < z < zRH
TRH
z
zRH
δ−8+2n
8−2n
s
ργ(Teq)
3M2
p z−3
2 (1+ω)
RD
zRH < z
Teqz
1−3ω
4
RH
z−1
p
ργ(TRH)
p
3Mp
z
zRH
−2 For the ω = 0 case, our generic results conform with those obtained in [70]. Approximate
analytic expressions for the variation of T(z) and H(z) in the various epochs are shown in
Tab. 2. 3
Dark matter freeze-in during matter domination In this section we discuss the impact of an EMD epoch with generalized dissipation of φ on the
freeze-in production of dark matter. Simply speaking, the entropy production during the non-
adiabatic phase of the EMD era induces a dilution in the relic abundance of the dark matter. However, T and z dependent dissipation rate of the meta-stable matter changes the amount
of dilution of the DM relic. Thus, a larger production rate for the DM may be required to
reproduce the observed abundance at the present time. To illustrate our point, we consider a simple setup where the DM (χ) is produced in pair
by the annihilation of two SM particles via the process SM + SM →X ∗→χ + χ. Here X acts
as a portal between the dark sector and the visible sector which can, for instance, be a dark
photon, a Z′, or a RH neutrino with extremely small couplings with the SM. The small coupling
with the SM is necessary to ensure that X is never in the thermal bath. Coupling between the
X and the DM can, however, be as large as O(1). The Boltzmann equation for the evolution of dark matter number density (nχ) is given by ˙
nχ + 3Hnχ = R(T),
(7) (7) where R(T) denotes the freeze-in production rate of the dark matter. The rate for the 2 →2
scattering SM + SM →X ∗→χ + χ can be written in its full glory as [77] here R(T) denotes the freeze-in production rate of the dark matter. The rate for the 2 →2
attering SM + SM →X ∗→χ + χ can be written in its full glory as [77] R(T) =
T
2048π6
Z
dspsK1
ps
T
v
u
t
1 −
4m2
χ
s
v
u
t
1 −
4m2
B
s
Z
dΩ|M|2 ,
(8) (8) where mB generically represents the mass of a SM particle in the thermal bath and |M|2 is the
amplitude square summed over initial and final states. In Eq. (8), we assume that both the
SM particles and the dark matter follow Maxwell-Boltzmann distribution and the interactions
between them are CP invariant. 2
Early matter domination with generalized dissipation The value of z at the beginning of the EMDNA can be calculated as For the ω = 0 case, our generic results conform with those obtained in [70]. Approximate
analytic expressions for the variation of T(z) and H(z) in the various epochs are shown in
Tab. 2. The value of z at the beginning of the EMDNA can be calculated as zNA =
1 +
2δργ(Teq)
1
2
p
3MpˆΓ(4 −n)(1 + ω)
2/δ
. (5) (5) (5) The temperature evolution during EMDNA era is sensitive to the exact temperature and scale
factor dependence in Eq. (1). For n ≥4 or δ < 0 the adiabatic evolution ends by an instan-
taneous reheating at TRH which we do not consider here. In Fig. 1, different cases of (n, k,ω)
lead to qualitatively different evolution of the temperature during the EMDNA epoch. During 5 SciPost Phys. 13, 022 (2022) the EMDNA era, the temperature remains constant for 2k = 3(1 + ω) (the (2,3/2,0) case in
Fig. 1), while T(z) grows with z for 2k > 3(1+ω) (the (3,3,0) case in Fig. 1). End of the EMD
era is characterized by the condition ργ(TRH) = ρφ(TRH), and the corresponding z is given as zRH =
2δργ(Teq)
1
2
p
3MpˆΓ(4 −n)(1 + ω)
4
2δ−(4−n)(1−3ω)
=
TRH
Teq
−
4
3(1+ω)
. (6) (6) The scale factor at today, expressed in terms of z is given by z0 = (Teq/T0)(TRH/Teq)
(3ω−1)
3(1+ω) . Note
that for ω ∈(−1,1/3), z0 is always greater than (Teq/T0) which would have been the value of
z0 in the absence of matter domination. The scale factor at today, expressed in terms of z is given by z0 = (Teq/T0)(TRH/Teq)
(3ω−1)
3(1+ω) . Note
that for ω ∈(−1,1/3), z0 is always greater than (Teq/T0) which would have been the value of
z0 in the absence of matter domination. 3
Dark matter freeze-in during matter domination The production rate given above can be parametrized in a
simple form depending on the hierarchy between the DM mass (mχ), the mediator mass (MX)
and the masses of the bath particles (mB) as R(T) ≃
λ2
1T pe−2mB
T ,
mB ≫MX,
MX ≫2mχ,
λ2
1T p ,
mB ≪MX ≪T,
MX ≫2mχ,
λ2
2M p
X
πT
ΓX
K1
MX
T
,
mB ≪MX ∼T,
MX ≫2mχ,
λ2
3
T p+4
M4
X e−2mB
T ,
mB, T ≪MX,
MX ≫2mχ,
λ2
1T pe−2
T max{mχ,mB} ,
MX ≪2mχ. (9) mB ≪MX ≪T,
MX ≫2mχ, (9) mB, T ≪MX,
MX ≫2mχ, λ2
1T pe−2
T max{mχ,mB} ,
MX ≪2mχ. MX ≪2mχ. 6 SciPost Phys. 13, 022 (2022) Here the exponent p = 4,6,8,... depends on the dimension of the operators describing the
interactions of X with the SM and the DM. For example, p = 4 arises for renormalizable
interactions while p > 4 comes from higher dimensional operators [97]. The coefficients
λ2
1,2,3, with dimension [M]4−p, vary depending on the details of specific model as well as
relative values of MX and T. We define a comoving number density of the DM as Yχ(z) ≡nχz3 and express the Boltz-
mann equation in terms of the dimensionless parameter z as dYχ(z)
dz
= z2R(T(z))
H(z)
. (10) (10) The present day relic density of dark matter is given by The present day relic density of dark matter is given by Ωχh2 =
mχnχ(T0)
ρc
=
mχYχ(z0)
ρcz3
0
,
(11) (11) where ρc denotes the critical density today and where ρc denotes the critical density today and Yχ(z0) = Yχ(zinf) +
Z z0
zinf
dz z2R(T(z))
H(z)
. (12) (12) Note that, during the EMD era z dependence of H(z) and T(z) are different compared to
radiation-dominated epoch, thereby also affecting the rate R(T). For the freeze-in DM pro-
duction, we assume that the initial DM abundance at the end of the inflation Yχ(zinf) = 0. The
effect of entropy injection during the EMDNA era is encoded into the z3
0 > (Teq/T0)3 factor, as
given in Eq. (11). 3
Dark matter freeze-in during matter domination Thus, in the presence of an EMD epoch larger value of Yχ(z0) is needed
to reproduce the observed Ωχh2 = 0.12, compared to the requirement in a purely radiation-
dominated universe. This in turn implies that even for the freeze-in production larger coupling
between the dark sector and the visible sector can be accommodated in the presence of EMD
era. The impact of matter domination on Yχ(z) depends on the temperature dependence of the
DM production rate. For R(T) ∝T p exp(−2mχ/T) with p > 4, majority of the DM is produced
at the ERD regime at higher temperatures, giving rise to the UV freeze-in. In this case, Yχ(z) is
not sensitive to the variation of (n, k,ω) during the EMD epoch since the DM number density
already saturates at much smaller z. Here we mention en passant that UV freeze-in of DM can
be boosted during an inflaton dominated epoch if the inflaton decays into the radiation with a
generalized rate as given in Eq. (1) [54]. On the other hand, IR freeze-in dominates for p = 4,
and majority of the DM production may happen during the EMD epoch depending on the DM
mass and the values of (n, k,ω). In both scenarios, however, the entropy production during
the final stage of matter domination dilutes the relic of the DM produced. For the sake of presentation, we assume p = 4, mχ = 1 GeV, MX = 10 GeV, and
λ1 = 2 × 10−14 = 2λ2 = λ3/8
p
34. In the left panel of Fig. 2, we show the evolution of
Yχ(z) for a fixed TRH = 1 GeV. The horizontal dashed lines represent the contours satisfying
Ωχh2 = 0.12 for different cases. We choose the parameters in such a way that the DM yield ap-
proximately saturates the Ωχh2 = 0.12 limit in a purely radiation dominated universe (dashed
cyan contours). In contrast, the DM is always under-abundant for the other scenarios in pres-
ence of an EMD epoch. 3
Dark matter freeze-in during matter domination The ratio of the relic densities of the DM in the presence of an EMD
era to that in a purely radiation dominated universe is given by Ωχh2
Ωχh2
RD
=
Yχ(z0)
Y RD
χ (z0)
zRD
0
z0
3
=
Yχ(z0)
Y RD
χ (z0)
TRH
Teq
1−3ω
1+ω
,
(13) (13) 4The ratios λ2
1/λ2
2 and λ2
1/λ2
3 can be fixed by performing the integral over s in Eq. (8) for a fixed value of p(= 4). 7 SciPost Phys. 13, 022 (2022) Figure 2: In the left panel, we show Yχ(z) as a function of z keeping a fixed value of
TRH = 1 GeV. The colored vertical lines denote the position of zNA for different cases. The horizontal dashed lines present the contours of Ωχh2 = 0.12. In the right panel,
we present the variation of Yχ(z) keeping ˆΓ fixed. In this case, the colored vertical
lines represent the corresponding values of zRH. In both panels we fix Tinf = 104 GeV
and Teq = 103 GeV. Figure 2: In the left panel, we show Yχ(z) as a function of z keeping a fixed value of
TRH = 1 GeV. The colored vertical lines denote the position of zNA for different cases. The horizontal dashed lines present the contours of Ωχh2 = 0.12. In the right panel,
we present the variation of Yχ(z) keeping ˆΓ fixed. In this case, the colored vertical
lines represent the corresponding values of zRH. In both panels we fix Tinf = 104 GeV
and Teq = 103 GeV. where the factor
TRH
Teq
1−3ω
1+ω encodes the entropy dilution. For the benchmark parameter choice
used in the left panel of Fig. 2, this ratio varies between ∼[0.1−0.001]. As a consequence, it is
evident that one would need a larger coupling between the DM and the SM particles to achieve
the observed relic abundance in presence of matter domination. Interestingly, for (3,3,0) and
(2,3/2,0), a second stage of DM production occurs, since the temperature starts to increase
during the EMDNA era. In the right panel of Fig. 2, we present the variation of Yχ(z) keeping ˆΓ = 10−17 GeV
fixed. Different values of (n, k,ω) thus lead to different values of TRH ranging from 3.5 MeV
to a few GeV (corresponding values of zRH are shown by colored vertical lines). 3
Dark matter freeze-in during matter domination Since the DM
production ceases below T ∼mχ, the saturation of Yχ(z) does not necessarily coincide with
T = TRH. For example, for the blue line in Fig. 2 corresponding to (1,0,0), Yχ(z) is saturated
much before the end of EMD era. In this case, the dilution due to entropy production during
the matter domination is maximum, thereby yielding an under-abundance of DM at present
day. In contrast, for (0,0,0) (red line), the DM number density is saturated just after the end
of EMD, causing less dilution compared to (1,0,0). 5Above the weak scale the U(1)D gauge boson has a kinetic mixing with the hypercharge Bµ which eventually
gives rise to the kinetic mixing term between the F ′
µν and Fµν below the electroweak symmetry breaking scale. In
addition to that, a mixing between A′
µ and the Zµ boson is also induced. However, for the dark matter phenomenol-
ogy discussed in this section with MA′ ≲1 GeV, the effect of the A′
µ −Zµ mixing is negligible. 4
Case study: dark photon portal to dark matter Finally, we focus on a specific model where a fermionic dark matter is produced by the freeze-in
mechanism via a dark photon portal interaction. In the presence of an EMD epoch with gen-
eralized dissipation of φ, this model comes under the scanner of terrestrial and astrophysical
experiments. We consider a Dirac fermion dark matter χ, charged under a dark gauged U(1)D with a
dark photon (A′
µ) which has a kinetic mixing with the SM photon. The relevant part of the 8 SciPost Phys. 13, 022 (2022) Lagrangian involving A′
µ and χ is given by ian involving A′
µ and χ is given by Lagrangian involving A′
µ and χ is given by L = −1
4 F′
µνF′µν −ε
2 F′
µνFµν + 1
2 M2
A′A′
µA′µ + ¯χ
i /∂−mχ
χ + gD ¯χγµA′
µχ ,
(14) (14) where ε and gD ≡
p
4παD denote the kinetic mixing parameter and the dark gauge coupling,
respectively.5 After diagonalizing the kinetic mixing term, the dark photon interacts with the
SM particles with an interaction strength proportional to εQ, where Q is the electric charge of
the corresponding SM field. We are primarily interested in the mass range MA′ ≲1 GeV, where
the DM production via freeze-in is dominated by the process f ¯f →A′ →χ ¯χ (f being the SM
fermions). The production rate for this process is given by [77] R(T) =
NcQ2
f αeαDε2T
6π3
Z
ds
v
u
u
t
1 −
4m2
χ
s
1 −
4m2
f
s
s5/2
1 +
2m2
χ
s
1 +
2m2
f
s
(s −M2
A′)2 + M2
A′Γ 2
A′
K1
ps
T
,
(15) (15) (15)
where Nc and Q f denote the number of colors and electric charge of the initial state fermions
respectively. The decay width of the dark photon ΓA′, dominated by its decay into a pair of DM
when MA′ > 2mχ, is given as [77] (
)
where Nc and Q f denote the number of colors and electric charge of the initial state fermions
respectively. The decay width of the dark photon ΓA′, dominated by its decay into a pair of DM
when MA′ > 2mχ, is given as [77] ΓA′ ≃αDMA′
3
1 +
2m2
χ
M2
A′
v
u
t1 −
4m2
χ
M2
A′
. 4
Case study: dark photon portal to dark matter 13, 022 (2022) Figure 3: The contours satisfying Ωχh2 = 0.12 are shown for MA′ = 10mχ (left
panel) and MA′ = 5mχ/3 (right panel). The dark grey region is not accessible for
the freeze-in production as the condition R(T)/neq
f H ≪1 is violated and as a conse-
quence DM comes in thermal equilibrium with the visible matter. The color coding
for different Ωχh2 contours are same as in Fig. 2. In the left panel, we overlap the
supernova cooling constraints [98] in the presence of non-zero branching ratios for
the decay of dark photon into dark matter. On the other hand, in right panel the
existing constraints from different beam dump experiments [99–104], supernova
bounds [98, 105, 106] and the projected bounds for future experiments like SHiP,
FASER, and SeaQuest [107–110] are superimposed assuming the dark photon de-
cays into SM particles only. Figure 3: The contours satisfying Ωχh2 = 0.12 are shown for MA′ = 10mχ (left
panel) and MA′ = 5mχ/3 (right panel). The dark grey region is not accessible for
the freeze-in production as the condition R(T)/neq
f H ≪1 is violated and as a conse-
quence DM comes in thermal equilibrium with the visible matter. The color coding
for different Ωχh2 contours are same as in Fig. 2. In the left panel, we overlap the
supernova cooling constraints [98] in the presence of non-zero branching ratios for
the decay of dark photon into dark matter. On the other hand, in right panel the
existing constraints from different beam dump experiments [99–104], supernova
bounds [98, 105, 106] and the projected bounds for future experiments like SHiP,
FASER, and SeaQuest [107–110] are superimposed assuming the dark photon de-
cays into SM particles only. As a consequence, all the contours satisfying Ωχh2 = 0.12 roughly overlap with the RD case
in that region of the parameter space. When MA′ < 2mχ, A′
µ decay to dark matter is kinematically suppressed. For an ultralight
dark photon with MA′ ≪mχ, the DM behaves as a millicharged particle which has been dis-
cussed in [30,111,112]. Here instead, we focus on the mass range mχ < MA′ < 2mχ, where
f ¯f →A′∗→χ ¯χ still dominates the DM production. In this case, we plot in the right panel of
Fig. 3, the contours satisfying Ωχh2 = 0.12 assuming MA′ = 5mχ/3, αD = 0.1 and TRH = 8 MeV. 4
Case study: dark photon portal to dark matter (16) (16) The rate given in Eq. (15) can be approximated depending on the hierarchy between MA′
and mχ, as shown in Eq. (9). Two different scenarios are of interest to us. In the first case,
MA′ ≫2mχ so that the A′
µ can be produced on-shell by s-channel resonance and subsequently
decay into a pair of DM particles. In the left panel of Fig. 3, we present the contours satisfying
Ωχh2 = 0.12 in the ε−MA′ plane with specific values of (n, k,ω). For the purpose of illustration,
we assume MA′ = 10mχ and αD = 10−3, as well as Tinf = 105 GeV, Teq = 104 GeV, and TRH = 8
MeV. We chose the sub-GeV mass range for MA′ and mχ to access the sweet-spot where the
effect of EMD epoch can push the freeze-in parameter space within the reach of experimental
observations. In this range of parameters, the dark photons produced by the inverse decay processes
SM + SM →A′
µ rapidly decays into dark matter with the rate given by Eq. (15), since it is
the dominant decay channel for A′
µ. As a result such inverse decays do not lead to a thermal
abundance of A′
µ. Thus the condition R(T) ≪neq
f H (where neq
f
is the equilibrium number
density of the SM fermions) is sufficient to guarantee that both the dark matter and the dark
photon are not in thermal equilibrium with the visible sector. The grey shaded area in Fig. 3
shows the region where the freeze-in condition R(T) ≪neq
f H is violated. Although, the freeze-
in condition depends on the values of (n, k,ω), we only show the most restrictive condition. We also overlap the constraints from the supernova cooling [98] on the dark photon mass and
the kinetic mixing. Note that for (n, k,ω) = (3,3,0) and (2,3/2,0), some part of the parameter
space is ruled out by the supernova observations. In the above plot we have set g∗(T) = 10. An actual variation of g∗(T) with respect to temperature does not change the overall picture
presented in this work. For MA′, mχ < TRH, majority of the DM is produced after the end of the
matter-dominated epoch since the freeze-in production is dominated by the IR contributions. 9 SciPost Phys. Acknowledgments The authors thank Marcos A. G. Garcia, Andrew J. Long, and Yann Mambrini for valuable
comments on the manuscript. A.B. acknowledges support from the Knut and Alice Wal-
lenberg foundation (Grant KAW 2017.0100, SHIFT project). This research of D.C. is sup-
ported by an initiation grant IITK/PHY/2019413 at IIT Kanpur and by a DST-SERB grant
SERB/CRG/2021/007579. 5
Conclusions The main message of this work is that the freeze-in production of the dark matter is significantly
altered in the presence of an early matter-dominated epoch prior to the BBN. Many motivated
BSM models predict late-decaying fields that can lead to a pre-BBN matter-dominated epoch. The transition from the matter domination to the usual radiation domination occurs through
the dissipation of the matter field into radiation. The generic dependence of the dissipation
rate on the instantaneous temperature of the thermal bath and the expansion rate of the uni-
verse leads to a qualitatively different evolution of the temperature and entropy of the thermal
plasma. These alterations of the background cosmology impacts the dark matter production rate
in the early universe. Due to an additional entropy injection during the matter-dominated
epoch, a larger coupling between the visible and the dark sector is essential to produce the
observed relic density of dark matter via the freeze-in mechanism. This leads to the interesting
possibility of probing the freeze-in dark matter models in experiments, which would otherwise
be impossible in the usual radiation-dominated cosmology. We would like to point out that majority of the analysis presented in this work is model
independent. We have elucidated the DM production via freeze-in for a number of well-
motivated cases with different temperature and scale factor dependence of the dissipation
rate. Indeed, our study quantitatively shows that orders of magnitude larger couplings are
required to produce the correct freeze-in relic abundance in the presence of early matter dom-
ination. Finally we concentrate on a concrete model consisting a fermionic dark matter interacting
with the SM via a dark photon portal to illustrate this phenomenon. We show that the relevant
parameter space for freeze-in DM production with a sub-GeV dark photon mediator comes
under the scanner of supernova observations and other terrestrial experiments. 4
Case study: dark photon portal to dark matter The constraints on the kinetic mixing parameter from supernova cooling [98,105,106] as well
as several beam dump experiments [99–104] rule out a significant region of the parameter
space with sub-GeV masses. Moreover, future experiments like SHiP [107,108,110] will also
probe a large part of the parameter space untouched by the current generation experiments. Similar to the left panel of Fig. 3, the grey shaded area in the right panel shows the region
where the freeze-in condition, R(T)/neq
f H ≪1, for the DM production via f ¯f →A′∗→χ ¯χ
process is violated. However, in this case we need to consider additional constraint on the ki-
netic mixing parameter to ensure that the dark photon, produced via inverse decay processes
SM + SM →A′
µ, can not achieve thermal equilibrium. To this end, we compare the inverse
decay rate Rinv(T) to the Hubble expansion rate (Rinv(T) ≪neq
SMH). As a consequence, one
can safely neglect other DM production channels such as A′
µ + A′
ν →χ + ¯χ. We observe that
this condition does not rule out any additional parameter space which was untouched by the
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Enhancing the generalization for Intent Classification and Out-of-Domain Detection in SLU
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Proceedings of the 59th Annual Meeting of the Association for Computational Linguistics
and the 11th International Joint Conference on Natural Language Processing, pages 2443–2453
August 1–6, 2021. ©2021 Association for Computational Linguistics Enhancing the generalization for Intent Classification
and Out-of-Domain Detection in SLU
Yilin Shen, Yen-Chang Hsu, Avik Ray, Hongxia Jin
Samsung Research America
{yilin.shen,yenchang.hsu,avik.r,hongxia.jin}@samsung.com
Abstract
assification is a major task in spoken
e understanding (SLU). Since most
are built with pre-collected in-domain
raining utterances, their ability to de-
upported out-of-domain (OOD) utter-
as a critical effect in practical use. Re-
rks have shown that using extra data
ls can improve the OOD detection per-
e, yet it could be costly to collect such
his paper proposes to train a model
IND d
hil
i
b h IND
I don’t like Thriller in playlist
Playlist deleted
I am too cold
Oven turned on
Figure 1: Failure Examples of Unsupported Skills in
AI Voice Assistants. The user’s utterances are out of
the designed domains of the assistant. Enhancing the generalization for Intent Classification
and Out-of-Domain Detection in SLU
Yilin Shen, Yen-Chang Hsu, Avik Ray, Hongxia Jin
Samsung Research America
{yilin.shen,yenchang.hsu,avik.r,hongxia.jin}@samsung.com
Abstract
assification is a major task in spoken
e understanding (SLU). Since most
are built with pre-collected in-domain
raining utterances, their ability to de-
upported out-of-domain (OOD) utter-
as a critical effect in practical use. Re-
rks have shown that using extra data
ls can improve the OOD detection per-
e, yet it could be costly to collect such
his paper proposes to train a model
IND d
hil
i
b h IND
I don’t like Thriller in playlist
Playlist deleted
I am too cold
Oven turned on
Figure 1: Failure Examples of Unsupported Skills in
AI Voice Assistants. The user’s utterances are out of
the designed domains of the assistant. on for Intent Classification
n Detection in SLU
su, Avik Ray, Hongxia Jin
earch America
k.r,hongxia.jin}@samsung.com
I don’t like Thriller in playlist
Playlist deleted
I am too cold
Oven turned on
Figure 1: Failure Examples of Unsupported Skills in
AI Voice Assistants. The user’s utterances are out of
the designed domains of the assistant. Enhancing the generalization for Intent Classification
and Out-of-Domain Detection in SLU I don’t like Thriller in playlist
Playlist deleted
I am too cold
Oven turned on
Figure 1: Failure Examples of Unsupported Skills in
AI Voice Assistants. The user’s utterances are out of
the designed domains of the assistant. Abstract Intent classification is a major task in spoken
language understanding (SLU). Since most
models are built with pre-collected in-domain
(IND) training utterances, their ability to de-
tect unsupported out-of-domain (OOD) utter-
ances has a critical effect in practical use. Re-
cent works have shown that using extra data
and labels can improve the OOD detection per-
formance, yet it could be costly to collect such
data. This paper proposes to train a model
with only IND data while supporting both IND
intent classification and OOD detection. Our
method designs a novel domain-regularized
module (DRM) to reduce the overconfident
phenomenon of a vanilla classifier, achieving
a better generalization in both cases. Besides,
DRM can be used as a drop-in replacement
for the last layer in any neural network-based
intent classifier, providing a low-cost strategy
for a significant improvement. The evalua-
tion on four datasets shows that our method
built on BERT and RoBERTa models achieves
state-of-the-art performance against existing
approaches and the strong baselines we cre-
ated for the comparisons. Figure 1: Failure Examples of Unsupported Skills in
AI Voice Assistants. The user’s utterances are out of
the designed domains of the assistant. during both training and testing. However, such
an assumption is not held in the practical use case
of PA systems, where the system is used under a
dynamic and open environment with personal ex-
pressions, new vocabulary, and unknown intents
that are out of the design scope. To address the challenges in open-world settings,
previous works adopt varied strategies. Shen et al. (2018a, 2019c) use a cold-start algorithm to gener-
ate additional training data to cover a larger variety
of utterances. This strategy relies on the software
developers to pre-build all possible skills. Shen
et al. (2019b,a) introduce a SkillBot that allows
users to build up their own skills. Recently, Ray
et al. (2018, 2019); Shen et al. (2018b, 2019d) en-
ables an SLU model to incorporate user personal-
ization over time. However, the above approaches
do not explicitly address unsupported user utter-
ances/intents, leading to catastrophic failures illus-
trated in Figure 1. Thus, it is critically desirable
for an SLU system to classify the supported intents
(in-domain (IND)) and reject unsupported ones
(out-of-domain (OOD)) correctly. 2.2
Related Work Intent Classification is one of the major SLU
components (Haffner et al., 2003; Wang et al.,
2005; Tur and De Mori, 2011). Various models
have been proposed to encode the user utterance
for intent classification, including RNN (Ravuri
and Stoicke, 2015; Zhang and Wang, 2016; Liu and
Lane, 2016; Kim et al., 2017; Wang et al., 2018;
Goo et al., 2018), Recursive autoencoders (Kato
et al., 2017), or enriched word embeddings (Kim
et al., 2016). Recently, the BERT model (Devlin
et al., 2019) was explored by (Chen et al., 2019) for
SLU. Our work also leverages the representation
learned in BERT. This paper proposes a strategy based on neural
networks to use only IND utterances and their la-
bels to learn both the intent classifier and OOD
detector. Our strategy modifies the structure of the
classifier, introducing an extra branch as a regu-
larization target. We call the structure a Domain-
Regularized Module (DRM). This structure is prob-
abilistically motivated and empirically leads to a
better generalization in both intent classification
and OOD detection. Our analysis focuses more
on the latter task, finding that DRM not only out-
puts a class probability that is a better indicator for
judging IND/OOD, but also leads to a feature rep-
resentation with a less distribution overlap between
IND and OOD data. More importantly, DRM is a
simple drop-in replacement of the last linear layer,
making it easy to plug into any off-the-shelf pre-
trained models (e.g. BERT (Devlin et al., 2019)) to
fine-tune for a target task. The evaluation on four
datasets shows that DRM can consistently improve
upon previous state-of-the-art methods. OOD Detection has been studied for many
years (Hellman, 1970). Tur et al. (2014) explores
its combination with intent classification by learn-
ing an SVM classifier on the IND data and ran-
domly sampled OOD data. Ryu et al. (2017) de-
tects OOD by using reconstruction criteria with an
autoencoder. Ryu et al. (2018) learns an intent clas-
sifier with GAN and uses the discriminator as the
classifier for OOD detection. Zheng et al. (2020)
leverages extra unlabeled data to generate pseudo-
OOD samples using GAN via auxiliary classifier
regularization. Tan et al. (2019) further incorpo-
rates the few-shot setting, learning the encoding
of sentences with a prototypical network that is
regularized with the OOD data outside a learn-
ing episode. 1
Introduction Spoken language understanding (SLU) systems
play a crucial role in ubiquitous artificially intelli-
gent voice-enabled personal assistants (PA). SLU
needs to process a wide variety of user utterances
and carry out user’s intents, a.k.a. intent classifica-
tion. Many deep neural network-based SLU mod-
els have recently been proposed and have demon-
strated significant progress (Guo et al., 2014; Liu
and Lane, 2016; Zhang and Wang, 2016; Wang
et al., 2018; Goo et al., 2018; Chen et al., 2019)
in classification accuracy. These models usually
apply the closed-world assumption, in which the
SLU model is trained with predefined domains, and
the model expects to see the same data distribution A straightforward solution is to collect OOD
data and train a supervised binary classifier on
both IND data and OOD data (Hendrycks et al.,
2018). However, collecting a representative set of
OOD data could be impractical due to the infinite 2443 compositionality of language. Arbitrarily select-
ing a subset could incur the selection bias, causing
the learned model might not generalize to unseen
OOD data. Ryu et al. (2017, 2018) avoid learning
with OOD data by using generative models (e.g.,
autoencoder and GAN) to capture the IND data
distribution, then judge IND/OOD based on the
reconstruction error or likelihood. Recently, Tan
et al. (2019) utilizes a large training data to enable
the meta-learning for OOD detection. Zheng et al. (2020) generates pseudo OOD data to learn the
OOD detector. The above-discussed approaches re-
quire additional data or training procedures beyond
the intent classification task, introducing significant
data collection effort or inference overhead. fixed but unknown IND distribution PIND(x, y). We aim to train an intent classifier model only on
IND training data DIND such that the model can
perform: (1) Intent Classification: classify the
intent class label y of an utterance x if x is drawn
from the same distribution PIND as the training set
DIND; (2) OOD Detection: detect an utterance
x to be an abnormal/unsupported sample if x is
drawn from a different distribution POOD. 2.2
Related Work Other researchers developed meth-
ods in computer vision based on the rescaling of
the predicted class probabilities (ODIN) (Liang
et al., 2017) or building the Gaussian model with
the features extracted from the hidden layers of
neural networks (Mahalanobis) (Lee et al., 2018). Recently, (Hsu et al., 2020) proposed Generalized-
ODIN with decomposed confidence scores. How-
ever, both approaches also heavily depend on the
image input perturbation to achieve good perfor-
mance. Unfortunately, such perturbation cannot be
applied to discrete utterance data in SLU. 2.1
Problem Definition In the application of intent classification, a user
utterance will be either an in-domain (IND) utter-
ance (supported by the system) or an out-of-domain
(OOD) utterance (not supported by the system). The classifier is expected to correctly (1) predict
the intent of supported IND utterances; and (2)
detect to reject the unsupported OOD utterances. The task is formally defined below. We are given
a closed world IND training set DIND = {x, y} =
{(xi, yi)}N
i=1. Each sample (xi, yi), an utterance
xi and its intent class label yi ∈{1 . . . C} for
C predefined in-domain classes, is drawn from a In the application of intent classification, a user
utterance will be either an in-domain (IND) utter-
ance (supported by the system) or an out-of-domain
(OOD) utterance (not supported by the system). The classifier is expected to correctly (1) predict
the intent of supported IND utterances; and (2)
detect to reject the unsupported OOD utterances. The task is formally defined below. We are given
a closed world IND training set DIND = {x, y} =
{(xi, yi)}N
i=1. Each sample (xi, yi), an utterance
xi and its intent class label yi ∈{1 . . . C} for
C predefined in-domain classes, is drawn from a 2444 Linear
!"
/
! logits
classification logits
domain logits
hidden
state
ℎ
Linear
!$
domain loss
classification
loss
sigmoid
Figure 2: The DRM involves a domain component and
a classification components for the IND classes. 3.1
Domain-Regularized Module (DRM) At the end, we obtain the final logits f to repre-
sent bp(y|d = 1, x) by putting fd and fc together
following the dividend-divisor structure of Equa-
tion 1: The motivation begins with introducing the domain
variable d (d = 1 means IND, while d = 0 means
OOD) following the intuition in (Hsu et al., 2020),
then rewrite the posterior of class y given x with
domain d as follows: f = fc/fd
(4) (4) where each element of fc is divided by the same
scalar fd. where each element of fc is divided by the same
scalar fd. bp(y|d = 1, x)
= bp(y, d = 1|x)
bp(d = 1|x)
=
bp(y|x)
bp(d = 1|x) −bp(y, d = 0|x)
bp(d = 1|x)
≈
bp(y|x)
bp(d = 1|x)
(1) 3
Our Method classification
loss domain loss domain loss Our method is inspired by the decomposed con-
fidence of Generalized-ODIN (Hsu et al., 2020),
but we leverage the fact that the training data are
all from IND to introduce an extra regularization. This regularization leads to a better generalization
(lower classification error) on the intent classifica-
tion. The improvement is in contrast to the original
Generalized-ODIN, which has its classification er-
ror slightly increased. Since the improved general-
ization is likely due to a more generalizable feature
representation, we leverage this observation, pro-
viding a modified Mahalanobis (Lee et al., 2018),
which we called L-Mahalanobis, for a transformer-
based model to detect OOD data. In the follow-
ing sections, we first describe the DRM and then
elaborate on using the outputs of a DRM-equipped
model to detect OOD data. sigmoid Figure 2: The DRM involves a domain component and
a classification components for the IND classes. Classification Logits fc models the probability
posterior bp(y|x) before normalization. It follows
the conventional linear projection from hidden state
h to the number of classes: fc = Wch + bc
(3) (3) where Wd ∈R|h|×C with C classes. where Wd ∈R|h|×C with C classes. 3.1.2
DRM Training We propose two training loss functions to train a
model with DRM. The first training loss aims to
minimize a cross-entropy between the predicted
intent class and ground truth IND class labels. (1) (1) where the last step holds since bp(y, d = 0|x) is
close to 0 with the intrinsic conflict between IND
classes y and random variable d = 0 for OOD. Lclassification ≜−
C
X
i=1
yi log bp(f)i
(5) (5) 3.1.1
DRM Design where bp(f) is the softmax of logits f: where bp(f) is the softmax of logits f: Motivated by the above Equation 1, we design the
DRM to mitigate overconfidence by decomposing
the final logits f into two branches. Figure 2 illus-
trates the architecture. bp(f) = softmax(f) The second training loss aims to ensure that the
domain component fd is close to 1 since all utter-
ances in the training set are IND. Domain Logits fd models bp(d = 1|x) before nor-
malization. It projects from hidden state h to a
scalar w.r.t. d: Ldomain ≜(1 −sigmoid(fd))2
(6) (6) fd = Wdh + bd
(2) (2) We first restrict fd between 0 and 1 by using sig-
moid activation function. Then, this loss function
encourages sigmoid(fd) close to 1 for training on
IND utterances. In order to avoid fd to be very
large values and affect the training convergence, where Wd ∈R|h|×1. Since bp(d = 1|x) is a proba-
bility between 0 and 1, Section 3.1.2 will describe
the training details of domain loss via the sigmoid
function. 2445 we further apply clamp function on fd before it
feeds to Equation 4: 3.2
IND Intent Classification Method where fℓ(x) represents the output features at the
ℓth-layer of neural networks; µi and Σ are the
class mean representation and the covariance ma-
trix. Thus, the overall score is their summation: Following Equation 1 and our DRM design, it
is straightforward to use the confidence score of
softmax(f) to predict the IND intent class. 3.3.2
Feature-based Method While our DRM confidence already outperforms
many existing methods (later shown in experi-
ments), we further design the feature-based Ma-
halanobis distance score, inspired by the recent
work (Lee et al., 2018) for detecting OOD images. fd =
(
fd
if −δ < fd < δ
δ
if fd <= −δ or fd >= δ Thus, we sum them up to optimize the model: Thus, we sum them up to optimize the model: We first recap the approach in (Lee et al., 2018)
which consists of two parts: Mahalanobis distance
calculation and input preprocessing. Mahalanobis
distance score models the class conditional Gaus-
sian distributions w.r.t. Gaussian discriminant anal-
ysis based on both low- and upper-level features of
the deep classifier models. The score on layer ℓis
computed as follows: L = Lclassification + Ldomain
(7) (7) Remarks: It is important to note that the de-
sign of Ldomain is to introduce extra regularization
to mitigate the overconfidence in standard poste-
rior probability bp(f). sigmoid(fd) is not used to
directly predict if an utterance is IND or OOD. Sℓ
Maha(x) = maxi −(fℓ(x) −µℓ
c)T Σ−1
ℓ(fℓ(x) −µℓ
c) 3.3
OOD Detection Methods SMaha(x) =
X
ℓ
SMaha(fℓ(x)) There are two types of strategies to utilize the out-
puts of a classifier to perform OOD detection. One
is based on the confidence which is computed from
logits, the other is based on the features. In the
below, we describe how to compute different OOD
scores with our DRM. In addition, the input preprocessing adds a small
controlled noise to the test samples to enhance the
performance. Although Mahalanobis distance score can be ap-
plied only to the last feature layer without input pre-
processing Slast
Maha(x), the analysis (Table 2 in (Lee
et al., 2018)) shows that either input preprocessing
or multi-layer scoring mechanism is required to
achieve decent OOD detection performance. Un-
fortunately, neither of the above two mechanisms is
applicable in the intent classifier for SLU. First, un-
like image data, noise injection into discrete natural
language utterances has been shown not to perform
well. Second, in most cutting-edge intent classi-
fier models, low- and upper-level network layers
are quite different. The direct application of multi-
layer Mahalanobis distance leads to much worse
OOD detection performance. (9) Since BERT-based models showed significant
performance improvement for intent classification
in SLU (Chen et al., 2019), we focus on designing
the multi-layer Mahalanobis score for BERT-based
classifier models. In existing BERT-based text clas-
sification models, such as BERT, RoBERTa, Distil-
BERT, ALBERT, etc., there are different designs
between the last transformer layer and the classifi-
cation layer. Figure 3 shows our generic design of with large T = 1000 (Liang et al., 2017). DRM Entropy Confidence Score:
ENTDRM = Entropy[softmax(fc)]
(10) 3.3.1
Confidence-based Methods Recent works (Liang et al., 2017) has shown that
the softmax outputs provide a good scoring for de-
tecting OOD data. In our DRM model, we use the
decomposed softmax outputs for the score. The log-
its fc w.r.t. the true posterior distribution in open-
world can be combined with varied approaches: DRM Confidence Score: ConfDRM = softmax(fc)
(8) ConfDRM = softmax(fc) (8) ODINDRM = softmax(fc/T)
(9) (9) DRM ODIN Confidence Score: ODINDRM = softmax(fc/T)
(9) with large T = 1000 (Liang et al., 2017).
DRM Entropy Confidence Score: ENTDRM = Entropy[softmax(fc)]
(10) (10) The OOD utterances have low ConfDRM,
ODINDRM scores and high ENTDRM score. 2446 Table 1: SLU Benchmark and In-House Dataset Statistics
Dataset
Domain
#Intents
#Train
#D
CLINC (Larson et al., 2019)
various domains in voice assistants
150
15,000
3,0
other out-of-scope domains
-
100
10
ATIS (Hemphill et al., 1990)
airline travel information domain
18
4,478
50
Snips (Coucke et al., 2018)
music, book, and weather domains
7
13,084
70
Movie (in-house)
movie QA domain
38
39,558
4,8
Transformer
Transformer
…
Transformer
tanh layer
Classification layer
tanh
tanh
!"#$#
%
!"#$#
&
!"#$#
'
∑
!"#$#
Figure 3: Multi-layer Mahalanobis Score Design for
BERT-based Classifier Model
Among all these datasets, the
CLINC dataset serves as a ben
detection in SLU. For the other
treat them mutually OOD due to
domains. We crowdsourced the in-hou
containing common questions t
regarding movies. This dataset m
queries a user may ask in the m
dataset consists of 38 different i
information, genre information, a
show trailer) and 20 slots or enti
award, release year). This data
using crowdsourcing as follow Table 1: SLU Benchmark and In-House Dataset Statistics Table 1: SLU Benchmark and In-House Dataset Statistics Dataset
Domain
#Intents
#Train
#Dev
#Test
CLINC (Larson et al., 2019)
various domains in voice assistants
150
15,000
3,000
4,500
other out-of-scope domains
-
100
100
1,000
ATIS (Hemphill et al., 1990)
airline travel information domain
18
4,478
500
893
Snips (Coucke et al., 2018)
music, book, and weather domains
7
13,084
700
700
Movie (in-house)
movie QA domain
38
39,558
4,897
4,926 Transformer
Transformer
…
Transformer
tanh layer
Classification layer
tanh
tanh
!"#$#
%
!"#$#
&
!"#$#
'
∑
!"#$#
Figure 3: Multi-layer Mahalanobis Score Design for
BERT-based Classifier Model Among all these datasets, the recently released
CLINC dataset serves as a benchmark for OOD
detection in SLU. For the other three datasets, we
treat them mutually OOD due to non-overlapping
domains. We crowdsourced the in-house Movie dataset
containing common questions that users may ask
regarding movies. This dataset mainly consists of
queries a user may ask in the movie domain. The
dataset consists of 38 different intents (e.g. rating
information, genre information, award information,
show trailer) and 20 slots or entities (e.g., director,
award, release year). This dataset was collected
using crowdsourcing as follows. 4
Experimental Evaluation We implemented our method using PyTorch on
top of the Hugging Face transformer library (Wolf
et al., 2019). We follow the hyperparameters in
the original models. For the only hyperparame-
ter δ, we experimented only on CLINC dataset with large T = 1000 (Liang et al., 2017).
DRM Entropy Confidence Score: At first, some
example template queries were generated by lin-
guistic experts for each intent, along with intent
and slot descriptions. Next, a generation crowd-
sourcing job was launched where a crowd worker
was assigned a random intent, a combination of
entities, and few slots generally associated with the
intent. To better understand the intent and slots,
the worker was asked to review the intent and slot
descriptions, and example template utterances. The
first task of the worker was to provide 3 different
queries corresponding to the given intent, which
also contains the provided entities. The second task
of the worker was to provide additional entities
corresponding to the same slot type. A subsequent
validation crowdsourcing job was launched where
these crowdsourced queries were rated by valida-
tion workers in terms of their accuracy with the
provided intent and entities. Each query was rated
by 5 different validation workers, and the final val-
idated dataset contains a subset of crowdsourced
queries with high accuracy score and high inter-
rater agreement. Figure 3: Multi-layer Mahalanobis Score Design for
BERT-based Classifier Model Mahalanobis score computation (blue) for various
BERT-based models. Our design is based on our extensive experiments
and understanding of the common insights in differ-
ent BERT-based models. Specifically, we use the
features from different layers between the last trans-
former layer and the classification layer. We em-
pirically found that the nonlinear tanh layer plays
an important role. Thus, to map the features of
each transformer layer and last layer into the same
semantic space, we pass the features of each layer
through tanh function and sum them up to compute
our Mahalanobis score: SL−Maha(x) = SMaha(fn(x))
+
X
1≤ℓ<n
SMaha(tanh(fℓ(x)))
(11) (11) where fℓand fn are the features of each layer ℓ
and last layer n in a BERT-based intent classifier
model. We refer to our proposed approach as L-
Mahalanobis. 4.3.2
OOD Detection Baselines We consider the existing OOD detection methods: We consider the existing OOD detection methods:
ConGAN (Ryu et al., 2018): a GAN-based
model based on given sentence representations
to generate OOD features with additional feature
matching loss. OOD utterances are expected to
have low discriminator confidence scores. EER (lower is better): (Equal Error Rate) mea-
sures the error rate when false positive rate (FPR)
is equal to the false negative rate (FNR). Here,
FPR=FP/(FP+TN) and FNR=FN/(TP+FN). EER (lower is better): (Equal Error Rate) mea-
sures the error rate when false positive rate (FPR)
is equal to the false negative rate (FNR). Here,
FPR=FP/(FP+TN) and FNR=FN/(TP+FN). FPR95 (lower is better): (False Positive Rate
(FPR) at 95% True Positive Rate (TPR)) can be
interpreted as the probability that an OOD utterance
is misclassified as IND when the true positive rate
(TPR) is as high as 95%. Here, TPR=TP/(TP+FN). Autoencoder (AE) (Ryu et al., 2017): first uses
an LSTM based classifier model to train sentence
representations; then train an autoencoder on the
above sentence embeddings. OOD utterances are
expected to have high reconstruction error. Detection Error (lower is better): measures
the misclassification probability when TPR is 95%. Detection error is defined as follows: ODIN (Liang et al., 2017): we only use the
temperature scaling on logits. OOD utterances are
expected to have a low scaled confidence score. ODIN (Liang et al., 2017): we only use the
temperature scaling on logits. OOD utterances are
expected to have a low scaled confidence score. min
δ {PIND(s ≤δ)p(x ∈PIND)
+POOD(s > δ)p(x ∈POOD)} Generalized-ODIN (G-ODIN) (Hsu et al.,
2020): we fine-tune on pre-trained BERT models
with replaced last layer and only use the decom-
posed confidence. We evaluate all three variations
proposed in the paper hI, hE and hC and report
the best one. OOD utterances are expected to have
low scaled confidence score. where s is a confidence score. We follow the same
assumption that both IND and OOD examples have
an equal probability of appearing in the testing set. AUROC (higher is better): (Area under the
Receiver Operating Characteristic Curve) The
ROC curve is a graph plotting TPR against the
FPR=FP/(FP+TN) by varying a threshold. Mahalanobis (Lee et al., 2018): we only use
the feature of BERT’s last layer to compute Ma-
halanobis distance score. OOD utterances are ex-
pected to have a low scaled confidence score. 4.1
Datasets We evaluate our proposed approach on three bench-
mark SLU datasets and one in-house SLU dataset. Table 1 provides an overview of all datasets. 2447 from 2.2 to 4 with uniform interval 0.2 (we try
10 values of δ) based on sigmoid(2.2) ≈0.9 and
sigmoid(4) ≈0.982. We used δ = 3 which
gives the best performance in our experiment for
all datasets. We train each model with 3 epochs
using 4 NVIDIA Tesla V100 GPUs (16GB) for
each training. We conducted experiments on two
transformer-based models, BERT (Devlin et al.,
2019) and RoBERTa (Liu et al., 2019). fier model to train the sentence representation and
then use them to further train an autoencoder. Thus,
En-ConGAN and En-AE are not existing baselines. Note that ERAEPOG (Zheng et al., 2020) and
O-Proto (Tan et al., 2019) are not comparable since
they require additional unlabeled data and labels. We only put the ERAEPOG results on CLINC
dataset (from the original paper) for reference. fier model to train the sentence representation and
then use them to further train an autoencoder. Thus,
En-ConGAN and En-AE are not existing baselines. Note that ERAEPOG (Zheng et al., 2020) and
O-Proto (Tan et al., 2019) are not comparable since
they require additional unlabeled data and labels. We only put the ERAEPOG results on CLINC
dataset (from the original paper) for reference. 4.4.2
OOD Detection Metrics We consider the strongest baseline BERT-Linear
(the last layer is linear) fine-tuned on the pre-trained
BERT-based models (Chen et al., 2019). we follow the evaluation metrics in literature (Ryu
et al., 2018) and (Liang et al., 2017; Lee et al.,
2018). Let TP, TN, FP, and FN denote true positive,
true negative, false positive, and false negative. We
use the following OOD evaluation metrics: 4.4.1
IND Intent Classification Metrics We evaluate IND performance using the classifica-
tion accuracy metric as in literature (Liu and Lane,
2016; Wang et al., 2018; Chen et al., 2019). 4.4
Evaluation Metrics Remarks: All experiments only use IND data
for both training and validation. We use the same
hyperparameters in all datasets and validate the
generalizability of our method. 4.3.2
OOD Detection Baselines AUPR (higher is better):
(Area under the
Precision-Recall Curve (AUPR)) The PR curve is a
graph plotting the precision against recall by vary-
ing a threshold. Here, precision=TP/(TP+FP) and
recall=TP/(TP+FN). AUPR-IN and AUPR-OUT is
AUPR where IND and OOD distribution samples
are specified as positive, respectively. For ConGAN and AE, we evaluate the model
in the original paper as well as customized BERT-
based backbone models as strong baselines. Specif-
ically, we customize En-ConGAN and En-AE as
follows: En-ConGAN uses BERT sentence repre-
sentation as input; En-AE applies a BERT classi- 2448 Table 2: Comprehensive OOD Detection Results on CLINC Dataset (CLINC Train/OOD) Model
Last Layer
OOD Method
OOD Evaluation
EER(↓)
FPR95(↓)
Detection Error(↓)
AUROC(↑)
AUPR In(↑)
AUPR Out(↑)
ConGAN
-
-
78.90§
94.40§
52.04§
52.22§
82.79§
23.54§
AE
-
-
18.13§
58.50§
23.94§
87.78§
96.98§
54.12§
ERAEPOG
-
-
12.04§
23.70§
11.67§
95.83§
99.05†
83.98§
BERT
En-ConGAN
75.20§
98.72§
49.95§
22.36§
69.86§
11.27§
En-AE
8.70§
13.03§
8.47§
96.12§
98.89§
88.38§
ODIN
9.01§
16.52§
8.66§
96.24§
98.73§
87.34§
G-ODIN
8.91§
12.99§
8.40§
95.81§
98.75†
88.81§
Linear
Confidence
11.31§
21.98§
11.00§
94.96§
98.52§
84.59§
Entropy
10.33§
17.99§
10.10§
95.65§
98.73§
87.20§
Mahalanobis
8.31§
12.68§
8.02§
96.90§
99.14†
88.19§
L-Mahalanobis*
7.21
10.18
6.92
97.52
99.41
89.37
DRM*
Confidence*
8.50
12.85
7.85
96.34
98.95
87.51
Entropy*
8.31
12.53
8.14
96.67
99.01
89.68
Mahalanobis*
7.01
10.88
6.88
97.43
99.37
90.36
L-Mahalanobis*
6.70
10.12
6.62
97.77
99.46
91.55
RoBERTa
En-ConGAN
80.26§
99.34§
49.95§
15.20§
66.64§
10.58§
En-AE
8.56§
12.38§
8.29§
96.82§
99.08†
90.06§
ODIN
9.11§
15.12§
8.68§
96.11§
98.84§
88.72§
G-ODIN
8.85§
12.26§
8.53§
96.74§
99.12†
89.95§
Linear
Confidence
10.81§
22.35§
10.38§
95.23§
98.58§
86.46§
Entropy
9.31§
14.81§
8.93§
95.89§
98.73§
88.70§
Mahalanobis
8.40§
11.82§
8.13§
96.92§
99.06§
90.37§
L-Mahalanobis*
6.90
9.53
6.71
97.94
99.50
92.47
DRM*
Confidence*
8.35
11.76
8.02
97.10
99.25
90.46
Entropy*
8.29
11.51
7.86
97.17
99.27
90.69
Mahalanobis*
6.31
7.80
6.13
98.07
99.53
92.86
L-Mahalanobis*
6.11
7.63
5.98
98.16
99.56
92.96
Our best method (DRM+L-Mahalanobis) is significantly better than each baseline model (without *) with p-value < 0.01 (marked by §) and p-value < 0.05
(marked by †) using t-test. All methods with * are our proposed methods. 4.4.3
Statistical Significance We also evaluate the statistical significance be-
tween all baselines and our best result (DRM +
L-Mahalanobis) on all the above metrics. We train
each model 10 times with different PyTorch ran-
dom seeds. We report the average results and t-test
statistical significance results. Our DRM methods (marked by *) are significantly better than baseline
model on all datasets with p-value < 0.05 (marked by †) using t-test. Our DRM methods (marked by *) are significantly better than baseline
model on all datasets with p-value < 0.05 (marked by †) using t-test. backbones, DRM models are significantly better
than conventional BERT-linear classification mod-
els with p-value < 0.05. backbones, DRM models are significantly better
than conventional BERT-linear classification mod-
els with p-value < 0.05. 4.3.2
OOD Detection Baselines Table 3: IND Intent Classification Results
Model
Last Layer
Datasets
CLINC
ATIS
Snips
Movie
BERT
Linear
96.19†
97.76†
97.97†
97.26†
DRM*
96.66
98.21
98.23
97.87
RoBERTa
Linear
96.82†
97.64†
98.07†
98.07†
DRM*
97.15
98.31
98.87
98.63
Our DRM methods (marked by *) are significantly better than baseline
model on all datasets with p-value < 0.05 (marked by †) using t-test. Table 3: IND Intent Classification Results Note that EER, detection error, AUROC, and
AUPR are threshold-independent metrics. 4.5.2
OOD Detection Results Table 3 reports the IND intent classification re-
sults on each dataset finetuned using BERT and
RoBERTa pre-trained models. It is interesting to
observe that all DRM combined models consis-
tently achieve better classification accuracy with
up to 0.8% improvement (reproduced ”No joint”
row in Table 3 in (Chen et al., 2019) on Snips
dataset). This is because the domain loss forces
sigmoid(fd) close to 1 and therefore also slightly
mitigates its impact to IND classification. Thus, the
true posterior distribution of IND data is also mod-
eled more precisely. For both BERT and RoBERTa Results on CLINC Dataset: Table 2 reports the
OOD detection results on CLINC dataset. This
result covers all existing work and our enhanced
baselines. We focus on analyzing the contribution
by each of our proposed techniques, DRM and
L-Mahalanobis. The first three rows report the
performance of existing approaches based on the
original designs in their papers (ERAEPOG in grey
uses additional unlabeled data). 4.5.2
OOD Detection Results Unfortunately, we
observe that their performance is even worse than
the simple confidence-based approach via BERT 2449 Table 4: OOD Detection Results on Snips/ATIS/Movie Datasets (RoBERTa Model Finetuning)
OOD Method
OOD Evaluation
EER(↓)
FPR95(↓)
Detection Error(↓)
AUROC(↑)
AUPR In(↑)
AUPR Out(↑)
IND dataset: Snips; OOD Datasets: CLINC OOD/ATIS/Movie
En-ConGAN
54.50§/63.05§/54.22§ 99.16§/99.87§/99.10§ 42.61§/49.10§/37.32§ 39.03§/30.88§/45.64§ 37.15§/34.47§/30.03§ 51.23§/45.70§/52.59§
Confidence
9.91§/17.83§/22.22§
14.94§/47.43§/51.85§
9.18§/11.17§/19.34§
96.09§/92.03§/87.44§ 94.78§/92.65§/97.67§ 97.21§/92.29§/55.16§
Entropy
10.21§/18.05§/23.15§ 14.54§/45.04§/52.68§
9.25§/10.77§/19.58§
96.32§/92.44§/87.12§ 94.90§/92.94§/97.60§ 97.53§/92.99§/52.27§
ODIN
10.01§/16.93§/23.15§ 14.22§/39.04§/58.33§
9.43§/9.64§/23.01§
96.46§/93.81§/83.58§ 94.59§/93.99§/96.63§ 97.75§/94.53§/47.36§
G-ODIN
9.65§/15.16§/22.02§
13.31§/37.86§/55.67§
8.32§/8.55§/21.82§
97.21§/94.73§/85.60§ 95.70§/95.04§/97.73§ 98.02§/95.44§/50.38§
En-AE
4.40§/4.37§/3.59§
4.18§/3.59§/3.08†
4.25§/4.00§/3.64†
98.56§/98.12§/88.96§ 97.41§/98.92†/94.39§ 98.12§/95.34§/86.84§
Maha
3.90§/1.81/11.11§
2.66§/2.23§/5.58§
3.47§/1.36†/10.21§
98.79†/99.74†/95.61§ 97.73§/99.75†/99.22§ 99.21§/99.77†/76.61§
DRM+L-Maha*
3.00/1.79/2.78
1.95/0.00/2.78
2.63/1.16/3.16
98.90/99.79/98.53
98.15/99.79/99.76
99.24/99.80/87.02
IND dataset: ATIS; OOD Datasets: CLINC OOD/Snips/Movie
En-ConGAN
21.60§/19.74§/23.28§ 81.52§/86.33§/93.77§ 15.51§/15.54§/16.03§ 82.34§/81.79§/79.32§ 84.52§/89.35§/58.36§ 72.74§/60.20§/89.14§
Confidence
10.21§/8.52§/10.19§
20.50§/12.92§/17.59§
9.28§/8.36§/9.33§
96.99§/97.84§/96.62§ 97.19§/98.57§/99.56† 97.04§/96.99§/84.27§
Entropy
9.91§/8.84§/10.12§
21.67§/13.75§/17.59§
9.11§/8.16§/9.38§
97.06§/97.93§/96.68§ 97.25§/98.62§/99.57† 97.11§/97.14§/85.02§
ODIN
9.11§/8.36§/10.08§
21.32§/14.39§/18.52§
7.50§/6.15§/9.37§
97.16§/98.00§/96.73§ 97.39§/98.68§/99.58† 97.16§/97.18§/84.88§
G-ODIN
8.75§/8.01§/9.97§
20.87§/13.44§/17.76§
7.31§/6.02§/8.98§
97.27§/98.11§/96.85§ 97.46§/98.76§/99.59† 97.28§/97.32§/85.90§
En-AE
4.00§/2.09/3.69§
2.20†/0.00/0.35†
3.45†/1.33/1.97†
99.41†/99.83/99.63§
99.43†/99.89/98.72§
99.43†/99.74/97.93§
Maha
4.00§/3.85§/6.48§
12.13§/8.06§/11.64§
3.76§/2.94§/5.04§
99.18§/99.47§/98.72† 98.78§/99.45§/99.71§
99.46/99.49/95.45§
DRM+L-Maha*
2.70/2.09/1.85
1.30/0.32/0.00
2.55/2.01/1.23
99.48/99.70/99.78
99.51/99.82/99.97
99.47/99.50/98.22
IND dataset: Movie; OOD Datasets: CLINC OOD/ATIS/Snips
En-ConGAN
45.90§/15.12§/41.09§ 44.05§/14.35§/39.59§
22.95§/7.56§/20.55§
43.85§/57.44§/45.78§ 85.21§/88.23§/90.40§ 14.68§/17.56§/10.09§
Confidence
19.22§/16.70§/18.81§ 36.81§/47.94§/47.52§ 18.51§/15.15§/18.53§ 91.65§/91.99§/90.53§ 98.11§/98.50§/98.68§ 76.78§/67.58§/59.63§
Entropy
19.12§/17.26§/19.13§ 34.64§/44.24§/44.80§ 18.25§/16.12§/18.87§ 91.79§/92.14§/90.72§ 98.11§/98.50§/98.69§ 78.66§/70.87§/63.96§
ODIN
19.42§/18.95§/19.94§ 34.43§/39.91§/39.38§ 18.24§/18.38§/19.33§ 91.34§/91.40§/90.03§ 97.96§/98.29§/98.53§ 78.56§/71.62§/65.18§
G-ODIN
18.61§/18.23§/19.25§ 34.19§/36.42§/37.03§ 18.15§/17.27§/18.91§ 91.86§/91.97§/90.63§ 98.21§/98.34§/98.70§ 78.98§/72.07§/66.79§
En-AE
13.70§/7.28§/16.05§
43.42§/16.05§/32.29§
11.00§/4.46§/11.87§
94.57§/93.56§/92.23§ 98.91§/99.58†/99.01† 77.12§/76.13§/68.75§
Maha
3.90§/3.41†/6.11§
6.02§/2.35§/15.40§
3.72§/3.02§/6.02§
99.37§/99.43§/98.63§
99.81†/99.89/99.82†
97.82†/97.27§/91.44§
DRM+L-Maha*
3.70/3.36/4.66
2.56/1.01/4.34
3.61/2.85/4.58
99.48/99.53/99.06
99.89/99.92/99.88
97.90/97.38/93.85
In each OOD method for an IND dataset, ”/” separates the results for different OOD datasets. Our method (*) is significantly better than baseline models with p-value < 0.01 (marked by §) and p-value < 0.05 (marked by †) using t-test in most cases. Table 4: OOD Detection Results on Snips/ATIS/Movie Datasets (RoBERTa Model Finetuning) finetuning baseline (row 5). Thus, we mainly focus
on comparing our method with strong baselines
with BERT and RoBERTa models. Ablation Study on CLINC Dataset: We analyze
how our two novel components, DRM model and
L-Mahalanobis, impact the performance. Ablation Study on CLINC Dataset: We analyze
how our two novel components, DRM model and
L-Mahalanobis, impact the performance. The rows with “DRM” in “Last Layer” column
of Table 2 show the performance of DRM model. As one can see, for all OOD methods, DRM consis-
tently performs better than the conventional “Lin-
ear” last layer. Specifically, the DRM and Confi-
dence combo also outperforms its closest baseline
G-ODIN. 4.6
Qualitative Analysis We provide a quantitative analysis by visualizing
our two methods, DRM and L-Mahalanobis. 5
Conclusion This paper proposes using only IND utterances to
conduct intent classification and OOD detection for
SLU in an open-world setting. The proposed DRM
has a structure of two branches to avoid overcon-
fidence and achieves a better generalization. The
evaluation shows that our method can achieve state-
of-the-art performance on various SLU benchmark
and in-house datasets for both IND intent classi-
fication and OOD detection. In addition, thanks
to the generic of our DRM design and with the
recent extensive use of BERT on different data
modalities, our work can contribute to improving
both in-domain classification robustness and out-
of-domain detection robustness for various classifi-
cation models such as image classification, sound
classification, vision-language classifications. 4.6.2
Feature Distribution Visualization model. When taking each dataset as IND, we
use the other two mutually exclusive datasets and
CLINC OOD as OOD datasets for evaluating OOD
detection performance. As one can see in Table 4,
our method outperforms other approaches on both
Snips and movie IND datasets. For ATIS IND
dataset, En-AE for Snips OOD dataset achieves
almost perfect performance. This is because ATIS
and Snips are almost completely non-overlapping
and ATIS is well designed with carefully selected
varieties and entities in the airline travel domain. When taking Snip as IND and ATIS as OOD, it
is interesting to see that our method achieves bet-
ter performance than En-AE. This is because that
Snips contains a large number of entities such that
the reconstruction error will be lower and become
less separable than that in ATIS OOD utterances. Figure 5 visualizes the utterance representations
learned with or without DRM. The red IND data are
tightly clustered within classes (totally 150 CLINC
IND classes), while the blue OOD data spread arbi-
trarily. As one can see, the blue dots in Figure 5(b)
have less overlap with red dots, indicating the DRM
helps to learn the utterance representation to better
disentangle IND and OOD data. (a) Conventional RoBERTa
(b) DRM RoBERTa
Figure 5: t-SNE Visualization of Utterance Representa-
tions on CLINC Dataset (Red: IND, Blue: OOD) For both Snips and Movie IND datasets,
DRM+L-Mahalanobis are significantly better than
baseline methods with p-value < 0.01 in most
cases for all OOD datasets. For ATIS IND dataset,
DRM+L-Mahalanobis shows similar behavior ex-
cept En-AE since it is easier to train an autoencoder
model for ATIS IND dataset due to its carefully col-
lected clean training utterances. Figure 5: t-SNE Visualization of Utterance Representa-
tions on CLINC Dataset (Red: IND, Blue: OOD) 4.5.2
OOD Detection Results This validates the effectiveness of our
disentangled logits design in DRM based on the
mathematical analysis of overconfidence. It also
shows that our new domain loss can indeed en-
hance the model awareness that all training data is
IND. For a given OOD detection method, we find that
their combinations with DRM consistently perform
better than those with standard models. The im-
provement is at least 1-2% for all metrics against
our enhanced baselines. Among all OOD detection
approaches, our proposed L-Mahalanobis OOD de-
tection approach achieves the best performance
for both linear and DRM combined BERT and
RoBERTa models. It is not surprising to observe
that our DRM method combined with a better pre-
trained RoBERTa model achieves larger OOD de-
tection performance improvement. Note that our
customized En-AE performs much better than most
other methods since we incorporated the enhanced
reconstruction capability with pre-trained BERT
models. However, En-AE cannot utilize all BERT
layers as our proposed L-Mahalanobis method, re-
sulting in worse performance. The rows with “L-Mahalanobis” in “OOD
Method” column of Table 2 outperform other OOD
methods with the same model and last layer. Com-
pared with its closest baseline Mahalanobis, the
better performance of L-Mahalanobis validates the
usefulness of all layers’ features in various models. Results on ATIS/Snips/Movie Datasets: Since
our strong baselines on pre-trained RoBERTa
model showed better results on CLINC, we next
evaluate other results finetuned on RoBERTa The rows with “L-Mahalanobis” in “OOD
Method” column of Table 2 outperform other OOD
methods with the same model and last layer. Com-
pared with its closest baseline Mahalanobis, the
better performance of L-Mahalanobis validates the
usefulness of all layers’ features in various models. Results on ATIS/Snips/Movie Datasets: Since
our strong baselines on pre-trained RoBERTa
model showed better results on CLINC, we next
evaluate other results finetuned on RoBERTa In addition, DRM+L-Mahalanobis models are
significantly better than existing methods and en-
hanced baselines with p-value < 0.01 on most
metrics for both BERT and RoBERTa backbones. 4.5.2
OOD Detection Results 2450 (a) Conventional Confidence Score
(b) DRM Confidence Score
(c) DRM L-Mahalanobis Score
Figure 4: Histogram of Detection Scores using Various Methods (Snips IND, ATIS OOD) (We choose this
IND/OOD combination to provide the best visualization for analysis) (b) DRM Confidence Score (c) DRM L-Mahalanobis Score (a) Conventional Confidence Score (a) Conventional Confidence Score Figure 4: Histogram of Detection Scores using Various Methods (Snips IND, ATIS OOD) (We choose this
IND/OOD combination to provide the best visualization for analysis) model. When taking each dataset as IND, we
use the other two mutually exclusive datasets and
CLINC OOD as OOD datasets for evaluating OOD
detection performance. As one can see in Table 4,
our method outperforms other approaches on both
Snips and movie IND datasets. For ATIS IND
dataset, En-AE for Snips OOD dataset achieves
almost perfect performance. This is because ATIS
and Snips are almost completely non-overlapping
and ATIS is well designed with carefully selected
varieties and entities in the airline travel domain. When taking Snip as IND and ATIS as OOD, it
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https://openalex.org/W3142773937
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https://journals.vilniustech.lt/index.php/Transport/article/download/14622/10476
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English
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GRIPPING DEVICES OF INDUSTRIAL ROBOTS FOR MANIPULATING OFFSET DISH ANTENNA BILLETS AND CONTROLLING THEIR SHAPE
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Transport
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cc-by
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Notations BGD – Bernoulli gripping device;
OM – object of manipulation;
RANS – Reynolds-averaged Navier–Stokes;
SST – shear stress transport. pneumatic grippers, namely BGD, have a minimal effect
on the lifting force from the curvature of the object to be
manipulated. BGD have a number of advantages – Ozcelik, Erzin-
canli (2002) and Ozcelik et al. (2003) did research for
transportation of non-rigid objects, for transportation
of food in production and finished products Davis et al. (2008) and Petterson et al. (2010), for transportation of
skin Dini et al. (2009). f Volodymyr SAVKIV1, Roman MYKHAILYSHYN2*, Pavlo MARUSCHAK3#,
Valerii KYRYLOVYCH4, Frantisek DUCHON5, Ľuboš CHOVANEC6 Volodymyr SAVKIV1, Roman MYKHAILYSHYN2*, Pavlo MARUSCHAK3#,
Valerii KYRYLOVYCH4, Frantisek DUCHON5, Ľuboš CHOVANEC6 1, 2, 3Dept of Automation Technological Processes and Production,
Ternopil Ivan Puluj National Technical University, Ukraine
4Dept of Automation and Computer-Integrated Technologies named after prof. B. B. Samotokin,
State University “Zhytomyr Polytechnic”, Ukraine
5, 6Dept of Robotics and Artificial Intelligence, Slovak University of Technology in Bratislava, Slovak Republic 1, 2, 3Dept of Automation Technological Processes and Production,
Ternopil Ivan Puluj National Technical University, Ukraine
4Dept of Automation and Computer-Integrated Technologies named after prof. B. B. Samotokin,
State University “Zhytomyr Polytechnic”, Ukraine
5, 6Dept of Robotics and Artificial Intelligence, Slovak University of Technology in Bratislava, Slovak Republic Submitted 13 May 2020; resubmitted 4 November 2020, 30 November 2020; accepted 31 December 202 Submitted 13 May 2020; resubmitted 4 November 2020, 30 November 2020; accepted 31 December 2020 Abstract. Invention proposes an adaptive gripping device of an industrial robot, which combines functions of capturing
different-shape manipulation objects with control of deviations from the shape of these objects. The device is a T-shaped
frame with three Bernoulli grips pivotally mounted thereon and a pneumatic sensor. Analytical dependencies are presented
for determination of design parameters of adaptive gripping device and calculation of required lifting force of each of
Bernoulli Gripping Device (BGD). Formula is derived for determining its position of pneumatic sensor on frame of grip-
ping devices. In the ANSYS-CFX software environment, numerical simulation of airflow dynamics in the gap between the
cooperating BGD surfaces and the offset mirror antenna plate blank. The simulation was based on the Reynolds-Averaged
Navier–Stokes (RANS) equations of viscous gas dynamics, the Shear Stress Transport (SST) model of turbulence, and the g
model of laminar–turbulent transition. As a result of the simulation, the effect of the curvature radius of the surface of the
plates of offset mirror antennas on the BGD power characteristics was determined. Keywords: Bernoulli gripping device, object manipulation, offset antenna, nozzle, radial flow, industrial robo TRANSPORT
ISSN 1648-4142 / eISSN 1648-3480
2021 Volume 36 Issue 1: 63–74
https://doi.org/10.3846/transport.2021.14622 TRANSPORT
ISSN 1648-4142 / eISSN 1648-3480
2021 Volume 36 Issue 1: 63–74
https://doi.org/10.3846/transport.2021.14622 #Editor of the TRANSPORT – the manuscript was handled by one of the Associate Editors, who made all decisions related to the manuscript (including the choice of referees
and the ultimate decision on the revision and publishing). 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 unre-
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. *Corresponding author. E-mail: mykhailyshyn@tntu.edu.ua #Editor of the TRANSPORT – the manuscript was handled by one of the Associate Editors, who made all decisions related to the manuscrip
and the ultimate decision on the revision and publishing). Copyright © 2021 The Author(s). Published by Vilnius Gediminas Technical University Introduction Modelling of dynamics
of course of air flow in step nozzle and in a radial inter-
val between the interacting flat surfaces of BGDs and OM
is carried out. For modelling based on RANS equations
of dynamics of viscous gas, SST model of turbulence and
γ-model of laminar and turbulent transition are used. As
a result of numerical modelling in the program ANSYS-
CFX (https://www.ansys.com/products/fluids/ansys-cfx) en-
vironment operational characteristics of BGD and static
characteristic of a measuring nozzle are defined. In paper by Dini et al. (2009) proposes the use of con-
tactless grippers instead of more traditional vacuum cups
or fingered grippers. In particular, the main objective of
this investigation is the measurement of the performance
of different gripper configurations whose lifting force is
generated by a high-speed air flow passing between the
gripper and the leather ply. Li and Kagawa (2014) investigated the pressure dis-
tribution and lifting force of BGD, are measured experi-
mentally. A theoretical model of the air flow between the
gripper and the workpiece is created, based on which the
theoretical formulas for calculating the pressure distribu-
tion and lifting force are derived by Shi and Li (2016). It
is found that the outer diameter of the gripper has a major
impact on the lifting force, and its design is closely related
to the gap height and the supply mass flow rate. Then,
the relationship between the outer diameter and the lifting
force and that between the gap height and the lifting force
are discussed, based on which a method for finding the
optimal outer diameter is presented. In paper by Shi and
Li (2018) study experimentally and theoretically investi-
gates the dynamic characteristics of the BGD. i
In this paper proposed a special gripping device capa-
ble of gripping a flat metal sheet for loading into a press,
and after it is pressed to grip a blank of a plate of an offset
mirror antenna, has acquired a parabolic shape. The main
elements of the proposed special gripping device are three
Bernoulli grips pivotally mounted on a T-shaped frame. In order to expand the functional capabilities of this grip-
ping device, a pneumatic measuring device is provided in
its design. The pneumatic measurement method provides
high accuracy and absence of contact with the controlled
object, high reliability and durability of operation, ease
of automation of the control process. Introduction As a result, it was observed that the non-contact end-
effector could be applied to handle different flat materials.h f
ppfl
The purpose of paper Petterson et al. (2010) is the
increase the flexibility of robots used for handling of 3D
(food) objects handling by the development and evalua-
tion of a novel 3D BGD. A new gripper technology have
been designed and evaluated. A deformable surface have
been used to enable individual product handling. The lift
force generated and the force exerted on the product dur-
ing gripping is measured using a material tester instru-
ment. Various products are tested with the gripper. An
experimental/theoretical approach is used to explain the
results. A deformable surface can be used to generate a
lift force using the Bernoulli principle on 3D objects. Us-
ing a small forming a significant increase in the lift force
generated is recorded. Increasing the forming further was
shown to have little or even negative effects. The forces
exerted on the product during forming was measured
to be sufficiently low to avoid product damage. The im-
provement of functional and structural integration of the
control system components by their integration into the
mechatronic module is considered in a paper by Aulin
et al. (2019). Gasdynamic analysis of the BGD interaction with the
surface of flat objects with displacement of the center of
mass carried out by Savkiv et al. (2018a) and Maruschak
et al. (2019). Also the papers by Savkiv et al. (2019b, 2017b,
2020a); Mykhailyshyn et al. (2019) deals with the topical
issue of reducing energy consumption for transportation
of industrial objects. The energy efficiency of the process
of OM with the use of the orientation optimization meth-
od while gripping with the help of different methods has
been studied. The economic efficiency of the use of the
optimal orientation of BGD while transporting the OM in
comparison to the transportation without re-orientation
has been proved. Influence of parameters of a gripping
system on power expenses of the industrial robot during
transportation is investigated Mykhailyshyn et al. (2018b).h The advantages of application in the transport and
loading systems of gripping devices with the integrated
functions of control of parameters of objects of transpor-
tation were substantiated in paper by Savkiv et al. (2020b). Modelling of BGDs with a possibility of dimensional
check and weight OM is offered. Introduction When automating handling operations using industrial
robots, the tasks of manipulating articles that change
shape during machining often arise. Most often, this prob-
lem is solved by cyclic automatic replacement of gripping
devices, or as Shameli et al. (2007) use magnetic levitation,
which generally reduces the productivity of the technolog-
ical operation. However, there are gripping devices that al-
low you to gripping objects with different active surfaces. Such gripping devices will reduce the time of transport
operations, by using one handling system to grip and hold
the part throughout the production cycle. In particular, A non-contact end-effector was applied by Ozcelik
et al. (2003) to lift three different materials, which have
different physical properties. These materials are mica (as
rigid material), carton (as semi-rigid material) and non-
rigid material (woven fabric). This end-effector operates
on the principle of generating a high-speed air flow be-
tween nozzles and the specimen surface thereby creating
a vacuum, which levitates the materials with no mechani-
cal contact. In this paper, the handling results of these 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 unre-
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://cr
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. V. Savkiv et al. Gripping devices of industrial robots for manipulating offset dish antenna billets ... 64 developed. The method of optimization of BGD orienta-
tion when performing transport operations on a rectilin-
ear and arc trajectory is provided by Savkiv et al. (2017a,
2017c, 2018b). Influence of force of front resistance of Q1,
Q2 on the minimum necessary lifting force is investigated
by Mykhailyshyn et al. (2018a). The description of experi-
mental installation and the analysis of the received experi-
mental results on application of a method of optimization
of BGD orientation is described by Mykhailyshyn et al. (2017). materials are compared with each other. The changes in
the physical behaviour of lifting materials were observed
during the experimental work. The effect of the various
air flow rates on the non-contact handling clearance gap
between the nozzle and the materials were also investigat-
ed. Introduction This measuring de-
vice allows to perform operational dimensional control of
stamped blanks of offset mirror antennas, and to establish
deviations from the required geometry. It is most of all investigated and introduced on pro-
duction BGD with cylindrical or circular nozzle and vor-
tex grippers. For the purpose of minimization of energy
consumption of BGD when performing handling opera-
tions by authors of the paper, the method of optimization
of gripper orientation in the course of manipulation was Transport, 2021, 36(1): 63–74 65 1. Development of the structure and principle
of operation of the gripping system pression spring 14 between the body 15 of the BGD and
T-shaped frame 4, the axle 5 together with the body 15 is
pressed into the extreme left position of the slot 6 (Figure
1c, 1d). However, the compression spring 16 comprises
the housing 15 in its extreme, counter clockwise inclined
position defined by the stop 17.tf The structure and cross-sections of the individual mecha-
nisms of the device for gripping and controlling the shape
of the plates of offset antennas are shown in Figure 1, and
the diagram of changing the shape of the antenna blank
during stamping in the mold in Figure 2. Gripping device
consists of three similar BGD 1, 2, 3 (Figure 1a, 1b, 1c)
hinged on T-shaped frame 4. BGD have the possibility of
mutual horizontal displacement by means of pins 5 rig-
idly connected to their upper part, which are aligned with
horizontal slots 6 (Figure 1d) of T-shaped frame. i
After forming the blank of the offset dish antenna 8 in
the mold (Figure 1b), the industrial robot introduces a T-
shaped frame 4 into the space above the blank. Positioning
of the frame is performed so that the first performance 10
of each of the Bernoulli grippers is set to the lower point
of the radial slot of the antenna blank. The robot then
performs a further downward shift of the frame, in which
each of the Bernoulli grippers is returned to a correspond-
ing angle until contact of the second projection 11 with the
antenna blank is reached. At the same time axis 5, is not
movably established in building 15 is displaced concerning
a groove 6 to the right, the spring 14 and 16 is unclenched
at necessary sizes, providing continuity of a kinematic
chain from performances 10, 11 to T-shaped frames 4.t g
p
Disk 7 is mounted in each BGD, in axial part of which
nozzle 9 is made along normal line to object of processing 8. Two performances 10 and 11 are arranged on different
sides of disc 7. The first step 10 in each BGD is arranged
around the periphery of the processing object 8, and the
second step 11 of each BGD is arranged in series towards
the center of the processed object 8. 2. Methodology Using the technique described in paper by Savkiv et al. (2019a), it is possible to determine the pressure distribu-
tion in the radial space for the case of interaction of the
BGD with the flat surface of the transport object. How-
ever, it is quite difficult to analytically evaluate the power
interaction of the BGD with the fragment of the parabolic
surface of the offset antenna. For this purpose, it is better
to use numerical simulation of airflow dynamics in the
chamber, nozzles of BGD, and in the interval between its
flat surface and the parabolic surface of the antenna blank.l Figure 2. Scheme for changing the shape of offset mirror
antenna blank during stamping in a mold at values of distance between interacting surfaces
/2
c
n
h
r
<
,
flow experiences greatest narrowing. At the point of great-
est narrowing, at excessive supply pressures of the pickup
by compressed air more than 30 kPa, the flow reaches the
critical speed equal to the speed of sound for these condi-
tions. As a result of further increase of radial flow area,
its supersonic speed increases, and static pressure on OM
surface decreases to value of lower atmospheric value. At
some distance from the nozzle center there is sharp brak-
ing of supersonic flow, with its subsequent transition to
subsonic flow, which is accompanied by formation of pres-
sure jump. As a result of further expansion, the subsonic
flow rate drops and the static pressure in the gap smoothly
increases to atmospheric air pressure pa. The effect of the
vacuum on the surface of the antenna blank results in an
lifting force towards the end portion of the disc 7. l
p
To simulate the airflow between the active surfaces of
the BGD and the antenna blank, we will use RANS equa-
tions (Snegirjov 2008; Garbaruk et al. 2016). At the same
time, the influence of mass forces is neglected. 2. Methodology Then the
system of basic equations will be as follows: »» equation of continuity of a stream: (
)
0;
j
j
V
t
x
∂r⋅
∂r +
=
∂
∂
(1) (1) »» impulse equation: »» impulse equation: ;
ij
i
i
j
j
j
V
V
V
t
x
x
∂t
∂
∂
r
+ r⋅
=
∂
∂
∂
(2) t
Control device 18 (Figure 1e) of this special grip is
made in the form of pneumatic size control sensor. It is
mounted on the T-shaped frame 4 normal to the offset
antenna blank at point 23 (Figure 2), which as a result
of the antenna manufacturing process, receives the maxi-
mum total strain Δ. (2) »» energy equation: (
);
j
j
ij
i
j
j
j
q
E
E
V
V
t
x
x
x
∂
∂
∂
∂
r
+ r⋅
= −
+
t ⋅
∂
∂
∂
∂
(3) (3) Thus, the T-frame is positioned in a well-defined posi-
tion with respect to the antenna blank. The pneumatic size
control device 18 attached to this frame is pre-adjusted to
zero count on the offset mirror antenna reference blank
with predetermined size parameters. The pneumatic device
measures the deviations of the highest point 23 (Figure 2)
of the surface profile of the antenna blank relative to the
adjusted zero display. This deviation is measured as fol-
lows. Compressed air is supplied to measuring chamber 21
under constant inlet pressure through connector 19 (Fig-
ure 1e). The air flowing through the measuring nozzle 22
depending on the gap between the end of the measur-
ing nozzle 22 and the surface of the offset mirror antenna
blank changes its flow characteristics, thereby changing
the air pressure in the measuring chamber 21. The value of
this pressure is measured by a sensor, which is connected
through a union 20, and after digitization of the measured
data, deviation of the profile of the antenna blank is deter-
mined. 1. Development of the structure and principle
of operation of the gripping system The axis of each pin 5
is perpendicular to the plane passing through the axis of
the respective nozzle and the center of the processing ob-
ject 8.i After all Bernoulli grippers are installed in the speci-
fied position, compressed air is supplied to chamber 12
through channel 13 (Figure 1c). Air flows through nozzle 9
into clearance between lower part of disk 7 and surface
of antenna blank. At that, at radius equal to nozzle radius rn, In the initial state (until the first step 10 enters the
radius slot of the blank offset dish antenna 8) with com- Figure 1. Design and cross-sections of individual mechanisms of the device for gripping
and controlling the shape of plates of offset mirror antennas
8
3
2
4
1
A
B
D
D
3
8
4
11
2
10
A-A
13
15
12
4
16
17
11
10
9
7
8
C
C
hc
B-B
5
6
14
4
4
6
15
C-C
19
20
4
18
21
22
D-D
b)
e)
d)
c)
a)
B 3
8
4
11
2
10
A-A
b)
d) 8
3
2
4
1
A
B
D
D
a)
B b) A-A 19
20
4
18
21
22
D-D
e) 13
15
12
4
16
17
11
10
9
7
8
C
C
hc
B-B
c) e) 5
6
14
4
4
6
15
C-C
d) d) B-B B-B c) 12 6 8 Figure 1. Design and cross-sections of individual mechanisms of the device for gripping
and controlling the shape of plates of offset mirror antennas V. Savkiv et al. Gripping devices of industrial robots for manipulating offset dish antenna billets ... V. Savkiv et al. Gripping devices of industrial robots for manipulating offset dish antenna billets ... 66 23
R
D
Figure 2. Scheme for changing the shape of offset mirror
antenna blank during stamping in a mold 2. Methodology This makes it possible for further transportation of
parts to carry out rose backing according to permissible
deviation of the outermost point of the profile.t »» ideal gas state equation: »» ideal gas state equation: ,
p
R T
r =
⋅
(4) ,
p
R T
r =
⋅ (4) where: i, j – indices, take values of 1, 2, 3; r – air density;
t – time; x – coordinate; V – vector of air velocity; tij –
stress tensor; E – the total energy of air; q – a heat flux
density vector taking into account heat transfer by heat
conduction and diffusion; R – gas constant; T – the abso-
lute air temperature; p – absolute gas pressure.h The system of Equations (1)–(4) must be supplement-
ed by a turbulence model. The SST is selected to describe
turbulence (Menter 1994). The SST model of turbulence
is supplemented by transition models Menter et al. (2002,
2006), which allow to predict the position of the lami-
nar–turbulent transition. These models are based on the
interleavability coefficient g, whose value at a given point
is determined by the ratio of the flow of which a turbulent
mode is observed to the total observation time.fi Sufficient accuracy of calculations is given by the lami-
nar–turbulent transition model with another differential
equation for the value g (Menter et al. 2015): i
After evaluating the variations in the shape of the an-
tenna blank, the supply of air to the measuring chamber
21 is stopped, and the industrial robot moves the final link
upwards. In the following, the industrial robot provides
movement of the antenna blank to the unloading zone and
stops the supply of compressed air to each of the BGD. Under the action of springs 14, 16 Bernoulli grippers re-
turn to initial position. 2. Methodology 2006) (for tur-
bulent flows of compressible gases moving at sound and
supersonic speeds), graphs of pressure distribution in the
interval between the flat end of the BGD and the spherical
surface were built for different values of radius R of this
surface at the distance from the nozzle to the spherical
surface hc = 0.2 mm – distance between the interacting
surfaces of the OM and the gripping device (Figure 4). (6) (7) The graphs in Figure 4 show that as the curvature ra-
dius of the surface of the OM increases, the size of the
supersonic vacuum zone on that surface increases, but the
amount of vacuum itself decreases. The radius of curva-
ture of the surface of the OM has minimal influence on
the value of the vacuum value in the subsonic zone. where: k – kinetic turbulent energy; w – the specific speed
of dissipation of kinetic energy of turbulence; Pk, Dk –
original generation and dissipation of the SST model, re-
spectively;
lim
k
P
– the additional part, which provides the
correct gain of turbulent viscosity in transitional area at
very low level of turbulent viscosity of the running stream;
nt – turbulent kinematic viscosity of gas; sk, a – empirical
constants of model; for details and constants see papers by
Menter (1994) and Menter et al. (2015). Figure 3. Airflow model limits
Figure 4. Plots of pressure p distribution in the interval
between the flat end of the BGD and the spherical surface r
for different values of radius R of this surface
–100000
0
100000
200000
300000
400000
0
5
10
15
20
25
30
r [mm]
p(r) [Pa]
R = 0.8 m
R = 1.2 m
R = 2 m
Inlet:
p = 400 kPa,
T = 5% for k and w
Walls:
smooth wall,
no-slip wall
Outlet:
p = 0 kPa,
T = 5% for k and w
u
u Figure 3. Airflow model limits
Inlet:
p = 400 kPa,
T = 5% for k and w
Walls:
smooth wall,
no-slip wall
Outlet:
p = 0 kPa,
T = 5% for k and w
u
u 2. Methodology (
)
(
)
j
j
p V
p
t
x
∂
⋅
g
∂
+
⋅
⋅g
=
∂
∂
t
j
j
P
E
x
x
g
g
g
m
∂
∂g
−
+
m +
∂
s
∂
,
(5) (5) where: Pg, Eg – generative and dissipation members of where: Pg, Eg – generative and dissipation members of Transport, 2021, 36(1): 63–74 67 managing directors of laminar and turbulent transition,
respectively; m – molecular dynamic viscosity of gas; mt –
turbulent dynamic viscosity of gas; sg = 1.0 – model con-
stant; g – interleavability coefficient. hi in the design area is 2.4…3.2 million. Mesh nodes are
combined into three-dimensional elements (tetrahedron
and prisms). The total number of volume elements of the
grid is 4.6…6.3 million. The total number of tetrahedron
is 2.3…3.4 million. In the simulation, it was set: air is the
ideal gas; thermodynamic process is adiabatic. The bound-
ary conditions for the airflow model are shown in Fig-
ure 3. The simulation was carried out for BGD with nozzle
radius rn = 3 mm and outer radius rg = 30 mm. The modified equations of the SST turbulence for
this g-model laminar–turbulent transition are as follows
(Menter et al. 2015): (
)
(
)
lim
j
k
k
j
k
V
k
P
P
t
x
∂
∂
r⋅
+
r⋅
=
+
−
∂
⋅
∂
(
)
k
k
t
j
j
k
D
x
x
∂
∂
+
m + s
m
∂
⋅
∂
;
(6)
(
)
(
)
j
j
V
t
x
∂
∂
r⋅w +
r⋅
∂
∂
⋅w =
k
t
P
D
C d
v
w
w
a⋅
−
+
⋅
+
(
)
t
j
j
x
x
w
∂
∂w
m + s
m
∂
∂
⋅
;
(7) g
Based on the results of the simulation using the deci-
sive sonicTurbFoam module (Menter et al. 4. Determination of the power characteristics
of the developed handling system By designing all forces on the z-axis,
we define the condition of antenna blank content: 1
1
2
cos
2
cos
lF
N
m g
⋅
⋅
a > ⋅
⋅
b +
⋅,
(8) (8) where: Fl – lifting force of gripping device; a – angle be-
tween the vertical and the axis of symmetry of the Ber-
noulli gripper; N1 – reaction, acting at the point of contact
of the tab 10 (Figure 1) with the surface of the antenna
blank; b1 – angle between the vertical and the normal
to the tangent to the parabolic surface of the antenna
blank at the point of contact of the tab 10 with its surface;
m – antenna mass; g – acceleration of gravity.h Analysis of these graphs shows that as the radius of
curvature of the surface of the OM increases from 0.6 to
1.8 m, the lifting force of this object by Bernoulli gripper
increases by 13...37%, and if the radius increases from 1.8
to 3 m only by 2...4%.ht Thus, after determining the required load capacity of
BGD 2 and 3 (Figure 1a), appropriate correction is made
for increase of their power characteristics depending on
diameter of offset antenna blank. The lifting force of the
BGD is generally increased by increasing the supply pres-
sure. The main requirement to rational design of BGD is
existence of smooth active surface provides smooth nar-
rowing and expansion of airflow and has no performanc-
es, which would interfere stream (Wagner et al. 2008). Providing smooth entrance and exit from nozzle reduces
losses of energy of air flow and reduces jog force in zone
opposite to nozzle (Savkiv et al. 2020a). The reaction N1 can be determined from the BGD
equilibrium condition. Taking into account the condition
of equilibrium of moments of forces relative to the axis of
rotation of the gripping device and the specified condition
N2 = 0, we will find: (
)
1
1
1
sin
cos
s
g
F a
m
g b
N
l
⋅−
⋅
⋅⋅
a
=
⋅
a −b
,
(9) (9) where: Fs is the elastic force of the spring 16 (Figure 1c);
mg – the mass of the gripping device with stops; l1 is the Figure 6. 3. Analysis of the power
characteristics of the BGD The lifting force by the BGD of the object of transporta-
tion is influenced by the following factors: the pressure of
supply of the gripping device with compressed air; geo-
metrical parameters of a nozzle; geometric parameters of
the surface of the transport object with which the gripping
device interacts. In order to assess the effect of curvature radius (con-
vexities) of fragments of parabolic surface of offset anten-
na blank on force characteristics of BGDs interacting with
these fragments, it is possible to replace parabolic surface
with equivalent spherical surface. Such change gives a
slight error, since within the fragment of the parabolic
surface with which the BGD interacts, the radius of cur-
vature of this surface varies by less than 4%. The radius of
curvature of the spherical surface shall be changed within
0.6...3.0 m, which corresponds to the dimensions of offset
antennas 0.85...2.15 m.l Figure 3. Airflow model limits Figure 3. Airflow model limits –100000
0
100000
200000
300000
400000
0
5
10
15
20
25
30
r [mm]
p(r) [Pa]
R = 0.8 m
R = 1.2 m
R = 2 m Numerical simulation of the dynamics of air flow in
the chamber, nozzles of the BGD and in the interval be-
tween its flat surface and the spherical surface of the ob-
ject of transportation was performed in the environment
of computational hydro-gas-dynamics ANSYS-CFX using
RANS and g-model turbulence. For modelling in this soft-
ware environment, an unstructured finite difference grid
is built in the simulation area. The total number of nodes Figure 4. Plots of pressure p distribution in the interval
between the flat end of the BGD and the spherical surface r
for different values of radius R of this surface V. Savkiv et al. Gripping devices of industrial robots for manipulating offset dish antenna billets .. 68 Figure 5. 3. Analysis of the power
characteristics of the BGD Dependence of the load capacity Fl of the BGD
on the radius R of curvature of the surface of the content
of the transportation object
15
20
25
30
35
0.6
1
1.4
1.8
2.2
2.6
3
h = 0.25 mm
h = 0.3 mm
F [N]
l
R [m] 15
20
25
30
35
0.6
1
1.4
1.8
2.2
2.6
3
h = 0.25 mm
h = 0.3 mm
F [N]
l
R [m] By means of additional rotation of the BGD, if there
are changes in the shape of the surface of the object to
be gripped, it is possible to provide the necessary gap be-
tween the lower surface of the disc 7 (Figure 1) and the
surface of the object, at which the lifting force of each of
the grippers would be maximum. 4. Determination of the power characteristics
of the developed handling system As can be seen from Figure 1a, 1c in static mode, the offset
dish antenna blank is sufficient to be held due to the force
of only BGD 2 and 3. At the same time BGD 1 is neces-
sary to ensure location of offset mirror antenna blank at
its gripping, as well as to prevent antenna rocking at accel-
eration (braking) of final link operation. Refer to Figure 6
for design diagrams for determining the required lifting
force of Bernoulli grippers.ht Figure 5. Dependence of the load capacity Fl of the BGD
on the radius R of curvature of the surface of the content
of the transportation object In order to determine the effect of the curvature radius
of the surface of the OM on the carrying capacity of the
Bernoulli gripping, numerical integration of the pressure
distribution data in the radial gap. As a result, it is revealed
that at the radius of curvature of the surface of the OM
more than 3 m the lifting force of the BGD reaches the
maximum value corresponding to the lifting force of flat
objects. It has also been found that when gripping an offset
antenna blank with dimensions of 0.85…1.05 m (the mini-
mum radius of curvature of the parabolic surface is 0.6 m),
the load capacity of the BGD will decrease by 39% com-
pared to the lifting force of the flat object. The results of
the calculations for the two values of the distance between
the BGD and the spherical surface are shown in Figure 5. The minimum lifting force of the BGD 2 and 3 (Figure 6)
required to contain the stamped antenna blank can be
determined from the equilibrium condition of all forces
(Figure 6b) acting on this blank at N2 = 0 (N2 – normal
reaction at the point of contact of the friction element of
the handling system). 4. Determination of the power characteristics
of the developed handling system (
)
0
2
d
gr
l
a
r
F
p
p
r r
= ⋅π⋅
−
⋅
∫
,
(14) (14) g pp
Elastic force of spring 16: Elastic force of spring 16: (
)
1
1
180
sF
k
a
π
=
⋅d + ⋅a −a ⋅
°
,
(10)ffi where: pa – atmospheric pressure.ht (10) The lifting force of the handling system Fgs can be de-
termined from Equation (12) by expressing from it the
component m g
⋅: where: k – spring stiffness coefficient; d1 is the value of
preliminary compression of spring 16, which provides
equilibrium state of BGD at frame positioning above off-
set antenna blank (Figure 6a); a1 is angle of inclination of
BGD axis, which corresponds to its equilibrium state in
initial position.h component m g
⋅: 1
2
2
2
cos
gs
l
eq
F
F
eq
= ⋅
⋅
a −
,
(15) (15) where: here:
(
)
1
1
1
2
2
180
eq
k a
a
π
= ⋅⋅⋅d + ⋅a −a
⋅
−
°
here:
(
)
1
1
1
2
2
180
eq
k a
a
π
= ⋅⋅⋅d + ⋅a −a
⋅
−
°
2
2
sin
g
m
g b
⋅
⋅
⋅⋅
a ;
(
)
2
1
2
1
cos
eq
l
=
⋅
a −b
. The initial angle of inclination of the BGD axis is
set by the previous compression of the spring 16 so that
(
)
1
2
5
7 °
a = a +
…
. Angle value a2 depends on geometric
parameters of offset antenna blank and design parameters
of gripping system. The relationship between the value of
the spring 16 and its coefficient of elasticity can be deter-
mined from Equations (9) and (10), taking into account
when positioning the BGD frame above the offset antenna
blank (Figure 6) N1 and N2 are zero. Then: 2
2
sin
g
m
g b
⋅
⋅
⋅⋅
a ;
(
)
2
1
2
1
cos
eq
l
=
⋅
a −b
. 4. Determination of the power characteristics
of the developed handling system To analyse the effect of the supply pressure of the Ber-
noulli grippers on the lifting force of the gripping system,
the simulation was performed in the following sequence: »» using the method presented in parts 2 and 3 of the
paper determined the pressure distributions pr on
the surface of offset antennas (diameters 0.85 and
1.2 m) at supply pressures of Bernoulli grippers 100,
200, 300, and 400 kPa; 1
1
sin
g
m
g b
k a
⋅
⋅⋅
a
d =
⋅
. (11)i (11) On the basis of Equations (8)–(10), find the required
lifting force of BGD 2 and 3 by substituting a = a2: »» according to Equation (14), plot the dependence of
Fl (p) (Figure 7a); »» according to Equation (14), plot the dependence of
Fl (p) (Figure 7a); 1
2
2
1
cos
2
l
eq
m g
F
eq
⋅
>
⋅
+
a
,
(12)
where:
(
)
1
1
1
2
180
eq
k a
a
π
=
⋅⋅d + ⋅a −a
⋅
−
°
2
sin
g
m
g b
⋅
⋅⋅
a ;
(
)
2
1
2
1
cos
eq
l
=
⋅
a −b . 1
2
2
1
cos
2
l
eq
m g
F
eq
⋅
>
⋅
+
a
,
(12) (12) »» according to Equation (15), plot the dependence of
Fgs (p) (Figure 7b). »» according to Equation (15), plot the dependence of
Fgs (p) (Figure 7b). where:
(
)
1
1
1
2
180
eq
k a
a
π
=
⋅⋅d + ⋅a −a
⋅
−
°
2
sin
g
m
g b
⋅
⋅⋅
a ;
(
)
2
1
2
1
cos
eq
l
=
⋅
a −b . 4. Determination of the power characteristics
of the developed handling system Diagrams for determination of required lifting force of BGD: a – positioning of frame with gripping devices
above offset antenna blank; b – grip of offset antenna blank; c – gripping of flat blank
m Чg
g
mЧg
z
bЧsin(a)
a
Fs
Fl
a = a1
a = a2
Fl
Fs
b2
z
x
x
mЧg
a
b1
N1
N2
l2
l1
Fl
Fs
N1
N2
x
mЧg
z
b)
a)
c) m Чg
g
mЧg
z
bЧsin(a)
a
Fs
Fl
a = a1
x
a) a = a2
Fl
Fs
b2
z
x
mЧg
a
b1
N1
N2
l2
l1
b) Fl
Fs
N1
N2
x
mЧg
z
c) a) c) Figure 6. Diagrams for determination of required lifting force of BGD: a – positioning of frame with gripping devices
above offset antenna blank; b – grip of offset antenna blank; c – gripping of flat blank Transport, 2021, 36(1): 63–74 Transport, 2021, 36(1): 63–74 69 In general, the lifting force by gripping the workpiece
can be determined by integrating the absolute pressure
distribution pr between their cooperating surfaces: distance from the grip axis to the center of the tab 10 (Fig-
ure 6b); a – the distance between the axis of the Bernoulli
gripper and the axis of the applied spring forces; b – the
distance between the axis of rotation and the center of
mass of the Bernoulli gripper. 5. Determining the location of the blank
antenna shape control sensor Formation of elliptic cutting from a paraboloid of rotation
is explained by the scheme is submitted in Figure 7. The
surface of the offset mirror antenna blank can be represent-
ed as being separate from the paraboloid of rotation P as a
result of its crossing by a cutting plane perpendicular to the
plane XOY and returned at an angle q with respect to the
axis OX. The cut plane passes through a point on the pa-
raboloid surface with coordinates (x0, y0, 0) of the original
XYZ system. An elliptical cut in the cutting plane will be
formed with a large axis of the ellipse L, a small axis of the
ellipse 2 B
⋅, and the maximum depth H of the offset anten-
na blank will correspond to the coordinate
max
(
, )
,
0
pH
x
H
. The maximum elliptical clipping width is
max
pB
x
.hi Figure 8. Diagram of elliptical cut-out from parabolic of rotation Figure 8. Diagram of elliptical cut-out from parabolic of rotation h
p
pp g
max
pB
The specified features of the shape of the working sur-
face of the offset antenna blank define the structural re-
quirements for gripping and the scheme of its dimensional
characteristics control. To provide offset mirror antenna
with greater spatial stiffness along its contour, radius bend
R is formed (Figure 2). At the same time, depending on
the technological conditions of forming the offset antenna
blank, there may be a different ratio of plastic and elas-
tic components of deformations for each of the following
blanks. This can lead to different changes in the shape of
D caused by instability of the process modes, significantly
complicates the task of positioning the invaders on the
variable spatial surface of the offset mirror antenna blank. The use of a contactless pneumatic dimensional control
sensor in this case is most advantageous, since the probe
of the contact measuring tip under the influence of the
measuring force can damage the controlled surface.f to the XOY plane and is returned at an angle q about the
OX axis. 4. Determination of the power characteristics
of the developed handling system Gripping devices of industrial robots for manipulating offset dish antenna billets ... Figure 8. Diagram of elliptical cut-out from parabolic of rotation
cutting plane
yP
y
xpHmax
p
H
xP
y0
x0
q
x
0
0
offset cutting
xpBmax
xP
B
L Data analysis of Figure 7 shows that the lifting force of
the handling system for offset antennas is only 3…7% low-
er than the total force of interaction of Bernoulli grippers
with the surface of these antennas. Prior to that, the lifting
force of the handling system based on Bernoulli grippers
is decrease with an increased diameter of offset antennas. 5. Determining the location of the blank
antenna shape control sensor The rotation of the axes in the XOY plane by the
angle q and the transfer of the origin of the reference sys-
tem in the x0, y0 forms the parabola equation in the new
system xp, yp as: 0
0
cos
sin ;
sin
cos ,
p
p
p
p
x
x
x
y
y
y
x
y
=
+
⋅
q −
⋅
q
=
+
⋅
q −
⋅
q
(17) (17) or subject to Equation (16): or subject to Equation (16): 2
0
0
cos
sin ;
4
sin
cos ,
p
p
p
p
y
x
x
y
F
y
y
x
y
=
+
⋅
q −
⋅
q
⋅
=
+
⋅
q +
⋅
q
. (18) (18) From the first equation of the system (Equation (18)),
the positive value: (
)
0
2
cos
sin
p
p
y
F
x
x
y
= ⋅
⋅
+
⋅
q −
⋅
q . (19) (19) As the practice of forming offset antennas shows, if the
existing shape deviations, they increase sequentially from
the periphery of the workpiece to the point of maximum
depth of profile H (Figure 8), that is, point 23 (Figure 2). It is at this point that it is necessary to mount a pneumatic
sensor to control the distance from the T-frame to the sur-
face of the workpiece in order to obtain the most complete
information about the distortion of the test profile of the
workpiece. The coordinates of this point are determined
according to the procedure below.h From the second equation of the system (Equation
(18)): (18)): 0
sin
cos
p
p
y
y
x
y
=
+
⋅
q +
⋅
q. (20) (20) Equating the right parts of Equations (19) and (20)
we get: (
)
0
2
cos
sin
p
p
F x
x
y
⋅
+
⋅
q −
⋅
q =
0
sin
cos
p
p
y
x
y
+
⋅
q +
⋅
q . 4. Determination of the power characteristics
of the developed handling system g
Modelling was performed for the following param-
eters: eters: »» gripping system: rn = 3 mm, rg = 30 mm, hc = »» gripping system: rn = 3 mm, rg = 30 mm, hc =
0.25 mm, l1 = 120 mm, a = 25 mm, b = 40 mm, k =
150 N/m, mg = 0.3 kg;f Find the required lifting force of BGD 2 and 3 for flat
blank content (Figure 6c) by substitution a = 0, b1 = 0 in
Equation (12): g
»» an offset antenna with a diameter of 0.85 m: a2 =
14°, d1 = 10 mm; g
»» an offset antenna with a diameter of 0.85 m: a2 =
14°, d1 = 10 mm; »» an antenna with a diameter of 1.2 m: a2 = 18°, d1 =
12.3 mm. 1
1
1
180
2
l
m g
k a
F
a
l
⋅
⋅
π
>
⋅d + ⋅a
+
°
. (13) (13) Figure 7. Dependences of the lifting force of offset antennas on the supply pressure: a – one Bernoulli gripper; b – gripping system
0
5
10
15
20
25
0
100
200
300
400
p [kPa]
antenna with a diameter of 0.85 m
antenna with a diameter of 1.2 m
0
10
20
30
40
50
0
100
200
300
400
F [N]
l
F [N]
gs
p [kPa]
a)
b) 0
5
10
15
20
25
0
100
200
300
400
p [kP ]
F [N]
l
a) 0
10
20
30
40
50
0
100
200
300
400
F [N]
gs
p [kPa]
b) antenna with a diameter of 0.85 m
antenna with a diameter of 1.2 m antenna with a diameter of 0.85 m antenna with a diameter of 1.2 m m
antenna with a diameter of 1.2 m Figure 7. Dependences of the lifting force of offset antennas on the supply pressure: a – one Bernoulli gripper; b – gripping system Figure 7. Dependences of the lifting force of offset antennas on the supply pressure: a – one Bernoulli gripper; b – gripping system Figure 7. Dependences of the lifting force of offset antennas on the supply pressure: a – one Bernoulli gripper V. Savkiv et al. Gripping devices of industrial robots for manipulating offset dish antenna billets ... 70 V. Savkiv et al. 5. Determining the location of the blank
antenna shape control sensor (23) pressure sensor
d0
d2
d1
p where: where: 2
cos
A =
q ; 2
cos
A =
q ; 0
4
sin
2
cos
B
F
y
=
⋅
⋅
q + ⋅
⋅
q + 2
sin
cos
p
x
⋅
⋅
q⋅
q;
2
0
0
2
sin
p
C
y
y
x
=
+ ⋅
⋅
q +
2
2
0
sin
4
p
x
F x
⋅
q −⋅
⋅
−
4
cos
p
F x
⋅
⋅
⋅
q. (25) (25) Positive solution of Equation (24): Positive solution of Equation (24): 2
4
2
p
B
B
A C
y
A
−
+
−⋅
⋅
=
⋅
. (26) (26) Figure 9. Construction of the antenna shape control sensor:
d0 – diameter of the active nozzle; d1 – inner diameter of the
measuring nozzle; d2 – outer diameter of the measuring nozzle The maximum yp will be when: d
0
d
p
p
y
x
= . d
0
d
p
p
y
x
= . (27) d
0
d
p
p
y
x
= . (27) (27) Walls:
smooth wall,
no-slip wall
0
0.0035
0.007 [m]
0.00175
0.00525
Inlet:
p = 400 kPa,
T = 5% for k and w
u
Outlet:
p = 0 kPa,
T = 5% for k and w
u
Figure 10. Airflow model limits of the antenna
shape control sensor
0
0.0025
0.005 [m]
0.00125
0.00375
5
3.857Ч10
5
3.576Ч10
5
3.295Ч10
5
3.015Ч10
5
2.734Ч10
5
2.453Ч10
5
2.173Ч10
5
1.892Ч10
5
1.611Ч10
5
1.331Ч10
5
1.050Ч10
4
7.696Ч10
4
4.889Ч10
4
2.083Ч10
3
–7.238Ч10
4
–3.530Ч10
4
–6.337Ч10
[Pa]
Pressure
contour 2
Figure 11. Distribution of pressure along the cross-section
of the measuring nozzle Walls:
smooth wall,
no-slip wall
0
0.0035
0.007 [m]
0.00175
0.00525
Inlet:
p = 400 kPa,
T = 5% for k and w
u
Outlet:
p = 0 kPa,
T = 5% for k and w
u
Figure 10. Airflow model limits of the antenna
shape control sensor Walls:
smooth wall,
no-slip wall
0
0.0035
0.007 [m]
0.00175
0.00525
Inlet:
p = 400 kPa,
T = 5% for k and w
u
Outlet:
p = 0 kPa,
T = 5% for k and w
u
Figure 10. 5. Determining the location of the blank
antenna shape control sensor Airflow model limits of the antenna
shape control sensor On the basis of Equation (25), taking into account
Equation (24), we determine the value of xp, which cor-
responds to the maximum value of yp: 4
2
1 sin
sin
cos
p
x
F
−
q
=
⋅
−
q⋅
q
0
0
sin
cos
y
x
⋅
q −
⋅
q. (28) (28) That is, according to the Equation (28), it is possible
to find the installation coordinate of the pneumatic sen-
sor
max
pH
x
(Figure 8), at which the depth of the cut-out
H will be maximum. 5. Determining the location of the blank
antenna shape control sensor (21) (21) The XOY plane of the original coordinate system (Fig-
ure 8) forms a parabola at the intersection with parabolic P: After converting the Equation (21): After converting the Equation (21): (
2
2
0
cos
4
sin
2
cos
p
y
F
y
⋅
q +
⋅
⋅
q + ⋅
⋅
q +
)
2
sin
cos
p
p
x
y
⋅
⋅
q⋅
q ⋅
+
(
2
2
2
0
0
2
sin
sin
p
p
y
y
x
x
+ ⋅
⋅
⋅
q +
⋅
q −
)
0
4
4
cos
0
p
F x
F x
⋅
⋅
−⋅
⋅
⋅
q = . 5. Determining the location of the blank
antenna shape control sensor (22) 2
4
y
x
F
=
⋅
,
(16) 2
4
y
x
F
=
⋅
, (16) where: F is the focus of the parabola.f To form an offset cut, a cut plane is drawn through
the x0, y0 coordinate point, which extends perpendicular (22) Transport, 2021, 36(1): 63–74 71 Reduce Equation (22) to the form:
(
2
2
0
cos
4
sin
2
cos
p
y
F
y
⋅
q +
⋅
⋅
q + ⋅
⋅
q +
)
2
sin
cos
p
p
x
y
⋅
⋅
q⋅
q ⋅
+
(
2
2
2
0
0
2
sin
sin
p
p
y
y
x
x
+ ⋅
⋅
⋅
q +
⋅
q −
)
0
4
4
cos
0
p
F x
F x
⋅
⋅
−⋅
⋅
⋅
q = ;
(23)
2
0
p
p
A y
B y
C
⋅
+
⋅
+
= ,
(24) Reduce Equation (22) to the form:
(
2
2
0
cos
4
sin
2
cos
p
y
F
y
⋅
q +
⋅
⋅
q + ⋅
⋅
q +
)
2
sin
cos
p
p
x
y
⋅
⋅
q⋅
q ⋅
+
(
2
2
2
0
0
2
sin
sin
p
p
y
y
x
x
+ ⋅
⋅
⋅
q +
⋅
q −
)
0
4
4
cos
0
p
F x
F x
⋅
⋅
−⋅
⋅
⋅
q = ;
(23)
2
0
p
p
A y
B y
C
⋅
+
⋅
+
= ,
(24) Reduce Equation (22) to the form:
(
2
2
0
cos
4
sin
2
cos
p
y
F
y
⋅
q +
⋅
⋅
q + ⋅
⋅
q +
)
2
sin
cos
p
p
x
y
⋅
⋅
q⋅
q ⋅
+
(
2
2
2
0
0
2
sin
sin
p
p
y
y
x
x
+ ⋅
⋅
⋅
q +
⋅
q −
)
0
4
4
cos
0
p
F x
F x
⋅
⋅
−⋅
⋅
⋅
q = ;
(23)
2
0
p
p
A y
B y
C
⋅
+
⋅
+
= ,
(24) Reduce Equation (22) to the form: measuring nozzle are shown in Figure 11 (p = 400 kPa,
hc = 0.2 mm).f Also, for different values of the radial interval hc, the
value of the pressure in the channel was determined using
a pressure sensor (Figure 9) and the static characteristics
of the measuring nozzle were constructed, which are pre-
sented in Figure 12. 6. Analysis of the characteristics
of the antenna shape control sensor Figure 10. Airflow model limits of the antenna
shape control sensor Figure 10. Airflow model limits of the antenna
shape control sensor Due to the instability of the modes of the technologi-
cal process of stamping offset antenna blanks and in the
course of wear of molds there is a deviation from the ref-
erence shape of the antenna. For this type of antenna, this
may shift the focal length and impair satellite signal recep-
tion. Therefore, it is proposed to use a pneumatic sensor to
monitor deviations from the shape of Figure 9. 0
0.0025
0.005 [m]
0.00125
0.00375
5
3.857Ч10
5
3.576Ч10
5
3.295Ч10
5
3.015Ч10
5
2.734Ч10
5
2.453Ч10
5
2.173Ч10
5
1.892Ч10
5
1.611Ч10
5
1.331Ч10
5
1.050Ч10
4
7.696Ч10
4
4.889Ч10
4
2.083Ч10
3
–7.238Ч10
4
–3.530Ч10
4
–6.337Ч10
[Pa]
Pressure
contour 2
Figure 11. Distribution of pressure along the cross-section
of the measuring nozzle Pressure
contour 2 Modelling of measuring characteristics of the pneu-
matic sensor was carried out according to the method
presented in Section 2 of the paper. The total number of
nodes in the design area is 0.05…0.3 million, the total
number of volume elements of the grid is 0.3…1.3 mil-
lion. During the simulation, the following was specified:
air is an ideal gas; thermodynamic process is adiabatic. The boundary conditions for the airflow model are pre-
sented in Figure 10. Simulations were performed for a pneumatic sensor
with the following parameters: d0 = 0.5 mm, d1 = 1.6 mm,
d2 = 4 mm, hc = 0…0.4 mm, p = 400…600 kPa. The results
of the pressure distribution along the cross-section of the Figure 11. Distribution of pressure along the cross-section
of the measuring nozzle V. Savkiv et al. Gripping devices of industrial robots for manipulating offset dish antenna billets ... 72 Figure 12. The dependence of the pressure ps in the measuring
channel of the pneumatic sensor on the distance hc
to the surface of the antenna
–100
0
100
200
300
400
500
600
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
h [mm]
c
p = 400 kPa
p = 600 kPa
p [kPa]
s –100
0
100
200
300
400
500
600
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
h [mm]
c
p = 400 kPa
p = 600 kPa
p [kPa]
s objects. Acknowledgements The results achieved were created in the framework of the
projects APVV-17-0214, APVV-16-0006, and “Robotické
pracovisko pre inteligentné zváranie maloobjemovej výroby
(IZVAR)”, code ITMS2014+: 313012P386, co-financed by
the European Regional Development Fund. At the same time control of position of the most in-
formative point of the surface of the captured object
makes it possible to assess its dimensional characteristics
in a comprehensive manner. hf The economic effect of the implementation of this
device is ensured by the possibility to use it for different
processing objects in different form, allows to increase the
productivity of the technological process. Savings are also
achieved by reducing scrap losses by being able to respond
quickly to control results and adjust the process accord-
ingly. Conclusions A BGDs have been proven to allow both flat and objects
with convex surfaces to be contained. This allows them to
be effectively used as elements of special gripping devices
of industrial robots for loading flat metal sheets into the
press and unloading stamped plates of offset mirror anten-
nas of parabolic shape. Funding The authors disclosed receipt of the following financial
support for the research, authorship of this paper. This work was supported by APVV-17-0214,
APVV-16-0006, and “Robotické pracovisko pre inteli-
gentné zváranie maloobjemovej výroby (IZVAR)”, code
ITMS2014+: 313012P386, co-financed by the European
Regional Development Fund. 6. Analysis of the characteristics
of the antenna shape control sensor It has also been found that when gripping a blank
of an offset antenna with dimensions of 0.85…1.05 m,
the load capacity of the BGD will decrease by 39% com-
pared to the lifting force of the flat object.t tl
It is established that the lifting force of the handling
system based on Bernoulli grippers decreases with the
increasing diameter of offset antennas. The reduction of
lifting force occurs only by 3…7% of the total force of
interaction of Bernoulli grippers with the antenna surface. It has been found that the pneumatic sensor for moni-
toring deviations in the shape of the offset antenna blank
must be located at the point of its maximum profile depth. The equation is proposed for the determination of its posi-
tion of a pneumatic sensor.h It is established that the lifting force of the handling
system based on Bernoulli grippers decreases with the
increasing diameter of offset antennas. The reduction of
lifting force occurs only by 3…7% of the total force of
interaction of Bernoulli grippers with the antenna surface. It has been found that the pneumatic sensor for moni-
toring deviations in the shape of the offset antenna blank
must be located at the point of its maximum profile depth. The equation is proposed for the determination of its posi-
tion of a pneumatic sensor.h Figure 12. The dependence of the pressure ps in the measuring
channel of the pneumatic sensor on the distance hc
to the surface of the antenna The reduction of errors in measuring the deviation of
the shape of the offset antenna is achieved due to the op-
eration of the pneumatic sensor on the linear section of
its measuring characteristics. This measuring range corre-
sponds to the distance between the pneumatic sensor and
the surface of the offset antenna 0.05…0.2 mm. In order to reduce errors in measuring the deviations
of the shape of the offset antenna, it is necessary to mount
the pneumatic sensor so that the working range of meas-
uring deviations corresponds to the linear section of the
dependence ps(hc), ie the range hc = 0.05…0.2 mm. In
practice, to ensure high accuracy in measuring the devia-
tions of the shape of the offset antenna, it is necessary to
optimize the design parameters of the pneumatic sensor
and use a higher level of compressed air supply pressure. Author contributions Roman Mykhailyshyn, Volodymyr Savkiv, Valerii Kyrylovy-
ch and Frantisek Duchon conceived the research and were
responsible for developing and developing data analysis. Roman Mykhailyshyn, Volodymyr Savkiv, Pavlo Mar-
uschak, Valerii Kyrylovych and Frantisek Duchon were re-
sponsible for data collection and analysis. As a result of the numerical simulation of the dynam-
ics of the airflow in the gap between the interacting BGD
surfaces and the plate blank of the offset mirror antenna,
it has been found that as the radius of curvature of the
surface of the OM increases, the size of the supersonic
vacuum zone on this surface increases, but the amount
of vacuum itself decreases. It has also been found that
the value of the vacuum value in the subsonic zone has
a minimal effect on the radius of curvature of the surface
of the OM. Roman Mykhailyshyn, Ľuboš Chovanec and Frantisek
Duchon were responsible for modelling in the ANSYS-
CFX software environment. t
Roman Mykhailyshyn, Pavlo Maruschak and Volody-
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respect to the research, authorship, and/or publication of
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The Evolutionary Relevance of Social Learning and Transmission in Non-Social Arthropods with a Focus on Oviposition-Related Behaviors
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Citation: Nieberding, C.M.;
Marcantonio, M.; Voda, R.; Enriquez,
T.; Visser, B. The Evolutionary
Relevance of Social Learning and
Transmission in Non-Social
Arthropods with a Focus on
Oviposition-Related Behaviors. Genes
2021, 12, 1466. https://doi.org/
10.3390/genes12101466 Citation: Nieberding, C.M.;
Marcantonio, M.; Voda, R.; Enriquez,
T.; Visser, B. The Evolutionary
Relevance of Social Learning and
Transmission in Non-Social
Arthropods with a Focus on
Oviposition-Related Behaviors. Genes
2021, 12, 1466. https://doi.org/
10.3390/genes12101466 Keywords: behavioral plasticity; communication; culture; Drosophila; fitness; herbivores; oviposition
site selection; natural selection The Evolutionary Relevance of Social Learning and
Transmission in Non-Social Arthropods with a Focus on
Oviposition-Related Behaviors eberding 1,*, Matteo Marcantonio 1, Raluca Voda 1, Thomas Enriquez 2 and Bertanne Visser 2 1
Evolutionary Ecology and Genetics Group, Earth and Life Institute, UCLouvain, 1348 Louvain-la-Neuve,
Belgium; matteo.marcantonio@uclouvain.be (M.M.); raluca.voda@uclouvain.be (R.V.)
2
Evolution and Ecophysiology Group, Earth and Life Institute, UCLouvain, 1348 Louvain-la-Neuve, Belgium; 1
Evolutionary Ecology and Genetics Group, Earth and Life Institute, UCLouvain, 1348 Louvain-la-Neuve,
Belgium; matteo.marcantonio@uclouvain.be (M.M.); raluca.voda@uclouvain.be (R.V.)
2
Evolution and Ecophysiology Group, Earth and Life Institute, UCLouvain, 1348 Louvain-la-Neuve, Belgium;
thomas.enriquez@uclouvain.be (T.E.); bertanne.visser@uclouvain.be (B.V.)
*
Correspondence: caroline.nieberding@uclouvain.be q
*
Correspondence: caroline.nieberding@uclouvain.be Abstract: Research on social learning has centered around vertebrates, but evidence is accumulating
that small-brained, non-social arthropods also learn from others. Social learning can lead to social
inheritance when socially acquired behaviors are transmitted to subsequent generations. Using ovipo-
sition site selection, a critical behavior for most arthropods, as an example, we first highlight the
complementarities between social and classical genetic inheritance. We then discuss the relevance
of studying social learning and transmission in non-social arthropods and document known cases
in the literature, including examples of social learning from con- and hetero-specifics. We further
highlight under which conditions social learning can be adaptive or not. We conclude that non-social
arthropods and the study of oviposition behavior offer unparalleled opportunities to unravel the
importance of social learning and inheritance for animal evolution. genes
G C A T
T A C G
G C A T genes
G C A T
T A C G
G C A T genes
G C A T
T A C G
G C A T genes
G C A T
T A C G
G C A T genes Citation: Nieberding, C.M.;
Marcantonio, M.; Voda, R.; Enriquez,
T.; Visser, B. The Evolutionary
Relevance of Social Learning and
Transmission in Non-Social
Arthropods with a Focus on
Oviposition-Related Behaviors. Genes
2021, 12, 1466. https://doi.org/
10.3390/genes12101466 1. Introduction In response to the social cue,
fly 1 changes its behavior, e.g., the female D. melanogaster reduces oviposition (fewer eggs are laid). The behavioral change proves
that the cue is perceived. Step II Social learning: Fly 1 has learned about the social cue and is now experienced, meaning that
the behavioral adjustment persists in time even when the social cue is no longer present, e.g., D. melanogaster females continue
laying fewer eggs even when the wasp has left the patch. Step III Transmission: The socially learned behavior is taken over by
naive fly 2 from experienced fly 1 (i.e., through visual and olfactory cues) that then changes its behavior. Step IV Transmission
across generations: The socially learned behavior spreads throughout the population and over subsequent generations, e.g.,
other Drosophila females (including those belonging to other species) perceive the behavioral change of individuals 1 or 2 and
also reduce their egg numbers (based on [22]). For social inheritance, naive flies belonging to the next generation should acquire
behaviors from experienced flies exhibiting socially learned behaviors. This remains to be tested explicitly in the example of
social learning of wasp threats in Drosophila. Of note, social inheritance can produce culture, based on additional criteria for
transmission of socially learned behaviors as described in [23]. In an intricate study by Danchin et al. [23], the authors use the fly D. melanogaster to
show that social inheritance (producing basic traditions or culture) can arise and spread
throughout subsequent generations. Female D. melanogaster made similar mate choice decisions
as the female fly they observed earlier when offered a choice between males with contrasting
phenotypes (colored pink or green) themselves. Transmission of color-based mate preference
also occurred when younger females observed older females, meaning that the acquired
preference could spread to a potential future generation as a tradition (i.e., step 4 in Figure 1). The authors further showed that long-term memory was involved, that mate preferences can
be transferred repeatedly over time, and that conformism was involved (i.e., taking over the
most common behavior), leading to a stable, cultural, mate choice preference in the population. This study provides a rare example of social inheritance in non-social insects (but see [24] that
consider D. melanogaster as moderately social; and [22,25,26] provide evidence for transmission
of socially learned behaviors, step 3 in Figure 1). 1. Introduction Step I Perception of social cues: Fly 1 perceives a social cue, e.g., the presence
of a parasitic wasp that can parasitize and kill the larvae of Drosophila melanogaster (based on [21]). In response to the social cue,
fly 1 changes its behavior, e.g., the female D. melanogaster reduces oviposition (fewer eggs are laid). The behavioral change proves
that the cue is perceived. Step II Social learning: Fly 1 has learned about the social cue and is now experienced, meaning that
the behavioral adjustment persists in time even when the social cue is no longer present, e.g., D. melanogaster females continue
laying fewer eggs even when the wasp has left the patch. Step III Transmission: The socially learned behavior is taken over by
naive fly 2 from experienced fly 1 (i.e., through visual and olfactory cues) that then changes its behavior. Step IV Transmission
across generations: The socially learned behavior spreads throughout the population and over subsequent generations, e.g.,
other Drosophila females (including those belonging to other species) perceive the behavioral change of individuals 1 or 2 and
also reduce their egg numbers (based on [22]). For social inheritance, naive flies belonging to the next generation should acquire
behaviors from experienced flies exhibiting socially learned behaviors. This remains to be tested explicitly in the example of
social learning of wasp threats in Drosophila. Of note, social inheritance can produce culture, based on additional criteria for
transmission of socially learned behaviors as described in [23]. Figure 1. The steps involved in social inheritance. Step I Perception of social cues: Fly 1 perceives a social cue, e.g., the presence
of a parasitic wasp that can parasitize and kill the larvae of Drosophila melanogaster (based on [21]). In response to the social cue
fly 1 changes its behavior, e.g., the female D. melanogaster reduces oviposition (fewer eggs are laid). The behavioral change proves
that the cue is perceived. Step II Social learning: Fly 1 has learned about the social cue and is now experienced, meaning that
the behavioral adjustment persists in time even when the social cue is no longer present, e.g., D. melanogaster females continue Figure 1. The steps involved in social inheritance. Step I Perception of social cues: Fly 1 perceives a social cue, e.g., the presence
of a parasitic wasp that can parasitize and kill the larvae of Drosophila melanogaster (based on [21]). 1. Introduction The emergence and spread of novel behaviors through social learning, or “learning from
others”, has been documented in a wide variety of animals, mainly in social vertebrates [1–5]. In
recent years, social learning has been demonstrated to act as the “second inheritance system”, called
“social inheritance”, that functions in parallel with classical genetic inheritance in a number of social
vertebrates in the wild. Social inheritance entails the perception of behaviors performed by others
that are subsequently taken over (e.g., by imitation, imprinting or teaching) and spread throughout
a population and subsequent generations [6–9] (see Figure 1 depicting the steps leading to social
inheritance). Aside from examples in humans, remarkable evidence for cultural evolution includes
the transmission of tool use in apes and song communication in birds and whales [8,10–14]. Academic Editors: Luigi Viggiano
and Renè Massimiliano Marsano Received: 31 July 2021
Accepted: 21 September 2021
Published: 22 September 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. p
g
Social vertebrates have been at the forefront of research on social learning, but studies using
small-brained and short-lived social invertebrates are increasing in number. In an exceptional
experiment with Bombus terrestris bumblebees, Alem et al. [15] showed that some individuals
can innovate by acquiring a non-natural, novel behavior for feeding: string pulling. Once
demonstrator individuals (previously trained to pull a string to reach a sugar source) were
observed by unexperienced individuals, these bees learned how to perform string pulling
themselves. The authors further showed that string pulling behavior could spread from a single
experienced individual (i.e., that perceived a social cue leading to a behavioral change) to other
bees, even when the original demonstrator was no longer present (completing step 1 to 4 that
demonstrate social inheritance as depicted in Figure 1; [15,16]). For invertebrates, most work has
been done with social insects and recent findings support the idea that insects have the cognitive
abilities necessary for transmission of socially learned behaviors [17–20]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/genes Genes 2021, 12, 1466. https://doi.org/10.3390/genes12101466 2 of 14 Genes 2021, 12, 1466 Figure 1. The steps involved in social inheritance. 2. Genetics, Epigenetics and Social Inheritance in the Context of
Oviposition Site Selection There are two non-mutually exclusive mechanisms by which socially learned behav-
iors can be transmitted to successive generations in a population. In his review, Whiten [7]
puts forth the parallels between genetic and social inheritance, where the former encom-
passes genetic changes that spread throughout populations, and the latter pertains to the
spread of socially learned behaviors over generations [8]. Genetic or epigenetic inheritance
is based on DNA, RNA or protein materials present in the parental germ cells that are
passed to the offspring when zygotes are formed. Social learning is transmitted inde-
pendently from the germ line material through perception and acquisition of behaviors
between individuals belonging to successive, yet overlapping generations. Genetic and
social inheritance can thus function alone or interact and act simultaneously ([45–48]; see
Figure 2 using oviposition site selection as an example). There is evidence that most behaviors and behavioral variation between individu-
als have some genetic basis [49–53]. For example, several candidate loci were identified
and associated with phenotypic variation for memorizing locations in the fly Drosophila
melanogaster [54]. The identification of candidate loci paves the way for finding the genetic
basis of complex behavioral traits, including spatial exploration ability and memory reten-
tion of spatial location (e.g., of suitable resources, including host plants for oviposition). Genetic variants with higher learning capacity and memory retention may thus become
more numerous in successive generations, when there is positive selection for oviposition
site selection (Figure 2). There is further evidence that learning ability itself has a genetic
basis and that there is genetic variation in learning ability between individuals in various
invertebrate, non-social taxa (reviewed in [54–58]). One gene whose allelic variation and
expression is associated with differential learning rate and memory retention is the foraging
gene (“for”), a pleiotropic gene that produces a cyclic GMP-dependent protein kinase (PKG),
a protein involved in many regulatory functions, including energy homeostasis [59–62]. Although the exact function of for in learning (and social learning) remains to be under-
stood [63], the existence of genetic variation for learning ability suggests that genetically
“better” learners can proportionally increase in subsequent generations, for example if
social learning of oviposition site selection from con-specifics is locally adaptive. Behaviors can also be transmitted epigenetically from parents to offspring, as was
found for multiple behaviors and species [64–66]. 1. Introduction While the potential fitness advantages of
mate-copying are clear [27,28], pink and green males do not occur in nature, meaning that
there is no ecological relevance and adaptive value of the artificial cue used in this study [29]. g
p
y
Social inheritance may play an important role in the evolution of non-social arthro-
pods. In this perspective, we discuss relevant examples of social learning in the context of
oviposition and related behaviors to illustrate the taxonomic diversity of observations in
non-social arthropods. We also highlight why studying non-social arthropods is both rele-
vant and timely. While learning of foraging, mating, host finding and other behaviors have
been discussed elsewhere [30–34], here we focus on the social transmission of oviposition
site selection. Oviposition site selection is a behavioral trait of key ecological significance
for the relationship between organisms and their habitat, as the decision on where to lay
eggs can have massive consequences for fitness and demography ([35] and references
therein). This is particularly true for herbivorous arthropods with limited mobility as
juveniles, because the egg-laying site is often also the offspring’s food source. Oviposition
is a critical behavior with which colonization of new suitable habitats is initiated [35]. We
start our perspective by illustrating the complementarities between genetic and social
heredity using the hypothetical example of oviposition site selection. Next, we show Genes 2021, 12, 1466 3 of 14 3 of 14 that social learning related to oviposition is reported by an increasing number of studies
(Table 1), adding to the ample evidence for non-social learning (i.e., learning solely from
previous experience, or “autonomous” learning) for oviposition in wasps, flies, moths and
butterflies (e.g., [36–44]). We then extend our discussion to cases where social learning
of oviposition-related behaviors occurs not only from interactions between con-specifics,
but also from hetero-specifics. Finally, we are paying particular attention to the evidence
for, and quantification of, the adaptive value of social learning using existing empirical
evidence for fitness effects. 2. Genetics, Epigenetics and Social Inheritance in the Context of
Oviposition Site Selection C: The increasing ability of individuals within a population to learn and remember the spatial location of resources,
such as host plants for oviposition, can be due to selection of (epi)genetic variants of the adaptive behavior, including
learning rate and memory retention, or due to social transmission of the spatial location of resources from older to younger
individuals leading to social inheritance. The accumulation of advantageous modifications of behavior in populations
across generations may produce differential local adaptation between populations in socially learned traits, based on local
environmental conditions and geography in much the same way as local adaptation through genetic differentiation does. Figure 2. (Epi)genetic and social inheritance for oviposition site selection can affect the colonization of new suitable
habitats with better host plant resources. A: Variation between individuals in oviposition site selection on host plants can
be due either to (epi)genetic variation or variation in social learning skills. Social learning can lead to the colonization
of new suitable habitats by naive individuals, for example by following experienced individuals towards a new habitat
patch. Here, social learning is based on imitation and can occur through horizontal, oblique or (more rarely so) vertical
transmission. Individuals not relying on social learning from conspecifics have a lower probability of finding new suitable
habitats for oviposition. B: More adaptive behavioral variants for finding a new suitable habitat for oviposition can be
transmitted through genetic or (epi)genetic variants (1). Transmission of social learning ability from parents to offspring
can be genetically based or (epi)genetically transmitted. In addition, social learners outperform individuals not using
social cues to learn about resource distribution in their environment (2). Social inheritance allows younger individuals to
locate new habitats based on social information provided by older conspecifics. When there is no (epi)genetic basis for
exploration, and learning and social learning does not occur, individuals have a lower probability of colonizing new habitats
(3). C: The increasing ability of individuals within a population to learn and remember the spatial location of resources,
such as host plants for oviposition, can be due to selection of (epi)genetic variants of the adaptive behavior, including
learning rate and memory retention, or due to social transmission of the spatial location of resources from older to younger
individuals leading to social inheritance. 2. Genetics, Epigenetics and Social Inheritance in the Context of
Oviposition Site Selection When there is no (epi)genetic basis for
exploration, and learning and social learning does not occur, individuals have a lower probability of colonizing new habitats
(3). C: The increasing ability of individuals within a population to learn and remember the spatial location of resources,
such as host plants for oviposition, can be due to selection of (epi)genetic variants of the adaptive behavior, including
learning rate and memory retention, or due to social transmission of the spatial location of resources from older to younger
individuals leading to social inheritance. The accumulation of advantageous modifications of behavior in populations
across generations may produce differential local adaptation between populations in socially learned traits, based on local
environmental conditions and geography in much the same way as local adaptation through genetic differentiation does. Figure 2. (Epi)genetic and social inheritance for oviposition site selection can affect the colonization of new suitable
habitats with better host plant resources. A: Variation between individuals in oviposition site selection on host plants can
be due either to (epi)genetic variation or variation in social learning skills. Social learning can lead to the colonization
of new suitable habitats by naive individuals, for example by following experienced individuals towards a new habitat
patch. Here, social learning is based on imitation and can occur through horizontal, oblique or (more rarely so) vertical
transmission. Individuals not relying on social learning from conspecifics have a lower probability of finding new suitable
habitats for oviposition. B: More adaptive behavioral variants for finding a new suitable habitat for oviposition can be
transmitted through genetic or (epi)genetic variants (1). Transmission of social learning ability from parents to offspring
can be genetically based or (epi)genetically transmitted. In addition, social learners outperform individuals not using
social cues to learn about resource distribution in their environment (2). Social inheritance allows younger individuals to
locate new habitats based on social information provided by older conspecifics. When there is no (epi)genetic basis for
exploration, and learning and social learning does not occur, individuals have a lower probability of colonizing new habitats
(3). 2. Genetics, Epigenetics and Social Inheritance in the Context of
Oviposition Site Selection For example, mice exposed to a neutral
fruity odor while receiving a mild electric shock adopt a startle behavior later in life while
only experiencing the odor, a behavior that is subsequently passed on to their children and
grandchildren when sensing the odor without ever experiencing the shock [67,68]. These
results pointed to the fixation of epigenetic variation affecting the expression of olfactory
genes [67,69–71]. There is, to the best of our knowledge, no evidence yet for epigenetic
transmission of spatial localization and memory of suitable resources, as depicted in our
example of Figure 2, nor for other behaviors typically related to oviposition site selection in
arthropods, such as transmission of preference for novel specific host plant species across
generations [68,72]. It will be important to tease apart the contribution of the genome,
epigenome, and social inheritance (described below), to understand how insects track
and potentially adapt to rarefying suitable habitats through oviposition site selection
behavior [73]. 4 of 14 Genes 2021, 12, 1466 pi)genetic and social inheritance for oviposition site selection can affect the colonization of new suitab
better host plant resources. A: Variation between individuals in oviposition site selection on host plants ca Figure 2. (Epi)genetic and social inheritance for oviposition site selection can affect the colonization of new suitable
habitats with better host plant resources. A: Variation between individuals in oviposition site selection on host plants can
be due either to (epi)genetic variation or variation in social learning skills. Social learning can lead to the colonization
of new suitable habitats by naive individuals, for example by following experienced individuals towards a new habitat
patch. Here, social learning is based on imitation and can occur through horizontal, oblique or (more rarely so) vertical
transmission. Individuals not relying on social learning from conspecifics have a lower probability of finding new suitable
habitats for oviposition. B: More adaptive behavioral variants for finding a new suitable habitat for oviposition can be
transmitted through genetic or (epi)genetic variants (1). Transmission of social learning ability from parents to offspring
can be genetically based or (epi)genetically transmitted. In addition, social learners outperform individuals not using
social cues to learn about resource distribution in their environment (2). Social inheritance allows younger individuals to
locate new habitats based on social information provided by older conspecifics. 2. Genetics, Epigenetics and Social Inheritance in the Context of
Oviposition Site Selection The accumulation of advantageous modifications of behavior in populations
across generations may produce differential local adaptation between populations in socially learned traits, based on local
environmental conditions and geography in much the same way as local adaptation through genetic differentiation does. Genes 2021, 12, 1466 5 of 14 The second main inheritance mechanism, social inheritance, is based on social learning of
behaviors between interacting individuals, such that learned behaviors can also be propagated
without a genetic or epigenetic material basis across generations (Figures 1 and 2). Social
inheritance has so far mainly been observed in social vertebrates and more recently in social
insects (e.g., [15]) and non-social insects (e.g., Drosophila; [23]). Social learning can increase local
adaptation of individuals relying on socially acquired information by increasing their chance of
finding a resource, or reducing the time or energetic cost these individuals need for finding and
remembering the location of a resource, such as host plants for oviposition in a new suitable
habitat (i.e., oviposition site selection; Figure 2A,B). Social learners may thus have overall
quicker and/or more access to suitable resources for survival and reproduction compared to
conspecifics that are not using or remembering social information. This, in turn, may lead
to increased reliance on social information across generations (Figure 2C), whether socially
acquired traits are transmitted over longer evolutionary times and multiple generations by
culture or not. Learning the location of a suitable plant for oviposition from a skilled con-specific
may represent an important evolutionary advantage compared to non-social learning of
host plant location. This is because non-social learners can be in a coevolutionary arms
race (i.e., Red Queen dynamics) with their host plants, given that plants are under strong
selection to avoid larval feeding using elusive traits for herbivorous arthropods (e.g., a
similar shape and color as non-host plants, and distinct morphologies such as “butterfly
egg mimicry” or apostatic selection) [35,74,75]. Social learners can thus avoid having to
“reinvent the wheel” when it comes to finding suitable host plants by following, copying or
imitating others. Two key aspects of social inheritance now need to be examined and tested
both in the laboratory and in the field. First, it will be important to quantify to what extent
social inheritance occurs throughout the diversity of evolving life, compared to genetic
inheritance (all living species have DNA or RNA and cell division), including in non-social
animals. 2. Genetics, Epigenetics and Social Inheritance in the Context of
Oviposition Site Selection Second, quantifying the adaptive value of social learning is of central importance
(as depicted in steps A and B of Figure 2), whether socially acquired traits are transmitted
over longer evolutionary times, or not. 4. Social Learning of Oviposition-Related Behavior from Con- and Hetero-Specifics Research on social learning in non-social organisms is becoming a burgeoning field and
progressively more evidence is being put forward. We focus on evidence for social learning
involved in oviposition behavior (Table 1; but see [30–33] for social learning of foraging, mating,
and other behaviors). The first step to show evidence of social learning is that a behavior is
modified in response to the perception of a social cue (step 1 in Figure 1). As a large number
of studies document the existence of step 1 in various non-social arthropods, we did not
include these studies in Table 1 (e.g., [35,83–93]). Historically, most studies on oviposition-
related behaviors have focused on parasitoid wasps (Hymenoptera) as model systems, where
oviposition takes place in or on the body of another arthropod [94]. These studies were
reviewed elsewhere [34] and we only cite a few representative case studies in Table 1. Many
wasps use previous experiences with a hetero-specific (i.e., the host) during development or as
adults as a social cue leading to a marked change in oviposition behavior compared to naive
individuals (Table 1). Table 1 summarizes the evidence of 11 key studies focusing on social
learning across 4 taxonomic orders within Arthropoda: the insect orders Hymenoptera (wasps),
Diptera (flies), and Coleoptera (beetles) and the arachnid order Trombidiformes (mites). We
thus see that modification of oviposition in response to earlier experience of social cues occurs
in diverse arthropod orders and we expect many other non-social arthropods to use social
learning, with a potential for social transmission and inheritance. Evidence for social learning of oviposition-related behaviors from con-specifics has
been particularly well-documented in Drosophila flies (Table 1), where a typical experiment
entails comparing fruit substrate preference for oviposition of flies with or without an
occasion to observe “trained” congeners displaying a strong preference for a specific
oviposition substrate. Training to develop a preference for a specific oviposition substrate
(i.e., strawberry) is obtained by associating another substrate (i.e., banana) to an oviposition
deterrent, such as quinine. Flies then develop a preference for another, simultaneously
available, substrate (i.e., strawberry). Adult female flies further learn to interpret and use a
wide variety of cues from con-specifics at different life stages when choosing an oviposition
site. 3. Relevance of Social Inheritance in Non-Social Arthropods Socially acquired behaviors cause social inheritance only if they are transmitted over
multiple generations. It is now timely to examine the extent of the transmission of socially
acquired behaviors as a second inheritance system in nature (step 4 in Figure 1, Figure 2C). Small-brained, non-social invertebrates are particularly relevant to study, because they make
up at least half of the species diversity on Earth [76,77]. The transmission of socially acquired
behaviors across generations requires that individuals of different life stages or age groups
live in contact with each other (Figure 1) [78]. For social inheritance to occur, generations
must therefore be overlapping. This is indeed the case for eusocial species (i.e., with a clear
reproductive division) that have overlapping generations by definition, but many non-social
insects also have overlapping generations [78]. Furthermore, several insect taxa have a social
population structure allowing the transmission of socially acquired behaviors over generations,
through maternal, paternal and biparental care [79–81]. Maternal and biparental care takes the
form of egg and/or offspring guarding, defense, nidification, and/or feeding facilitation or
progressive provisioning and underpins the single most widespread form of sociality found in
“non-eusocial” insects. These behaviors have been reported for >40 insect families belonging to
12 orders, as well as several non-insect arthropod groups, such as spiders, scorpions, opiliones,
mites, chilopodes, and amphipod crustaceans [79]. Moreover, in a diverse array of mainly
hemimetabolous arthropods, including treehoppers, true bugs, thrips, cockroaches and social
spiders [82], mixed supercolonies of adults and immatures are found. While historically
social inheritance has not actively been looked for in most insect taxa to date, the social
structure of many insect species provides opportunities for transmission and inheritance of
socially acquired behaviors far beyond the few documented cases in well-known, emblematic,
social insects. Genes 2021, 12, 1466 6 of 14 4. Social Learning of Oviposition-Related Behavior from Con- and Hetero-Specifics This study is exceptional, because
observations and experiments were conducted in the field using wild bees [95]. The value of social information from hetero-specifics has also been studied in Drosophila. Particularly noteworthy is the flow of social information in the genus Drosophila related to
the presence of a parasitoid observed by Kacsoh and co-authors [22]. The divergence in
social cues that evolved between different species led to the formation of species-specific
communication patterns (referred to as “dialects”). The magnitude of divergence in species-
specific communication patterns was found to be correlated with the phylogenetic distance
between species. Kacsoh et al. [22] exploited this system to test whether the degree of
hetero-specific social information transfer between Drosophila species was related to their
relative phylogenetic distance, hypothesizing that phylogenetically close species are more
successful in sharing social information. Similar to earlier experiments by Kacsoh et al. [21]
(Figure 1), Drosophila females were presented with visual cues of parasitic wasps that
led to a reduction in the number of eggs laid. When the experienced fly belonged to a
different species, Kacsoh et al. [22] observed the same decrease in number of eggs laid. While closely related Drosophila species were able to efficiently communicate information
about the presence of the parasitoid, species that were phylogenetically more distant had
limited to no communication abilities. Interestingly, multi-species communities enhanced
inter-specific communication, allowing Drosophila to learn multiple dialects. This indicates
a degree of plasticity in learning abilities that could be adaptive in nature when Drosophila
species occur in sympatry [22]. This study represents a rare empirical test for socially
learned behaviors can be transmitted to others in non-social invertebrates (i.e., up to step 3
in Figure 1). g
)
Evidence for social learning has been based on at least three experimental setups:
some studies compare the behavior of individuals before (test a), during (test b) and after
(test c) experiencing the social cue. Evidence for social learning becomes apparent when the
behaviors observed in tests b and c are similar, but different from the behavior displayed
in test a. Another, better design, takes ageing (and its potential confounding effect) into
account by comparing groups of naive individuals with experienced individuals (that
had an earlier experience with the social cue) of similar age. 4. Social Learning of Oviposition-Related Behavior from Con- and Hetero-Specifics Visual cues, such as the presence of con-specific eggs and/or larvae on oviposition
substrates, interactions with more experienced female demonstrators, as well olfactory
cues produced by con-specifics have been shown to positively influence female oviposition
decisions after the original cue has been removed. This suggests that the benefits of con-
specific attraction in oviposition site selection may outweigh the costs of competition in the
wild [85,86]. In the context of research on social learning in Drosophila, the large knowledge-
base on cues used for oviposition site selection, as well as the documented evidence for
social learning (Table 1), make it an excellent model for testing whether social learning of
oviposition sites can be inherited socially. Acquiring social information from other species can be an efficient way to increase
fitness. This is particularly true for non-social insects with limited access to information
from con-specifics (such as for early dispersers, insects with small population sizes, and/or
species with low con-specific encounter rates. Such species can use information from other
species sharing aspects of their ecological niche to make nest choice decisions [95]. An
interesting example of hetero-specific social learning can be found in the parasitic wasp
Trichogramma evanescens [96]. Like its congener T. brassicae, this wasp uses the pheromones
of its adult host, the butterfly Pieris brassicae to identify mated females that will subse-
quently lay eggs suitable for parasitism by the wasp. By using this information, the wasp
will hitch-hike along for the ride to a new oviposition opportunity (i.e., the egg laying site
of P. brassicae), but unlike T. brassicae, T. evanescens needs to learn through an oviposition
experience that both host pheromones (to identify adult hosts) and hitch-hiking (towards
host eggs) lead to a suitable oviposition site [96]. Several solitary bee species provide an-
other example of social learning from hetero-specifics [95]. The cavity-nesting mason bees,
although the possibility that nest selection behavior is innate and not due to social learning
could not be ruled out completely. Osmia caerulescens and O. leaiana examine the nests
of another congener, O. bicornis, for evidence of brood cell parasites. Though associative Genes 2021, 12, 1466 7 of 14 learning of nest site quality of congeners (using geometric symbols), O. caerulescens and O. leaiana preferred to start their own nest at sites associated with healthy nests of O. bicornis
and rejected sites associated with brood cell parasites. 4. Social Learning of Oviposition-Related Behavior from Con- and Hetero-Specifics The behaviors of the naive
and experienced groups should differ in the absence of the social cue to show evidence
of social learning in the experienced group. A third setup consists of associating a social
cue to another cue (that does not need to be social, i.e., color, symbols etc.), and comparing
the behavior of a group of naive individuals with a group that experienced the social
and the associated cue, in the presence of only the associated cue. Evidence for social
learning is then based on a significant difference in behavior between the two groups in
the presence of the associated (but not the social) cue, for the experienced group. These
experimental set-ups, when carefully designed, allow to discriminate beyond any doubt
socially learned behaviors from behaviors that are innate or learned as consequence of
interactions with abiotic cues. As such, they provide excellent opportunities to study social
learning, transmission, and inheritance of oviposition-related (and other) behaviors in a
wide range of non-social arthropods. In light of the accumulating evidence for widespread
social learning, these experimental designs can greatly contribute to our understanding of
the role of social learning in evolution. 8 of 14 Genes 2021, 12, 1466 Table 1. List of studies on non-social arthropods where social cue perception, social learning, and transmission of socially learned oviposition-related behaviors was quantified. Only
studies that document social learning are included (i.e., from step 2 of Figure 1 onwards), as there is a large body of literature covering cue perception (i.e., step 1 of Figure 1). The table
includes the species, the order (Diptera = D, Hymenoptera = H, C = Coleoptera, Trombidiformes = T), the type of social cue and the behavior under study, con- (c) or hetero- (h) specific
social learning, the steps towards social inheritance (as in Figure 1) and if effects on fitness were quantified in the study. Studies concerned with foraging, mating, host finding and other
behaviors, including in non-insect invertebrates, have been discussed elsewhere [30–34]. Species
Order
Social Cue
Behavior
Learning from con- (c)
or Hetero- (h) Specifics
Step Towards Social
Inheritance
Fitness Tested
Reference
D. melanogaster
D
Experienced females with
preferred oviposition site
Site selection
c
1, 2, 3
y
[25]
D. melanogaster
D
Parasitoid presence (i.e., threat
to offspring survival)
Clutch size
c
1, 2
y
[21]
Drosophila spp. 5. The Adaptive Value of Social Learning Social learning is an important mechanism in evolution even when transmission of socially
acquired behaviors is limited to a few generations within a season, such that social inheritance
will not be maintained over long evolutionary times (step 4 in Figure 1). Indeed, we suggest
that building expertise during a lifetime by social experiences can increase the adaptation
rate of populations that are using and memorizing social information, for example for the
spatial location of essential resources, even if every adult individual dies at the end of the
reproductive season. This is, for example, because social information allows individuals to
avoid unfavorable oviposition sites, to reach an oviposition site earlier or at lower exploratory
costs, compared to individuals that explore and spatially navigate without this information. In
this regard, most current evidence for social learning, including in non-social insects, concerns
behaviors such as foraging and host location, which are based on resources that vary rapidly
in space and time notably due to seasonal changes. Related social information is thus of
ephemeral relevance as well and it needs to be updated constantly, suppressing the emergence
of any form of longer-term social inheritance. Rupture of socially transmitted behaviors can
also take place because most representatives of insect populations die seasonally, for example
during winter in temperate regions. In the latter case, social information about resources can
be acquired and exchanged socially de novo at the beginning of the new reproductive season
each year, starting from newly emerged naive individuals in spring that learn about resource
distribution in their surrounding environment. g
The adaptive value of learned behaviors is documented in some vertebrates [4,5],
but experimental evidence for the adaptive value of socially learned behaviors in ecolog-
ically relevant conditions currently remains unquantified for the vast majority of living
taxa [17,111], including non-social insects [112]. Social learning can increase the fitness
of individuals and as such be under positive selection in rapidly changing environments. Yet, this is not necessarily the case as negative effects on fitness were documented from
partially or incorrectly interpreted social cues that caused increased energy expenditure
in basic tasks, such as foraging [108]. The costs associated with social learning, including
energetic costs and time constraints, and the environmental parameters under which social
learning becomes adaptive, have been explored both experimentally [113] and through
modeling work [114,115]. 4. Social Learning of Oviposition-Related Behavior from Con- and Hetero-Specifics D
Parasitoid presence (i.e., threat
to offspring survival)
Clutch size
c + h
1, 2, 3
y
[22]
D. melanogaster
D
Mated females
Site selection
c
1, 2
y
[97]
Leptopilina boulardi
H
Host insect
Site selection
h
1, 2
n
[98]
Necremnus tutae
H
Host insect and plant species
Host species preference
h
1, 2
n
[99]
Osmia sp.*
H
Nest site parasitism
Site selection
h
1, 2
n
[95]
Trichogramma evanescens
H
Host adult and eggs
Phoresy to oviposition
substrate
h
1, 2
n
[96]
Anisopteromalus
calandrae
H
Host insect
Host preference +
host-finding +
parasitism rates
h
1, 2
y
[100]
Phratora vulgatissima
C
Adult females
Distance between
clutches
c
1, 2
y
[101]
Tetranychus urticae, T. kanzawai
T
Predator
Site selection (leaf
surface vs web)
h
1, 2
n
[102]
* Tested under field conditions. * Tested under field conditions. Genes 2021, 12, 1466 9 of 14 9 of 14 Evidence for hetero-specific social learning has also been found for behaviors other
than oviposition. Social learning in non-social arthropods was first reported in a cricket,
Nemobius sylvestris, that changed its predator avoidance behavior based on observations,
and memory of such observations, of either predator presence (spiders) or of congener
crickets that had already experienced the presence of spiders [26]. Hetero-specific social
information can thus also be transmitted from experienced to naive crickets [26], which
can decrease predation risk. Hetero-specific social information was also found to increase
the efficiency of locating food sources [103–106]. Although social information from hetero-
specifics is ubiquitous, it can be challenging to decode, for example because the cue may
have had a different original meaning or purpose than what is interpreted by the receiving
species [107–110]. 5. The Adaptive Value of Social Learning These studies have revealed that social learning is not necessar-
ily adaptive under all conditions and that learning can lead to evolutionary traps under
rapidly changing environmental conditions [116]. A study with D. melanogaster convincingly suggested that social learning has adaptive
value also in the context of oviposition-related behaviors in non-social insects [21]. Here,
the authors exposed ovipositing D. melanogaster females to a parasitoid wasp that lays
eggs inside D. melanogaster larvae, which are subsequently consumed from the inside out
by the developing parasitoid. Having been faced with a serious threat to the survival of
their offspring [117], female D. melanogaster reduced the number of eggs laid in the next
clutch [118]. When a parasitoid-experienced fly was then observed by a naive female fly,
the latter also reduced her clutch size, even though the original social cue, the wasp, was
no longer present [21]. Within an ecological context, reducing egg numbers in the face Genes 2021, 12, 1466 10 of 14 10 of 14 of an immediate threat to offspring survival can have a clear adaptive value, also under
natural conditions. Indeed the wasp species used in this study actively searches for host
patches in the environment [119,120], where mating, oviposition or feeding Drosophila
larvae generate perceivable olfactory cues for the wasp [121]. It remains to be tested
whether social learning in D. melanogaster females can be transmitted from one generation
to the next (as was found in [23]). 6. Conclusions Perception of social cues, social learning and transmission are the stepping stones
towards social inheritance. While perception of social cues is now well known to induce
behavioral changes in multiple arthropods (e.g., [35,83–93]), we need to increase our
understanding of social learning in non-social arthropods and determine its prevalence,
both in the laboratory and in the field. Due to its inherent link to fitness, oviposition site
selection offers unparalleled opportunities to study social learning and transmission, also
in systems other than Drosophila. The increasing number of studies on social learning in
non-social arthropods (see Table 1) offer promising possibilities for empirical tests of social
transmission and inheritance. Author Contributions: Conceptualization, C.M.N. and B.V.; writing—original draft preparation,
C.M.N., M.M., R.V., T.E., B.V.; writing—review and editing, C.M.N., M.M., R.V., T.E., B.V.; visualiza-
tion, C.M.N., R.V., T.E.; supervision, C.M.N.; project administration, C.M.N.; funding acquisition,
C.M.N., T.E. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by UCLouvain, the Fédération Wallonie-Bruxelles, and by the
Fonds National de Recherche Scientifique (F.R.S.-FNRS). R.V. was supported by the F.R.S.-FNRS
grant number T.0169.21 of C.M.N.; M.M. was supported by the “Action de Recherche concertée”
grant number 17/22-086 of C.M.N. TE. was funded by the Fyssen foundation. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Whiten, A.; Caldwell, C.A.; Mesoudi, A. Cultural diffusion in humans and other animals. Curr. Opin. Psychol. 2016, 8, 15–21
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The Solution Behavior of Dopamine in the Presence of Mono and Divalent Cations: A Thermodynamic Investigation in Different Experimental Conditions
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Biomolecules
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Citation: Gigliuto, A.; Cigala, R.M.;
Irto, A.; Felice, M.R.; Pettignano, A.;
Milea, D.; Materazzi, S.; De
Stefano, C.; Crea, F. The Solution
Behavior of Dopamine in the Presence
of Mono and Divalent Cations: A
Thermodynamic Investigation in
Different Experimental Conditions. Biomolecules 2021, 11, 1312. https://
doi.org/10.3390/biom11091312 Academic Editors: Valeria M. Nurchi
and Jan Aaseth Received: 2 August 2021
Accepted: 2 September 2021
Published: 5 September 2021 Received: 2 August 2021
Accepted: 2 September 2021
Published: 5 September 2021 Keywords: chemical speciation; metal complexes; catechol; sequestration; stability constants Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Antonio Gigliuto 1, Rosalia Maria Cigala 1
, Anna Irto 1
, Maria Rosa Felice 1, Alberto Pettignano 2
,
Demetrio Milea 1
, Stefano Materazzi 3
, Concetta De Stefano 1 and Francesco Crea 1,* Antonio Gigliuto 1, Rosalia Maria Cigala 1
, Anna Irto 1
, Maria Rosa Felice 1, Alberto Pettignano 2
,
Demetrio Milea 1
, Stefano Materazzi 3
, Concetta De Stefano 1 and Francesco Crea 1,* 1
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università degli Studi di
Messina, V.le F. Stagno d’Alcontres, 31, I-98166 Messina, Italy; agigliuto@unime.it (A.G.);
rmcigala@unime.it (R.M.C.); airto@unime.it (A.I.); mrfelice@unime.it (M.R.F.); dmilea@unime.it (D.M.);
cdestefano@unime.it (C.D.S.) 1
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università degli Studi di
Messina, V.le F. Stagno d’Alcontres, 31, I-98166 Messina, Italy; agigliuto@unime.it (A.G.);
rmcigala@unime.it (R.M.C.); airto@unime.it (A.I.); mrfelice@unime.it (M.R.F.); dmilea@unime.it (D.M.);
cdestefano@unime.it (C.D.S.) 2
Dipartimento di Fisica e Chimica, Università degli Studi di Palermo, V.le delle Scienze, ed. 17,
I-90128 Palermo, Italy; alberto.pettignano@unipa.it y
p
g
p
3
Dipartimento di Chimica, Università “La Sapienza” di Roma, Piazzale A. Moro 5, I-00185 Rome, Italy;
stefano.materazzi@uniroma1.it *
Correspondence: fcrea@unime.it; Tel.: +39-0906765761 Abstract: The interactions of dopamine [2-(3,4-Dihydroxyphenyl)ethylamine, (Dop−)] with methylmer-
cury(II) (CH3Hg+), magnesium(II), calcium(II), and tin(II) were studied in NaCl(aq) at different ionic
strengths and temperatures. Different speciation models were obtained, mainly characterized by mononu-
clear species. Only for Sn2+ we observed the formation of binuclear complexes (M2L2 and M2LOH (charge
omitted for simplicity); M = Sn2+, L = Dop−). For CH3Hg+, the speciation model reported the ternary
MLCl (M = CH3Hg+) complex. The dependence on the ionic strength of complex formation constants was
modeled by using both an extended Debye–Hückel equation that included the Van’t Hoff term for the
calculation of enthalpy change values of the formation and the Specific Ion Interaction Theory (SIT). The
results highlighted that, in general, the entropy is the driving force of the process. The sequestering ability
of dopamine towards the investigated cations was evaluated using the calculation of pL0.5 parameter. The
sequestering ability trend resulted to be: Sn2+ > CH3Hg+ > Ca2+ > Mg2+. For example, at I = 0.15 mol
dm−3, T = 298.15 K and pH = 7.4, pL0.5 = 3.46, 2.63, 1.15, and 2.27 for Sn2+, CH3Hg+, Ca2+ and Mg2+
(pH = 9.5 for Mg2+), respectively. For the Ca2+/Dop−system, the precipitates collected at the end of the
potentiometric titrations were analyzed by thermogravimetry (TGA). The thermogravimetric calculations
highlighted the formation of solid with stoichiometry dependent on the different metal:ligand ratios and
concentrations of the starting solutions. biomolecules 1. Introduction In the context of biological
and environmental monitoring, speciation involves three different levels of study: (1) spe-
cies analysis of a specific element; (2) subdivision through specific chemical analysis of
organic and inorganic species; (3) application to the analysis of information about the dif-
ferences in the distribution of the different species of an element [1–3]. Dopamine [2-(3,4-Dihydroxyphenyl)ethylamine (see Scheme 1)] is a neurohormone
released by the hypothalamus. Its main function as a hormone is to inhibit the release of
prolactin from the anterior pituitary lobe [4–7]. ferences in the distribution of the different species of an element [1 3]. Dopamine [2-(3,4-Dihydroxyphenyl)ethylamine (see Scheme 1)] is a neurohormone
released by the hypothalamus. Its main function as a hormone is to inhibit the release of
prolactin from the anterior pituitary lobe [4–7]. Scheme 1. 2-(3,4-Dihydroxyphenyl)ethylamine [dopamine]. Scheme 1. 2-(3,4-Dihydroxyphenyl)ethylamine [dopamine]. Scheme 1. 2-(3,4-Dihydroxyphenyl)ethylamine [dopamine]
Scheme 1. 2-(3,4-Dihydroxyphenyl)ethylamine [dopamine]. Scheme 1. 2-(3,4-Dihydroxyphenyl)ethylamine [dopamine]. Scheme 1. 2-(3,4-Dihydroxyphenyl)ethylamine [dopamine]. Dopamine is biosynthesized in the body (especially in the nervous tissue and adrenal
medulla) by a mechanism that primarily involves the hydroxylation of the amino acid L-
tyrosine (normally found in the diet) into L-DOPA through the enzyme tyrosine 3-
monooxygenase, and subsequent decarboxylation of L-DOPA by the DOPA decarbox-
ylase, which will remove the carboxyl group (-COOH) from the DOPA side chain. Dopamine is biosynthesized in the body (especially in the nervous tissue and adrenal
medulla) by a mechanism that primarily involves the hydroxylation of the amino acid
L-tyrosine (normally found in the diet) into L-DOPA through the enzyme tyrosine 3-
monooxygenase, and subsequent decarboxylation of L-DOPA by the DOPA decarboxylase,
which will remove the carboxyl group (-COOH) from the DOPA side chain. In some neurons, dopamine is transformed into noradrenaline by dopamine beta-
hydroxylase and after synthesis; it is here “packaged” into synaptic vesicles, which are
then released into the synapses in response to a presynaptic action potential. The storage
inside the vesicles has the purpose of protecting the molecule from degradation by the
monoamine oxidase and is indispensable for the release of the neurotransmitter in the
synaptic space by the nerve impulse. In some neurons, dopamine is transformed into noradrenaline by dopamine beta-
hydroxylase and after synthesis; it is here “packaged” into synaptic vesicles, which are
then released into the synapses in response to a presynaptic action potential. 1. Introduction The storage
inside the vesicles has the purpose of protecting the molecule from degradation by the
monoamine oxidase and is indispensable for the release of the neurotransmitter in the
synaptic space by the nerve impulse. y
p
p
y
p
As a medicinal, it is used in the treatment of neuropathology, such as Parkinson’s
and schizophrenia. Specifically: Parkinson’s disease is associated with a decrease in the
concentration of dopamine in the brain; therefore, it requires the use of drugs containing
levodopa (LD) combined with a certain amount of carbidopa (CD), which makes LD avail-
able for transport to the brain and subsequently converted to dopamine in the basal gan-
glia. Schizophrenia is associated with an increase in dopamine concentration in the brain. In this case, the drug used blocks dopamine receptors in the brain [8]. y
p
p
y
p
As a medicinal, it is used in the treatment of neuropathology, such as Parkinson’s
and schizophrenia. Specifically: Parkinson’s disease is associated with a decrease in the
concentration of dopamine in the brain; therefore, it requires the use of drugs containing
levodopa (LD) combined with a certain amount of carbidopa (CD), which makes LD
available for transport to the brain and subsequently converted to dopamine in the basal
ganglia. Schizophrenia is associated with an increase in dopamine concentration in the
brain. In this case, the drug used blocks dopamine receptors in the brain [8]. I
i
a e,
e
ug u e
o
opa
i e e ep o
i
e
ai
[ ]
Many years ago, this research group started a systematic investigation on the acid-
base properties, solubility, and interaction of molecules of biological and pharmaceutical
relevance towards different metal ions [9–19]. Many years ago, this research group started a systematic investigation on the acid-
base properties, solubility, and interaction of molecules of biological and pharmaceutical
relevance towards different metal ions [9–19]. relevance towards different metal ions [9 19]. The acid-base properties of dopamine in NaCl aqueous solutions and the distribution
between NaCl solutions/organic solvent at different experimental conditions (ionic
strengths and temperatures) were already studied [10]
The acid-base properties of dopamine in NaCl aqueous solutions and the distribu-
tion between NaCl solutions/organic solvent at different experimental conditions (ionic
strengths and temperatures) were already studied [10]. strengths and temperatures) were already studied [10]. 1. Introduction Speciation is the process of formation of chemical species through a series of reactions
(formation of complexes) that lead to the characterization of a component in a natural or
artificial system. The more complicated the system where the interactions are investigated,
the greater the degree of difficulty in fully defining the effective distribution of the chemical
species. Therefore, a complete speciation model of a fluid can be formulated if all of the
possible interactions among the components dissolved are considered. Each constituent of
the natural and biological fluids assumes its own thermodynamic behavior, i.e., different
speciation, as a function of different variables, such as temperature, ionic medium, ionic
strength, component concentrations, and ligand:metal molar ratios [1–3]. Speciation stud-
ies, in the fields of clinical and environmental chemistry, are of great importance, since the
polluting, toxic, or curative action of a given component depends on the specific species
to which it originates in that natural fluid. In the context of biological and environmental Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 1312. https://doi.org/10.3390/biom11091312 Biomolecules 2021, 11, 1312 2 of 28
eat im- monitoring, speciation involves three different levels of study: (1) species analysis of a
specific element; (2) subdivision through specific chemical analysis of organic and inor-
ganic species; (3) application to the analysis of information about the differences in the
distribution of the different species of an element [1–3]. the specific species to which it originates in that natural fluid. In the context of biological
and environmental monitoring, speciation involves three different levels of study: (1) spe-
cies analysis of a specific element; (2) subdivision through specific chemical analysis of
organic and inorganic species; (3) application to the analysis of information about the dif-
ferences in the distribution of the different species of an element [1–3]. monitoring, speciation involves three different levels of study: (1) species analysis of a
specific element; (2) subdivision through specific chemical analysis of organic and inor-
ganic species; (3) application to the analysis of information about the differences in the
distribution of the different species of an element [1–3]. the specific species to which it originates in that natural fluid. 1. Introduction As a further contribution to the speciation studies of this molecule, the interactions
towards different metal ions, such as CH3Hg+, Ca2+, Mg2+ and Sn2+, were investigated. g
y
As a further contribution to the speciation studies of this molecule, the interactions
towards different metal ions, such as CH3Hg+, Ca2+, Mg2+ and Sn2+, were investigated. towards different metal ions, such as CH3Hg , Ca , Mg and Sn , were investigated. The studies were carried out by means of potentiometric titrations in NaCl(aq) in a
wide range of component concentrations and ligand/metal molar ratios, at different ionic
strengths and temperatures
The studies were carried out by means of potentiometric titrations in NaCl(aq) in a
wide range of component concentrations and ligand/metal molar ratios, at different ionic
strengths and temperatures. strengths and temperatures. The dependence of the complex formation constants on the ionic strength was inves-
tigated by means of an extended Debye–Hückel equation that included a Van’t Hoff term
for the calculation of the enthalpy change values of formation of the metal/dopamine com-
plexes. The Specific Ion Interaction Theory (SIT) approach was used to calculate the spe-
cific ion interaction parameters [20 22]
The dependence of the complex formation constants on the ionic strength was in-
vestigated by means of an extended Debye–Hückel equation that included a Van’t Hoff
term for the calculation of the enthalpy change values of formation of the metal/dopamine
complexes. The Specific Ion Interaction Theory (SIT) approach was used to calculate the
specific ion interaction parameters [20–22]. cific ion interaction parameters [20–22]. The effective sequestering ability of dopamine towards the different metal ions here
considered was quantified at selected ionic strengths, pHs, and temperatures, by using a
widely tested approach [23,24]. For Ca2+/Dop−system, some of the precipitates collected at the end of the titrations
were analyzed by means of thermogravimetry (TGA). Biomolecules 2021, 11, 1312 3 of 28 2.1. Chemicals Dopamine solutions were prepared by weighing the corresponding hydrochloric salt
without further purification. The purity was checked potentiometrically by alkalimetric
titrations and resulted to be >99.5%. Sodium chloride aqueous solutions were prepared
at different ionic strengths from dilution of a concentrated solution obtained by weighing
pure salt previously dried in an oven (Thermo Fisher Scientific, Waltham, MA, USA) at
T = 383.15 K for 2 h. Sodium hydroxide and hydrochloric acid solutions were prepared
from concentrated ampoules and standardized against potassium hydrogen phthalate and
sodium carbonate, respectively. p
y
Standard stock solutions of the metals (CH3Hg+; Ca2+; Mg2+, and Sn2+) were prepared
from the corresponding chloride salts and were used without further purification. The
standard stock solutions of SnCl2 were acidified with HCl to pH~2 and a piece of metallic
tin was added to the solutions after the preparation, to prevent the oxidation of Sn2+ to
Sn4+ and to hamper the formation of soluble and sparingly soluble hydrolytic species. The concentration of the metal ions in the aqueous solutions were determined by
means of complexometric titrations with ethylenediaminetetraacetic acid sodium salt
(EDTA) [25]. All of the products were purchased from Sigma-Aldrich (Milano, Italy). All solutions
were prepared with analytical grade water (ρ = 18 MΩcm−1) using grade A glassware and
preserved from atmospheric CO2 by means of soda lime traps. Further details on the chemicals used for the investigation here carried out are reported
in Table 1. able 1. Chemicals used in this work, purchased from Sigma-Aldrich, Italy. Table 1. Chemicals used in this work, purchased from Sigma-Aldrich, Italy. Chemical. CAS Number
Purification Method
Purity (% wt.)
Purity Check
Hydrochloric acid
7647-01-0
none
≥99%
Volumetric titrations
with Na2CO3
Potassium phthalate
monobasic
877-24-7
none
≥99.5%
-
Sodium carbonate
497-19-8
none
≥99.5%
-
Sodium chloride
7647-14-5
none
≥99%
-
Sodium hydroxide
1310-73-2
none
≥99%
Volumetric titrations
with potassium
phthalate monobasic
CH3HgCl
115-09-3
none
≥99%
-
MgCl2·6H2O
7791-18-6
none
≥99%
Volumetric titrations
with EDTA
CaCl2·2H2O
10035-04-8
none
≥99%
Volumetric titrations
with EDTA
SnCl2·2H2O
10025-69-1
none
≥99%
Volumetric titrations
with EDTA
Dopamine·HCl
62-31-7
none
≥99.5%
Volumetric titrations 2.2. Apparatus and Procedure 2.2.1. Potentiometric Titrations The interactions of dopamine with the selected cations were studied potentiometrically
by means of an apparatus consisting of an 809 model Metrohm Titrando system (Metrohm,
Varese, Italy), connected to a half-cell Ross Type glass electrode (model 8101 from Thermo
Fisher Scientific, Waltham, MA, USA), and coupled with a standard Ag/AgCl reference
electrode. The system was connected to a personal computer and the parameters of
the titrations were controlled by means of the Metrohm TiAMO 2.2 computer program
(Metrohm, Varese, Italy), which allowed performing automatic titrations, by addition of
desired amounts of titrant when the equilibrium state was reached and recording the e.m.f. Biomolecules 2021, 11, 1312 4 of 28 of the solution under investigation. The estimated accuracy was ±0.15 mV and ±0.003 mL
for e.m.f and titrant volume readings, respectively. The measurements were carried out under continuous stirring and pure N2 flow in a
thermostatted cell, connected to a Model D1-G-Haake thermocryostat (Gebrüder HAAKE
GmbH, Karlsruhe, Germany), by means of water circulation in the outer chamber of the
titration cell. Investigations were performed from T = 288.15 to 310.15 K; only for the
CH3Hg+/Dop−system, measurements were also carried out at T = 318.15 K. g
p
y
Concerning the dependence of the stability constants on the ionic strength, interactions
were studied at 0.15 ≤I/mol dm−3 ≤1.0. For Ca2+ and Sn2+/Dop−systems, at T = 288.15
and 310.15 K, only the ionic strength I = 0.15 mol dm−3 was investigated. y
g
g
The titrated solutions consisted of different amounts of the cation, dopamine·HCl, an
excess of hydrochloric acid and NaCl, for obtaining the desired ionic strength values. To
investigate the possible formation of both mono- and/or polynuclear species, solutions
were prepared in a wide range of cation to ligand molar ratios and were titrated with
standard carbonate free NaOH, up to alkaline pH values, or to the formation of sparingly
soluble species. Table 2 reports the experimental conditions employed in the investigations
of the systems (components concentration, ligand:cation molar ratios). Table 2. Experimental conditions used for the Mn+/Dop−systems in NaCl aqueous solutions. cM (mmol dm−3)
cL (mmol dm−3)
M:L
1
1.5
1:1.5
1
4
1:4
1.5
3
1:2
1.5
5
1:3
2
5
1:2.5
2
6
1:3
M = generic metal ion; L = dopamine. Table 2. Experimental conditions used for the Mn+/Dop−systems in NaCl aqueous solutions. Computer Programs All parameters of the potentiometric titrations (standard electrode potential (E0), liquid
junction potential coefficient (ja), ionic product of the water (Kw), analytical concentration
of reagents and formation constants) were determined by using the non-linear least square
minimization method and the BSTAC program (further details are reported in ref [26]), by
solving the component mass balance equations to obtain explicit functions for the average
number of ligand bound to the metal. g
By using the BSTAC program, we minimized the error square sum in the electromotive
force (E = e.m.f.) U = ∑W·(Eexp −Ecalcd)2
(1) U = ∑W·(Eexp −Ecalcd)2 (1) Weights for each point of the titration curves are given as (s2 = variance) W = 1/s2
(2)
s2 = sE2 + ((∂Ei)/(∂Vi))2·sV2
(3) (2) (2)
(3) s2 = sE2 + ((∂Ei)/(∂Vi))2·sV2
(3) (3) where sE2 and sV2 are the estimated variances for the e.m.f. and volume, respectively. where sE and sV are the estimated variances for the e.m.f. and volume, respectively. The program uses a Gauss–Newton technique for the refinement of the equilibrium
constants: The program uses a Gauss–Newton technique for the refinement of the equilibrium
constants: s = (AT·W·A)−1·(AT·W)·e
(4) (4) where, A = matrix of the partial derivatives ∂y/∂p (y is an independent variable and p
the ith point of the potentiometric measurement); W = weight matrix; e = residuals vector;
s = shifts vector. Different independent variables, y, can be used for the refinement of the
equilibrium constants from the potentiometric data, such as electromotive force (e.m.f.), v
(titrant volume), X (analytical concentrations), n (average number of ligands bound to the
metal), Z (average number of protons displaced per metal ion), etc. The LInear And Nonlinear Analysis (LIANA) computer program, which minimizes
the error square sum in y of an equation y = f(xi) (a generic function given by the user),
by using the general Levenberg–Marquardt–Gauss–Newton Method, was employed for
the determination of the equilibrium constants at infinite dilution, and the corresponding
parameters for the dependence on the ionic strength, and of Specific Ion Interaction Theory
parameter of the ion-pairs [26]. p
p
The ES4ECI [27] or Hyss [28] programs were employed for the calculation of the
formation percentages of the species present in solution at the equilibrium and to draw
both the distribution and the sequestration diagrams in different conditions. 2.2.1. Potentiometric Titrations For each titration, the total number of potentiometric experimental points collected
varied between 60 and 100, in dependence on the possible formation of a sparingly soluble
species. At least two titrations were performed for each experimental condition. Indepen-
dent titrations of strong acid (HCl) solutions with NaOH solutions were carried out at the
same experimental conditions of the systems investigated, with the aim of determining
the electrode potential (E0) and the acidic junction potential (Ej = ja [H+]). In this way, the
pH scale used was the free concentration scale, pH ≡−log [H+], where [H+] is the free
proton concentration (not activity). The reliability of the calibration in the alkaline range
was checked by calculating the ionic product of water (pKw). Figure 1 reports the titration curves of dopamine and of the metal/dopamine systems
at I = 0.15 mol dm−3 and T = 298.15 K, where it is possible to observe quite different curve
profiles, especially for the Sn2+ containing solution, due to the different acid-base properties
of the metal ions and the strength of the interaction towards dopamine. 5 of 30 0.0
0.5
1.0
1.5
2.0
2.5
2
4
6
8
10
12
Dop
-
CH3Hg
+
Sn
2+
Ca
2+
Mg
2+
pH
cm
3 Titrant (NaOH 0.1005 mol dm
-3)
Figure 1. Titration curves of dopamine and of the different metal/Dop- systems at T = 298.15 K and
I = 0.15 mol dm−3. (Experimental conditions: cDop- = 3 mmol dm−3; cMn+= 1.5 mol dm−3). 2 2 2 Thermogravimetric Measurements
Figure 1. Titration curves of dopamine and of the different metal/Dop−systems at T = 298.15 K and
I = 0.15 mol dm−3. (Experimental conditions: cDop−= 3 mmol dm−3; cMn+= 1.5 mol dm−3). 0.0
0.5
1.0
1.5
2.0
2.5
2
4
6
8
10
12
Dop
-
CH3Hg
+
Sn
2+
Ca
2+
Mg
2+
pH
cm
3 Titrant (NaOH 0.1005 mol dm
-3) Figure 1. Titration curves of dopamine and of the different metal/Dop- systems at T = 298.15 K and
I = 0.15 mol dm−3. (Experimental conditions: cDop- = 3 mmol dm−3; cMn+= 1.5 mol dm−3). Figure 1. Titration curves of dopamine and of the different metal/Dop−systems at T = 298.15 K and
I = 0.15 mol dm−3. (Experimental conditions: cDop−= 3 mmol dm−3; cMn+= 1.5 mol dm−3). 5 of 28 Biomolecules 2021, 11, 1312 2.2.2. Thermogravimetric Measurements 2.2.2. Thermogravimetric Measurements A thermo-analytic characterization was performed by a PerkinElmer TGA7 equipment
(PerkinElmer, Milano, Italy). The investigated samples (approximately 2–10 mg) were
heated in platinum crucibles in the temperature range 293–1123 K, under an atmosphere
of air (gaseous mixture of nitrogen and oxygen with 80% and 20%, v/v, respectively) at a
flow rate of 100 mL min−1 and a scanning rate of 10 K min−1. These conditions allowed
the best possible resolution of the thermogravimetric curves. 2.4. Dependence of the Stability Constants on the Ionic Strength and Temperature 2.4. Dependence of the Stability Constants on the Ionic Strength and Temperature The dependence of the formation constants on ionic strength was studied both by
means of the Extended Debye–Hückel (EDH) equation and specific ion interaction theory
(SIT) approach (general information can be found, e.g., in [20–22,29,30]. 2.4.1. Extended Debye–Hückel (EDH) 2.4.1. Extended Debye–Hückel (EDH) The dependence of the formation constants on the ionic strength and temperature
was modeled by means of an extended Debye–Hückel (EDH) equation, allowing the
calculation of the complex formation constants at infinite dilution and the enthalpy change
of formation, when measurements were also carried out at different temperatures: logβpqr = logTβpqr −z*·A·√I/(1 + 1.5·√I) + C·I + L·(1/298.15 −1/T)·52.23
(6) (6) where 52.23 is 1/(R·ln10) and C is the adjustable parameter for the dependence of formation
constants on ionic strength in the molar concentration scale; this parameter can be expressed
by the following equation: C = c0p* + c1z*; p* = ∑preactants −∑pproducts and z* = ∑zreactants2
−∑zproducts2, where z and p are the charge and the stoichiometric coefficient, respectively. LogTβpqr is the formation constant at infinite dilution and A is the Debye–Hückel term,
expressed with respect to its dependence on the temperature, as reported in the Equation (7),
where 0.856 and 0.00385 are empirical parameters, T = 298.15 K is the reference temperature
and T’ the desired one, expressed in kelvin (K): where 52.23 is 1/(R·ln10) and C is the adjustable parameter for the dependence of formation
constants on ionic strength in the molar concentration scale; this parameter can be expressed
by the following equation: C = c0p* + c1z*; p* = ∑preactants −∑pproducts and z* = ∑zreactants2 p
−∑zproducts2, where z and p are the charge and the stoichiometric coefficient, respectively. LogTβpqr is the formation constant at infinite dilution and A is the Debye–Hückel term,
expressed with respect to its dependence on the temperature, as reported in the Equation (7),
where 0.856 and 0.00385 are empirical parameters, T = 298.15 K is the reference temperature
and T’ the desired one, expressed in kelvin (K): A = (0.51 + (0.856·(T’ −298.15) + 0.00385·(T −298.15)2)/1000)
(7) (7) and and L = (∆Hn0 −z*·(1.5 + 0.024·(T’ −298.15)·√I)/(1 + 1.5·√I))
(8) (8) By using Equation (8), it is possible to calculate, the standard enthalpy changes value
of formation of the species at infinite dilution ∆Hn0 and in the investigated ∆T range. Equation (8) takes also into account the variation of the ∆Hn0 with I/mol dm−3. By using Equation (8), it is possible to calculate, the standard enthalpy changes value
of formation of the species at infinite dilution ∆Hn0 and in the investigated ∆T range. Equation (8) takes also into account the variation of the ∆Hn0 with I/mol dm−3. 2.4.2. 2.4.1. Extended Debye–Hückel (EDH) Specific Ion Interaction Theory (SIT) Approach Computer Programs The HySS
program allows considering the formation of the sparingly soluble species, by using the
solubility product of the species in the speciation model. Within the manuscript, if not differently specified, hydrolysis (q = 0, r < 0) constants
of metal cations, protonation (p = 0) constants of the ligands (Lz−), and complex formation
constants are given according to the overall equilibrium: logβpqr: pMn+ + qLz−+ rH+ = MpLqHr(np−zq+r)
(5 (5) logβpqr: pMn+ + qLz−+ rH+ = MpLqHr(np−zq+r) Biomolecules 2021, 11, 1312 6 of 28 The errors associated to formation constants, enthalpy, and entropy change values and
parameters for the dependence on ionic strength are expressed as ± standard deviation
(Std. Dev.). 2.4.2. Specific Ion Interaction Theory (SIT) Approach When the stability constants and the ionic strengths are expressed in the molal concen-
tration scale, Equation (6) becomes (neglecting the last term containing ∆Hn0) the classical
and widely used Specific Ion Interaction Theory (SIT) equation [20–22], in which C is
replaced by ∆ε, as follows: logβpqr = logTβpqr −z* (A√(Im))/(1 + 1.5·√Im) + ∆ε·Im + jlogaw
(9) (9) where Im = ionic strength in the molal concentration scale, log aw is the activity coefficient
of water (log aw = 0.015), j = number of water molecules involved in the equilibrium. where Im = ionic strength in the molal concentration scale, log aw is the activity coefficient
of water (log aw = 0.015), j = number of water molecules involved in the equilibrium. ∆ε = ∑εreactants −∑εproducts
(10) (10) ε is the SIT coefficient for the interaction of the ionic species involved in the considered
equilibrium with the ion (of opposite sign) of the supporting electrolyte (i.e., NaCl). ε is the SIT coefficient for the interaction of the ionic species involved in the considered
equilibrium with the ion (of opposite sign) of the supporting electrolyte (i.e., NaCl). q
pp
g
pp
g
y
In all the equilibria involving uncharged species, the activity coefficient must also be
taken into account by means of the Setschenow equation [31]. Log γ = kc,m · I
(11) (11) Biomolecules 2021, 11, 1312 7 of 28 where kc and km are the Setschenow coefficients of the neutral species in a given medium,
in the molar and molal concentration scales, respectively. where kc and km are the Setschenow coefficients of the neutral species in a given medium,
in the molar and molal concentration scales, respectively. If all interactions between the ligand and the metal ion are taken into account, the
generic ∆ε parameter of Equation (9) can be explicated to obtain the ion-pairs SIT coefficient
for all species involved in the equilibrium of formation of the complexes. 3. Results 3.1. Acid-Base Properties of Dopamine and Hydrolytic Constants of the Investigated Cations The hydrolytic constants of the cations here investigated, and the protonation con-
stants of dopamine were already investigated. In a previous paper [10], the protonation constants of dopamine in aqueous solutions
of sodium chloride were determined at different ionic strengths and temperatures. g
p
The hydrolysis constants of CH3Hg+ in NaCl at 0 < I/mol dm−3 ≤1.0 were taken from
previous investigations [23,32]. In chloride media, the acid-base behavior of methylmer-
cury(II) is characterized by the formation of a stable CH3HgCl complex that forms in
significant amount and represents the main species up to pH~7–8. For Mg2+, the hydrolytic constants were taken from [33]; whilst for Ca2+ from [34]. 2 For Sn2+, the acid-base properties were already studied in a previous work, from an
accurate analysis of literature data and experimental investigations, at different conditions (i.e.,
I/mol dm−3, T/K, etc.) in NaCl aqueous solutions [35], by also determining the stability of
the weak complexes with chloride anion. The literature data for the protonation constants of
dopamine, the hydrolysis of the metal ions and the corresponding stability constants of the
complexes formed with Cl−are reported in Tables S1 and S2 of the Supplementary Material. 3.2. CH3Hg+/Dop−System 2.4.2. Specific Ion Interaction Theory (SIT) Approach As an example,
in the case of the ML species, we have for M2+ (Ca2+, Mg2+) or M+ (CH3Hg+) cations: M2+: ∆ε = ε(M2+,Cl−) + ε(L−,Na+) −ε(ML+,Cl−)
(12)
M+: ∆ε = ε(M+,Cl−) + ε(L−,Na+) −km(ML0)
(13) (12) (13) For the MLH species and M2+ cation, we have as an example: For the MLH species and M2+ cation, we have as an example: ∆ε = ε(M2+,Cl−) + ε(L−,Na+) + ε(H+,Cl−) −ε(MLH2+,Cl−)
(14) (14) For MLOH species and M2+ cation, if the following equilibrium of formation is
considered, M2+ + L−+ H2O = MLOH0 + H+ The specific ion interaction coefficients can be calculated by: ∆ε = ε(M2+,Cl−) + ε(L−,Na+) −km(MLOH0) −ε(H+,Cl−)
(15) (15) In this case, the activity coefficient of water must be considered (log aw = 0.015). 3.2. CH3Hg+/Dop−System The determination of the best speciation model for the metal/dopamine systems and
of the corresponding stability constants was obtained by using some general rules and
guidelines, already applied in previous papers [18,24,36]. Investigations regarding the methylmercury/dopamine system proved to be partic-
ularly complex. These complexities can be explained by taking into consideration two
factors: the first is related to the experimental measurements carried out in aqueous so-
lutions of NaCl, the second one to the tendency of this metalloid to form a complex of
high stability with the chloride, avoiding the hydrolysis of CH3Hg+, up to rather high pH
values (~8). The interactions of dopamine towards methylmercury(II) was investigated in quite
wide experimental conditions, from T = 288.15 to 318.15 K and from I = 0.15 to 1.0 mol dm−3. In order to check the formation of all the possible cation/ligand complexes, solutions at
different metal:ligand molar ratios were prepared (Table 2). Biomolecules 2021, 11, 1312 8 of 28 The potentiometric titrations were carried out up to pH~10, without observing the
formation of sparingly soluble species. The stability constants of CH3Hg+/Dop−species
at different ionic strengths and temperatures are reported in Table 3. The potentiometric titrations were carried out up to pH~10, without observing the
formation of sparingly soluble species. The stability constants of CH3Hg+/Dop−species
at different ionic strengths and temperatures are reported in Table 3. Table 3. Experimental formation constants of the CH3Hg+/Dop−species in NaCl aqueous solutions a
strengths and temperatures. tal formation constants of the CH3Hg+/Dop−species in NaCl aqueous solutions at different ionic
ratures Table 3. Experimental formation constants of the CH3Hg+/Dop−species in NaCl aqueous solutions at different ionic
strengths and temperatures. 3.2. CH3Hg+/Dop−System I/mol dm−3
logβML a)
logβMLH a
logβMLCl a
logβMLOH a
T = 288.15 K
0.151
10.89 ± 0.05 b
19.48 ± 0.08
11.42 ± 0.04
1.99 ± 0.06
0.502
10.87 ± 0.05
19.36 ± 0.09
11.32 ± 0.04
1.97 ± 0.06
0.751
10.91 ± 0.06
19.33 ± 0.11
11.31 ± 0.05
1.96 ± 0.06
1.004
10.95 ± 0.07
19.31 ± 0.13
11.30 ± 0.06
1.95 ± 0.07
T = 298.15 K
0.151
10.43 ± 0.03
19.43 ± 0.05
11.21 ± 0.02
1.99 ± 0.04
0.502
10.40 ± 0.03
19.30 ± 0.07
11.10 ± 0.03
1.98 ± 0.04
0.748
10.44 ± 0.04
19.27 ± 0.09
11.09 ± 0.04
1.97 ± 0.05
1.002
10.48 ± 0.06
19.25 ± 0.11
11.08 ± 0.05
1.96 ± 0.06
T = 310.15 K
0.152
9.91 ± 0.05
19.37 ± 0.03
10.96 ± 0.01
2.01 ± 0.02
0.496
9.88 ± 0.05
19.24 ± 0.06
10.86 ± 0.03
1.99 ± 0.03
0.751
9.91 ± 0.06
19.20 ± 0.09
10.84 ± 0.04
1.98 ± 0.03
0.998
9.95 ± 0.07
19.18 ± 0.12
10.83 ± 0.05
1.97 ± 0.05
T = 318.15 K
0.150
9.58 ± 0.07
19.33 ± 0.04
10.81 ± 0.02
2.01 ± 0.02
0.495
9.54 ± 0.07
19.19 ± 0.06
10.70 ± 0.03
1.99 ± 0.02
0.751
9.57 ± 0.07
19.16 ± 0.09
10.68 ± 0.04
1.99 ± 0.03
1.002
9.61 ± 0.08
19.13 ± 0.12
10.67 ± 0.06
1.98 ± 0.05
a Refers to the general equilibrium: pMn+ + qLz−+ rH+ + Cl−= MpLqHrCl(np−zq+r−1); b ±Std. Dev. As already observed in previous investigations [18,23,32], the speciation of methylmer-
cury(II) in sodium chloride aqueous solutions is characterized by the formation of the
stable MCl species (Table S2 of the Supplementary Material), so that in the presence of
dopamine, the formation of the ternary MLCl complex, together with the ML, MLH, and
MLOH ones, is observed, as reported in the speciation diagram in Figure 2. 9 of 30 5
6
7
8
9
10
0
20
40
60
80
100
5b,c
5a
2a,b,c
3c
3b
3a
6a,b,c
4c
4b
4a
1c
1b
1a
% CH3Hg
+
pH
Figure 2. Distribution diagram for the CH3Hg+/Dop− species at T = 298.15 K and different ionic
strengths. Species: 1. MCl; 2. MOH; 3. ML; 4. MLH; 5 MLCl; 6. MLOH. a: I = 0.151 mol dm−3; b: I =
0.502 mol dm−3; c: I = 1.002 mol dm−3. 3.2. CH3Hg+/Dop−System 3
At I = 0.15 mol dm−3, and between pH~6–9, the MLH is the main species of the
CH3Hg+/Dop− system, reaching a formation of 55–65% at pH~8. Increasing the ionic
t e
th the
a i
u
of fo
atio
of MLH hift at lo
e
H
ea hi
70% at I
1 0 p
g
The other CH3Hg+/Dop−species form with a yield of 15–20% for the ML in depen
dence on the ionic strength, and of ~90–95% for the MLOH at pH~9.5–10. At I = 0.15 mol dm−3, and between pH~6–9, the MLH is the main species of the
CH3Hg+/Dop− system, reaching a formation of 55–65% at pH~8. Increasing the ionic
t
th th
i
f f
ti
f MLH hift
t l
H
hi
70% t I
1 0 At I = 0.15 mol dm−3, and between pH~6–9, the MLH is the main species of the
CH3Hg+/Dop−system, reaching a formation of 55–65% at pH~8. Increasing the ionic
strength, the maximum of formation of MLH shifts at lower pH, reaching ~70% at I = 1.0
mol dm−3. The profile of the MLOH curves is instead almost unchanged. This behavior
could seem strange, but it can be easily explained considering that the overall formation
constant of this species is characterized by z* and p* values equal to zero (see Section 2.4.1). strength, the maximum of formation of MLH shifts at lower pH, reaching ~70% at I = 1.0
mol dm−3. The profile of the MLOH curves is instead almost unchanged. This behavior
could seem strange, but it can be easily explained considering that the overall formation
constant of this species is characterized by z* and p* values equal to zero (see Section
2.4.1.). The effect of the temperature on the speciation and distribution of the CH3Hg+/Dop−
i
i
h
i
h Fi
3 The effect of the temperature on the speciation and distribution of the CH3Hg+/Dop−
species is shown in the Figure 3. species is shown in the Figure 3. 5
6
7
8
9
10
0
20
40
60
80
100
3b
3a
2b
% CH3Hg
+
pH
1a
1b
2a
4a
4b
5a
5b
6b
6a
Figure 3. Distribution diagram for the CH3Hg+/Dop− species at I = 0.151 mol dm−3 and different tem-
peratures. Species: 1. MCl; 2. MOH; 3. MLH; 4. ML; 5 MLCl; 6. MLOH. a: T = 298.15 K; b: T = 318.15
K. 3.3. Metal2+/Dop−Systems The complexing ability of dopamine towards the bivalent cations (Mg2+, Ca2+, and
Sn2+) was investigated at different component concentrations and metal:ligand molar
ratios, as already reported in Table 2. Owing to the quite different acid-base properties
of Ca2+ and Mg2+ with respect to Sn2+, different speciation models and stability of the
complex species were obtained. 3.2. CH3Hg+/Dop−System (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP− = 1.5 mmol dm−3) [M = CH3Hg+; L = Dop−]. Figure 3. Distribution diagram for the CH3Hg+/Dop−species at I = 0.151 mol dm−3 and different
temperatures. Species: 1. MCl; 2. MOH; 3. MLH; 4. ML; 5 MLCl; 6. MLOH. a: T = 298.15 K; b: T =
318.15 K. (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP−= 1.5 mmol dm−3) [M = CH3Hg+;
L = Dop−]. 5
6
7
8
9
10
0
20
40
60
80
100
3b
3a
2b
% CH3Hg
+
pH
1a
1b
2a
4a
4b
5a
5b
6b
6a Figure 3. Distribution diagram for the CH3Hg+/Dop− species at I = 0.151 mol dm−3 and different tem-
peratures. Species: 1. MCl; 2. MOH; 3. MLH; 4. ML; 5 MLCl; 6. MLOH. a: T = 298.15 K; b: T = 318.15
K. (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP− = 1.5 mmol dm−3) [M = CH3Hg+; L = Dop−]. Figure 3. Distribution diagram for the CH3Hg+/Dop−species at I = 0.151 mol dm−3 and different
temperatures. Species: 1. MCl; 2. MOH; 3. MLH; 4. ML; 5 MLCl; 6. MLOH. a: T = 298.15 K; b: T =
318.15 K. (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP−= 1.5 mmol dm−3) [M = CH3Hg+;
L = Dop−]. Some important aspects can be discussed; the first one is that the MCl species un-
dergoes a considerable shift on the maximum of formation, changing the temperature, as
well as the corresponding formation percentage. As an example, at pH ~ 6.5, we have
percentages of ~18% at T = 318.15 K and ~90% at T = 298.15 K. The opposite trend is
observed for the ternary MLOH and MLH species. In the case of ML and MLCl, the pH
ranges of formation remain almost unchanged, while the percentages of formation are
lower increasing the temperature. 3.2. CH3Hg+/Dop−System (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP− = 1.5
mmol dm−3) [M = CH3Hg+; L = Dop−]. Figure 2. Distribution diagram for the CH3Hg+/Dop−species at T = 298.15 K and different ionic strengths. Species: 1. MCl; 2. MOH; 3. ML; 4. MLH; 5 MLCl; 6. MLOH. a: I = 0.151 mol dm−3; b: I = 0.502 mol dm−3;
c: I = 1.002 mol dm−3. (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP−= 1.5 mmol dm−3)
[M = CH3Hg+; L = Dop−]. 5
6
7
8
9
10
0
20
40
60
80
100
5b,c
5a
2a,b,c
3c
3b
3a
6a,b,c
4c
4b
4a
1c
1b
1a
% CH3Hg
+
pH Figure 2. Distribution diagram for the CH3Hg+/Dop− species at T = 298.15 K and different ionic
strengths. Species: 1. MCl; 2. MOH; 3. ML; 4. MLH; 5 MLCl; 6. MLOH. a: I = 0.151 mol dm−3; b: I =
0.502 mol dm−3; c: I = 1.002 mol dm−3. (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP− = 1.5
mmol dm−3) [M = CH3Hg+; L = Dop−]. Figure 2. Distribution diagram for the CH3Hg+/Dop−species at T = 298.15 K and different ionic strengths. Species: 1. MCl; 2. MOH; 3. ML; 4. MLH; 5 MLCl; 6. MLOH. a: I = 0.151 mol dm−3; b: I = 0.502 mol dm−3;
c: I = 1.002 mol dm−3. (Experimental conditions: cCH3Hg+ = 0.5 mmol dm−3; cDOP−= 1.5 mmol dm−3)
[M = CH3Hg+; L = Dop−]. Biomolecules 2021, 11, 1312 9 of 28 In particular, we observe an increase of the formation percentage of the MLCl species
with increasing the chloride concentration (i.e., the ionic strength) in the solution, and a
formation percentage that reaches ~20% at I = 1.0 mol dm−3. formation percentage that reaches ~20% at I = 1.0 mol dm−3. The other CH3Hg+/Dop− species form with a yield of 15–20% for the ML in depend-
ence on the ionic strength, and of ~90–95% for the MLOH at pH~9.5–10. At I
0 15
l d
3
d b t
H 6 9 th
MLH i
th
i
i
f th p
g
The other CH3Hg+/Dop−species form with a yield of 15–20% for the ML in depen-
dence on the ionic strength, and of ~90–95% for the MLOH at pH~9.5–10. 3.3.1. Ca2+/Dop−System a: I = 0.143 mol dm−3; b: I
(Experimental conditions: cCa2+ = 1.5 mmol dm−3; cDOP− = 5 m
As it can be highlighted in Figure 4, the useful ra
it
f
H
i
t
H 10 th f
ti
f
i
l igure 4. Distribution diagram for the Ca2+/Dop− species at T = 298.15 K and different ionic strengths. pecies: 1 ML; 2 MLH; 3 MLOH. a: I = 0.143 mol dm−3; b: I = 0.472 mol dm−3; c: I = 0.933 mol dm−3. Experimental conditions: cCa2+ = 1.5 mmol dm−3; cDOP− = 5 mmol dm−3) [M = Ca2+; L = Dop−]. Figure 4. Distribution diagram for the Ca2+/Dop−species at T = 298.15 K and different ionic strengths. Species: 1 ML; 2 MLH; 3 MLOH. a: I = 0.143 mol dm−3; b: I = 0.472 mol dm−3; c: I = 0.933 mol dm−3. (Experimental conditions: cCa2+ = 1.5 mmol dm−3; cDOP−= 5 mmol dm−3) [M = Ca2+; L = Dop−]. units of pH, since at pH ~10, the formation of an insoluble species is observed. The distribution of the Ca2+/Dop− species at different temperatures is reported in Fig-
ure 5. We can observe a significant variation on the distribution of the species, both in
terms of the pH of maximum formation and formation percentages. As it can be highlighted in Figure 4, the useful range for the complexation is just two
units of pH, since at pH ~10, the formation of an insoluble species is observed. The distribution of the Ca2+/Dop− species at different temperatures is reported in Fig-
ure 5. We can observe a significant variation on the distribution of the species, both in
terms of the pH of maximum formation and formation percentages. 8.0
8.5
9.0
9.5
10.0
0
20
40
60
80
100
% Ca
2+
pH
2c
2a
1b
3a
3b
3c
1a
8.0
8.5
9.0
9.5
10.0
0
20
40
60
80
100
% Ca
2+
pH
2c
2a
1b
3a
3b
3c
1a
Figure 5. Distribution diagram for the Ca2+/Dop− species at I~0.15 mol dm−3 and different tempera-
tures. Species: 1 ML; 2 MLH; 3 MLOH.a: T = 288.15 K; b: T = 298.15 K; c: T = 310.15 K. (Experimental
conditions: cCa2+ = 1.5 mmol dm−3; cDOP− = 5 mmol dm−3) [M = Ca2+; L = Dop−]. 3 3 2 Mg2+/Dop−System
Figure 5. 3.3.1. Ca2+/Dop−System 11 of 30
60
80
a
2+
1a
1c 8.0
8.5
9.0
9.5
10.0
0
20
40
60
80
100
% Ca
2+
pH
1a
1b
1c
2a
2b
2c
3c
3a
3b
Figure 4. Distribution diagram for the Ca2+/Dop− species at T = 298.15 K and different ionic strengths. Species: 1 ML; 2 MLH; 3 MLOH. a: I = 0.143 mol dm−3; b: I = 0.472 mol dm−3; c: I = 0.933 mol dm−3. (Experimental conditions: cCa2+ = 1.5 mmol dm−3; cDOP− = 5 mmol dm−3) [M = Ca2+; L = Dop−]. Figure 4. Distribution diagram for the Ca2+/Dop−species at T = 298.15 K and different ionic strengths. Species: 1 ML; 2 MLH; 3 MLOH. a: I = 0.143 mol dm−3; b: I = 0.472 mol dm−3; c: I = 0.933 mol dm−3. (Experimental conditions: cCa2+ = 1.5 mmol dm−3; cDOP−= 5 mmol dm−3) [M = Ca2+; L = Dop−]. 8.0
8.5
9.0
9.5
10.0
0
20
40
% C
pH
1b
2a
2b
2c
3c
3a
3b
Figure 4. Distribution diagram for the Ca2+/Dop− species at T = 298.15 K and different ionic strengths. Species: 1 ML; 2 MLH; 3 MLOH. a: I = 0.143 mol dm−3; b: I = 0.472 mol dm−3; c: I = 0.933 mol dm−3. (Experimental conditions: cCa2+ = 1.5 mmol dm−3; cDOP− = 5 mmol dm−3) [M = Ca2+; L = Dop−]. As it can be highlighted in Figure 4, the useful range for the complexation is just two
units of pH, since at pH ~10, the formation of an insoluble species is observed. The distribution of the Ca2+/Dop− species at different temperatures is reported in Fig-
ure 5. We can observe a significant variation on the distribution of the species, both in
terms of the pH of maximum formation and formation percentages. 8.0
8.5
9.0
9.5
10.0
0
20
40
60
80
100
% Ca
2+
pH
1a
1b
1c
2a
2b
2c
3c
3a
3b
8.0
8.5
9.0
9.5
10.0
0
20
40
% C
pH
1b
2a
2b
2c
3c
3a
3b
Figure 4. Distribution diagram for the Ca2+/Dop− species at T
Species: 1 ML; 2 MLH; 3 MLOH. 3.3.1. Ca2+/Dop−System For the calcium interactions with dopamine, the speciation model, which gave the best
results in terms of statistical parameters, was characterized by only the MLH and MLOH
species. At T = 298.15 K, the higher number of available experimental data collected at
different ionic strengths also allowed the determination of the ML species, which is formed
in smaller percentages if compared with the other two MLH and MLOH complexes. Biomolecules 2021, 11, 1312 10 of 28 10 of 28 The formation constants for the Ca2+/Dop−species are reported in Table 4; in particu-
lar, data at T = 298.15 K, refer to different ionic strengths, whilst at T = 288.15 and 310.15 K,
only to I = 0.15 mol dm−3. From the results, we can observe a low stability for each species. The formation constants for the Ca2+/Dop−species are reported in Table 4; in particu-
lar, data at T = 298.15 K, refer to different ionic strengths, whilst at T = 288.15 and 310.15 K,
only to I = 0.15 mol dm−3. From the results, we can observe a low stability for each species. The formation constants for the Ca2+/Dop−species are reported in Table 4; in particu-
lar, data at T = 298.15 K, refer to different ionic strengths, whilst at T = 288.15 and 310.15 K,
only to I = 0.15 mol dm−3. From the results, we can observe a low stability for each species. Table 4. Experimental formation constants of the Ca2+/Dop−species in NaCl aqueous solutions. Table 4. Experimental formation constants of the Ca2+/Dop−species in NaCl aqueous solutions. T/K
I/mol dm−3
logβML a
logβMLH a
logβMLOH a
288.15
0.150
-
13.60 ± 0.03 b
−6.97 ± 0.08 b
298.15
0.143
4.83 ± 0.08 b
13.43 ± 0.08
−5.78 ± 0.16
298.15
0.472
3.65 ± 0.07
13.28 ± 0.06
−7.78 ± 0.10
298.15
0.692
3.95 ± 0.07
13.65 ± 0.05
−6.69 ± 0.09
298.15
0.933
5.26 ± 0.10
14.44 ± 0.06
−6.49 ± 0.15
310.15
0.150
-
13.51 ± 0.23
−5.60 ± 0.22
a Refers to the general equilibrium: pMn+ + qLz−+ rH+ = MpLqHr(np−zq+r); b ±Std. Dev. Biomolecules 2021, 11, x
11 of 30
100 Some interesting observations can be made from the distribution diagrams given in
Figures 4 and 5. The complexation takes place at pH > 7.5, except for the ionic strength
I = 1.0 mol dm−3, where it begins at lower pH. 3.3.2. Mg2+/Dop−System The Mg2+/Dop−interactions were investigated at different of ionic strengths
(I range = 0.15–1.0 mol dm−3) and temperatures (from T = 288.15 to 310.15 K). The stoi-
chiometry and stability constants of the Mg2+/Dop−species were determined following
the procedure already reported in the previous sections. The selected speciation model that
gave the best results in terms of statistical parameters considers the ML and MLOH species. Table 5 reports the formation constants at different temperatures and ionic strengths
in NaCl aqueous solution. Table 5. Experimental formation constants of the Mg2+/Dop−species in NaCl aqueous solutions at
different ionic strengths and temperatures. I/mol dm−3. logβML a)
logβMLOH a)
T = 288.15 K
0.151
3.459 ± 0.068 b)
−6.278 ± 0.040 b)
0.503
3.259 ± 0.078
−6.676 ± 0.030
0.753
3.267 ± 0.084
−6.905 ± 0.032
1.004
3.467 ± 0.092
−7.336 ± 0.012
T = 298.15 K
0.148
3.034 ± 0.044
−6.111 ± 0.026
0.501
2.797 ± 0.052
−6.531 ± 0.020
0.753
2.777 ± 0.056
−6.825 ± 0.022
0.996
2.951 ± 0.092
−7.324 ± 0.008
T = 310.15 K
0.150
2.561 ± 0.064
−5.926 ± 0.040
0.496
2.281 ± 0.074
−6.369 ± 0.030
0.748
2.232 ± 0.082
−6.736 ± 0.034
1.003
2.375 ± 0.092
−7.311 ± 0.012
a) Refers to the general equilibrium: pMn+ + qLz−+ rH+ = MpLqHr(np−zq+r); b) ±Std. Dev. Table 5. Experimental formation constants of the Mg2+/Dop−species in NaCl aqueous solutions at
different ionic strengths and temperatures. a) Refers to the general equilibrium: pMn+ + qLz−+ rH+ = MpLqHr(np−zq+r); b) ±Std. Dev. The formation of the metal–ligand species occurs at pH~8.5, and up to this pH value
magnesium is present as free metal ion, and only at higher pH, the MLOH(aq) complex
becomes the predominant one, and ML reaches ~10%. p
As an example, the distribution of the Mg2+/Dop−species at T = 310.15 K and I =
0.15 mol dm−3 is reported in Figure S1 of the Supplementary Material. 2 Unlike the Ca2+/Dop−system, the formation of the MLH species was not observed. This can be probably due to the different behavior of the two metals in term of hy-
drolytic constants. y
As an example, from the distribution diagrams of the two metal/Dop systems, we
observe that the Ca2+ and dopamine interaction begins at pH~7.5, whilst for Mg2+, at about
8.5; this aspect can explain why, in the second case, we did not obtain the formation of the
MLH species. 3.3.1. Ca2+/Dop−System Distribution diagram for the Ca2+/Dop−species at I~0.15 mol dm−3 and different tempera-
tures. Species: 1 ML; 2 MLH; 3 MLOH.a: T = 288.15 K; b: T = 298.15 K; c: T = 310.15 K. (Experimental
conditions: cCa2+ = 1.5 mmol dm−3; cDOP−= 5 mmol dm−3) [M = Ca2+; L = Dop−]. As it can be highlighted in Figure 4, the useful range
nits of pH, since at pH ~10, the formation of an insolubl
The distribution of the Ca2+/Dop− species at different
re 5. We can observe a significant variation on the dis
erms of the pH of maximum formation and formation p
40
60
80
100
% Ca
2+
2c
2a
3
1a
8.0
8.5
9.0
9.5
10.0
0
20
40
60
80
100
% Ca
2+
pH
2c
2a
1b
3a
3b
3c
1a 8.0
8.5
9.0
9.5
10.0
0
20
1b
3a
3b
3c
Figure 5. Distribution diagram for the Ca2+/Dop− species at I~0.15 mol dm−3 and different tempera-
tures. Species: 1 ML; 2 MLH; 3 MLOH.a: T = 288.15 K; b: T = 298.15 K; c: T = 310.15 K. (Experimental
conditions: cCa2+ = 1.5 mmol dm−3; cDOP− = 5 mmol dm−3) [M = Ca2+; L = Dop−]. Figure 5. Distribution diagram for the Ca2+/Dop−species at I~0.15 mol dm−3 and different tempera-
tures. Species: 1 ML; 2 MLH; 3 MLOH.a: T = 288.15 K; b: T = 298.15 K; c: T = 310.15 K. (Experimental
conditions: cCa2+ = 1.5 mmol dm−3; cDOP−= 5 mmol dm−3) [M = Ca2+; L = Dop−]. Biomolecules 2021, 11, 1312 11 of 28 11 of 28 As it can be highlighted in Figure 4, the useful range for the complexation is just two
units of pH, since at pH~10, the formation of an insoluble species is observed. p
p
p
The distribution of the Ca2+/Dop−species at different temperatures is reported in
Figure 5. We can observe a significant variation on the distribution of the species, both in
terms of the pH of maximum formation and formation percentages. 3.3.2. Mg2+/Dop−System 3.3.3. Sn2+/Dop−System Sn2+ has different acid-base properties and behaviors with respect to calcium and mag-
nesium, since it tends to form polynuclear hydrolytic species also at low pH values [18,37–39]. Moreover, Sn2+ forms, SnCli (i = 1–3) species of significant stability and a mixed SnOHCl
hydrolytic complex in chloride solutions. As already reported in a previous investigation [35],
the formation of the Sn(OH)2(s) sparingly soluble species at pH values is dependent on the Biomolecules 2021, 11, 1312 12 of 28 12 of 28 metal concentration, ionic medium and ionic strength and must be considered. All the equilibria
of formation of these species, together with the dopamine protonation constants, were used as
input in the speciation scheme. Applying the already cited criteria of selection of the speciation
model, the best results were obtained when the following complexes were considered: M2L2,
ML2, MLOH, and M2LOH, whose stability constants are reported in Table 6. Table 6. Experimental formation constants of the Sn2+/Dop−species in NaCl aqueous solutions. T/K. I/mol dm−3
logβM2L2 a
logβML2 a
logβMLOH a
logβM2LOH a
288.15
0.150
35.21 ± 0.09 b
24.51 ± 0.01 b
9.54 ± 0.01 b
15.43 ± 0.06 b
298.15
0.147
34.83 ± 0.01
24.12 ± 0.01
9.49 ± 0.01
15.67 ± 0.03
298.15
0.472
34.35 ± 0.01
23.10 ± 0.01
9.10 ± 0.01
15.55 ± 0.02
298.15
0.709
33.95 ± 0.01
22.51 ± 0.01
8.74 ± 0.01
15.96 ± 0.01
298.15
0.954
31.68 ± 0.07
20.75 ± 0.03
7.63 ± 0.02
15.60 ± 0.05
310.15
0.150
32.54 ± 0.09
20.89 ± 0.02
8.47 ± 0.06
14.90 ± 0.01
a Refers to the general equilibrium: pMn+ + qLz−+ rH+ = MpLqHr(np−zq+r); b ±Std. Dev. Biomolecules 2021, 11, x
13 of 30
298.15
0.954
31.68 ± 0.07
20.75 ± 0.03
7.63 ± 0.02
15.60 ± 0.05
310.15
0.150
32.54 ± 0.09
20.89 ± 0.02
8.47 ± 0.06
14.90 ± 0.01
a Refers to the general equilibrium: pMn+ + qLz− + rH+ = MpLqHr(np−zq+r); b ±Std. Dev. e 6. Experimental formation constants of the Sn2+/Dop−species in NaCl aqueous solutions. From the distribution diagrams shown in Figure 6, we can observe that all species are
formed in high percentages, covering the entire range of pH investigated. Moreover, the
free metal ion is present in very lower percentages (~5% and at pH < 4) with respect the
other two M2+/Dop−systems. 3.3.3. Sn2+/Dop−System The M2L2 species is present in almost all of the investigated
pH ranges, representing the main complexes up to pH~6.5, beyond which the species
MLOH and ML2 become the most important ones. From the distribution diagrams shown in Figure 6, we can observe that all species
are formed in high percentages, covering the entire range of pH investigated. Moreover,
the free metal ion is present in very lower percentages (~5% and at pH < 4) with respect
the other two M2+/Dop− systems. The M2L2 species is present in almost all of the investi-
gated pH ranges, representing the main complexes up to pH ~6.5, beyond which the spe-
cies MLOH and ML2 become the most important ones. 2
3
4
5
6
7
8
0
20
40
60
80
100
8b
7b
6a
5a
4b
3b
2b
1b
8a
7a
6b
5b
4a
3a
2a
1a
% Sn
2+
pH
Figure 6. Distribution diagram for the Sn2+/Dop− species a T = 298.15 K and different ionic strengths. a: I = 0.147 mol dm−3; b: I = 0.954 mol dm−3. Species: 1. MCl; 2. MCl2; 3. MCl3; 4. MCl(OH); 5. M2L2; 6. ML2; 7. MLOH; 8. M2LOH. (Experimental conditions: cSn2+ = 1.5 mmol dm−3; cDOP− = 5 mmol dm−3) [M
= Sn2+; L = Dop−]. Figure 6. Distribution diagram for the Sn2+/Dop−species a T = 298.15 K and different ionic strengths. a: I = 0.147 mol dm−3; b: I = 0.954 mol dm−3. Species: 1. MCl; 2. MCl2; 3. MCl3; 4. MCl(OH); 5. M2L2; 6. ML2; 7. MLOH; 8. M2LOH. (Experimental conditions: cSn2+ = 1.5 mmol dm−3; cDOP−= 5
mmol dm−3) [M = Sn2+; L = Dop−]. 2
3
4
5
6
7
8
0
20
40
60
80
100
8b
7b
6a
5a
4b
3b
2b
1b
8a
7a
6b
5b
4a
3a
2a
1a
% Sn
2+
pH pH Figure 6. Distribution diagram for the Sn2+/Dop− species a T = 298.15 K and different ionic strengths. a: I = 0.147 mol dm−3; b: I = 0.954 mol dm−3. Species: 1. MCl; 2. MCl2; 3. MCl3; 4. MCl(OH); 5. M2L2; 6. ML2; 7. MLOH; 8. M2LOH. (Experimental conditions: cSn2+ = 1.5 mmol dm−3; cDOP− = 5 mmol dm−3) [M
= Sn2+; L = Dop−]. Figure 6. Distribution diagram for the Sn2+/Dop−species a T = 298.15 K and different ionic strengths. 3.3.3. Sn2+/Dop−System For the modeling of the Ca2+ and Sn2+ systems, Equation (6) was
employed, but without using the Van’t Hoff term, since experiments at different ionic strengths
were only carried out at T = 298.15 K. g
y
For these last two systems, the enthalpy change values of formation of the complex
ecies were only calculated at I = 0.15 mol dm−3 by means of the classical formulation of
e Van’t Hoff equation:
For these last two systems, the enthalpy change values of formation of the complex
species were only calculated at I = 0.15 mol dm−3 by means of the classical formulation of
the Van’t Hoff equation: logβpqrT’ = logβpqrT + 1/(R·2.303)·∆Hθ·(1/T − 1/T’)
(16)
logβpqrT’ = logβpqrT + 1/(R·2.303)·∆Hθ·(1/T −1/T’)
(16) 6) (16) here T and T’ are the reference and desired temperatures, respectively, expressed in kel-
n (K); Hθ, is the temperature coefficient valid for the investigated temperature interval,
at can be related to the standard enthalpy change values. For CH3Hg+ and Mg2+ systems, the simplified Equation (6) (without considering the
where T and T’ are the reference and desired temperatures, respectively, expressed in kelvin
(K); ∆Hθ, is the temperature coefficient valid for the investigated temperature interval, that
can be related to the standard enthalpy change values. For CH3Hg+ and Mg2+ systems, the simplified Equation (6) (without considering the here T and T’ are the reference and desired temperatures, respectively, expressed in kel-
n (K); Hθ, is the temperature coefficient valid for the investigated temperature interval,
at can be related to the standard enthalpy change values. For CH3Hg+ and Mg2+ systems the simplified Equation (6) (without considering the
where T and T’ are the reference and desired temperatures, respectively, expressed in kelvin
(K); ∆Hθ, is the temperature coefficient valid for the investigated temperature interval, that
can be related to the standard enthalpy change values. 2 For CH3Hg+ and Mg2+ systems, the simplified Equation (6) (without considering the
n’t Hoff term) was also applied at each single temperature (Tables 7 and 8). For CH3Hg+ and Mg2+ systems, the simplified Equation (6) (without considering the
Van’t Hoff term) was also applied at each single temperature (Tables 7 and 8). Table 7. Formation constants at infinite dilution and different temperatures of the CH3Hg+/Dop− species. 3.3.3. Sn2+/Dop−System For the overall stability constants of MLOH and M2LOH,
the differences are smaller, namely about 1 and 0.5 order of magnitudes, respectively. 14 of 30 2
4
6
8
0.0
0.2
0.4
0.6
0.8
1.0
T = 318.15 K
T = 310.15 K
T = 298.15 K
T = 288.15 K
x
pL
(a)
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
pH = 10.5
pH = 8.2
pH = 7.4
x
pL
(b)
gure 7. Sequestering diagram of dopamine towards CH3Hg+ in NaCl aqueous solution. (a) I = 0.15
ol dm−3, pH = 7.4 at different temperatures; (b) I = 0.15 mol dm−3, T = 310.15 K and different pH
alues. pL0.5 values: (a) 2.36 (T = 288.15 K) < 2.63 (T = 298.15 K) < 3.74 (T = 310.15 K) < 4.50 (T = 318.15
).(b) 3.74 (pH = 7.4) < 4.41 (pH = 8.2) < 6.15 (pH = 10.5). Figure 7. Sequestering diagram of dopamine towards CH3Hg+ in NaCl aqueous solution. (a) I = 0.15 mol
dm−3, pH = 7.4 at different temperatures; (b) I = 0.15 mol dm−3, T = 310.15 K and different pH values. pL0.5 values: (a) 2.36 (T = 288.15 K) < 2.63 (T = 298.15 K) < 3.74 (T = 310.15 K) < 4.50 (T = 318.15 K). (b) 3.74
(pH = 7.4) < 4.41 (pH = 8.2) < 6.15 (pH = 10.5). 2
4
6
8
0.0
0.2
0.4
0.6
0.8
1.0
T = 318.15 K
T = 310.15 K
T = 298.15 K
T = 288.15 K
x
pL
(a) 2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
pH = 10.5
pH = 8.2
pH = 7.4
x
pL
(b) gure 7. Sequestering diagram of dopamine towards CH3Hg+ in NaCl aqueous solution. (a) I = 0.15
ol dm−3, pH = 7.4 at different temperatures; (b) I = 0.15 mol dm−3, T = 310.15 K and different pH
lues. pL0.5 values: (a) 2.36 (T = 288.15 K) < 2.63 (T = 298.15 K) < 3.74 (T = 310.15 K) < 4.50 (T = 318.15
(b) 3.74 (pH = 7.4) < 4.41 (pH = 8.2) < 6.15 (pH = 10.5). Figure 7. Sequestering diagram of dopamine towards CH3Hg+ in NaCl aqueous solution. 3.3.3. Sn2+/Dop−System (a) I = 0.15 mol
dm−3, pH = 7.4 at different temperatures; (b) I = 0.15 mol dm−3, T = 310.15 K and different pH values. pL0.5 values: (a) 2.36 (T = 288.15 K) < 2.63 (T = 298.15 K) < 3.74 (T = 310.15 K) < 4.50 (T = 318.15 K). (b) 3.74
(pH = 7.4) < 4.41 (pH = 8.2) < 6.15 (pH = 10.5). 4. Modeling of the Stability Constants with Respect to Ionic Strength and Temperature
3.4. Modeling of the Stability Constants with Respect to Ionic Strength and Temperature . Modeling of the Stability Constants with Respect to Ionic Strength and Temperature
3.4. Modeling of the Stability Constants with Respect to Ionic Strength and Temperature As already reported in the Section 2.4., two different approaches can be used to model
e dependence of the stability constants on the ionic strength. As already reported in the Section 2.4, two different approaches can be used to model
the dependence of the stability constants on the ionic strength. As already reported in the Section 2.4., two different approaches can be used to model
e dependence of the stability constants on the ionic strength. As already reported in the Section 2.4, two different approaches can be used to model
the dependence of the stability constants on the ionic strength. The first approach uses an extended Debye–Hückel equation (Equation (6) of Section
4.1.), which can also allow the modeling of the dependence of the stability constants on
e temperature by the calculation of the enthalpy change values of formation of the
etal/ligand complexes, when measurements at different T/K are performed. This equa-
on was applied for the CH3Hg+/Dop− and Mg2+/Dop− systems, investigated at 288.15 ≤
/K ≤ 318.15 and 0.15 ≤ I/mol dm−3 ≤ 1.0. For the modeling of the Ca2+ and Sn2+ systems,
quation (6) was employed, but without using the Van’t Hoff term, since experiments at
fferent ionic strengths were only carried out at T = 298.15 K. The first approach uses an extended Debye–Hückel equation (Equation (6) of Section 2.4.1),
which can also allow the modeling of the dependence of the stability constants on the tem-
perature by the calculation of the enthalpy change values of formation of the metal/ligand
complexes, when measurements at different T/K are performed. This equation was applied
for the CH3Hg+/Dop−and Mg2+/Dop−systems, investigated at 288.15 ≤T/K ≤318.15 and
0.15 ≤I/mol dm−3 ≤1.0. 3.3.3. Sn2+/Dop−System a: I = 0.147 mol dm−3; b: I = 0.954 mol dm−3. Species: 1. MCl; 2. MCl2; 3. MCl3; 4. MCl(OH); 5. M2L2; 6. ML2; 7. MLOH; 8. M2LOH. (Experimental conditions: cSn2+ = 1.5 mmol dm−3; cDOP−= 5
mmol dm−3) [M = Sn2+; L = Dop−]. The strength of the Sn2+/Dop− complexes avoids the formation of high amounts of the
Snp(OH)q species, reaching about 5% of formation (not reported in Figure 7), even if the
SnOHCl one at I = 0.15 mol dm−3 and pH ~3, achieves about 20% of formation. The pH
limit investigated (pH~8–9) is dependent on the different used experimental conditions
and on the formation of an insoluble species. The effect of the ionic strength on the stability
of the complexes can be highlighted observing the distribution of species at different pH
values (Figure 6) and the corresponding formation percentages. Different formation per-
centages and a shift of the maximum of formation are observed when the ionic strength
and the NaCl concentration increases from I = 0.15 to 1.0 mol dm−3. A similar observation
can be made for the effect of the temperature; in this case, for the overall stability constants
of M2L2 and ML2 species, differences of about 3 orders of magnitudes between T = 288.15
The strength of the Sn2+/Dop−complexes avoids the formation of high amounts of
the Snp(OH)q species, reaching about 5% of formation (not reported in Figure 7), even if the
SnOHCl one at I = 0.15 mol dm−3 and pH~3, achieves about 20% of formation. The pH limit
investigated (pH~8–9) is dependent on the different used experimental conditions and on
the formation of an insoluble species. The effect of the ionic strength on the stability of the
complexes can be highlighted observing the distribution of species at different pH values
(Figure 6) and the corresponding formation percentages. Different formation percentages
and a shift of the maximum of formation are observed when the ionic strength and the NaCl
concentration increases from I = 0.15 to 1.0 mol dm−3. A similar observation can be made
for the effect of the temperature; in this case, for the overall stability constants of M2L2 and
ML2 species, differences of about 3 orders of magnitudes between T = 288.15 and 310.15 K at 13 of 28 Biomolecules 2021, 11, 1312 13 of 28 I = 0.15 mol dm−3 were observed. 3.3.3. Sn2+/Dop−System logTβML b)
logTβMLOH b)
T = 288.15 K
I→0 a)
3.895 ± 0.040 c)
−5.291 ± 0.090 c)
C d)
0.190 ± 0.072
−0.692 ± 0.144
T = 298.15 K
I→0
3.560 ± 0.036
−5.366 ± 0.108
C
0.140 ± 0.090
−0.612 ± 0.152
T = 310.15 K
I→0
3.007 ± 0.170
−4.796 ± 0.094
C
−0.007 ± 0.223
−1.239 ± 0.146
a) I/mol dm−3; b) refers to the general equilibrium: pMn+ + qLz−+ rH+ = MpLqHr(np−zq+r); c) ±Std. Dev.;
d) parameter for the dependence of logβpqr on I/mol dm−3. Table 8. Formation constants at infinite dilution and different temperatures of the Mg2+/Dop−species. By using the approaches above reported, the stability constants at infinite dilu-
tion and the parameters for the dependence on ionic strength were calculated, con-
sidering as reference temperature T = 298.15 K. These data are reported in Table 9 to-
gether with the enthalpy and entropy change values of formation of the species. We
remember that the ∆H/kJ mol−1 and T∆S/kJ mol−1 of Ca2+ and Sn2+ systems refer to
I = 0.15 mol dm−3. Table 9. Formation constants at infinite dilution and T = 298.15 K for the Mn+/Dop−species and enthalpy and entropy
change values. 3.3.3. Sn2+/Dop−System logTβML b
logTβMLH b
logTβMLCl b
logTβMLOH b
T = 288.15 K
I→0 a)
10.80 ± 0.02 c
19.52 ± 0.03 c
11.44 ± 0.02 c
1.99 ± 0.06 c
C d
1.58 ± 0.04
−0.40 ± 0.03
1.68 ± 0.04
−0.16 ± 0.03
T = 298.15 K
I→0
10.62 ± 0.04
19.65 ± 0.05
11.42 ± 0.02
2.00 ± 0.04
C
0.30 ± 0.02
2.18 ± 0.11
0.07 ± 0.06
0.99 ± 0.14
T = 310.15 K
I→0
10.48 ± 0.02
19.43 ± 0.01
11.16 ± 0.02
2.01 ± 0.03
C
−0.62 ± 0.02
0.49 ± 0.04
−0.49 ± 0.03
−0.24 ± 0.04
T = 318.15 K
Table 7. Formation constants at infinite dilution and different temperatures of the CH3Hg+/Dop−species. logTβML b
logTβMLH b
logTβMLCl b
logTβMLOH b
T = 288.15 K
I→0 a
10.80 ± 0.02 c
19.52 ± 0.03 c
11.44 ± 0.02 c
1.99 ± 0.06 c
Cd
1.58 ± 0.04
−0.40 ± 0.03
1.68 ± 0.04
−0.16 ± 0.03
T = 298.15 K
I→0
10.62 ± 0.04
19.65 ± 0.05
11.42 ± 0.02
2.00 ± 0.04
C
0.30 ± 0.02
2.18 ± 0.11
0.07 ± 0.06
0.99 ± 0.14
T = 310.15 K
I→0
10.48 ± 0.02
19.43 ± 0.01
11.16 ± 0.02
2.01 ± 0.03
C
−0.62 ± 0.02
0.49 ± 0.04
−0.49 ± 0.03
−0.24 ± 0.04
T = 318.15 K
I→0
9.79 ± 0.08
19.56 ± 0.02
11.50 ± 0.02
2.02 ± 0.02
C
−0.68 ± 0.10
−0.49 ± 0.03
−0.38 ± 0.02
−0.10 ± 0.01
a I/mol dm−3; b refers to the general equilibrium: pMn+ + qLz−+ rH+ + Cl−= MpLqHrCl(np−zq+r−1); c ±Std. Dev.; d parameter for the
dependence of logβpqr on I/mol dm−3. onstants at infinite dilution and different temperatures of the CH3Hg+/Dop− species. n constants at infinite dilution and different temperatures of the CH3Hg+/Dop−species. Table 7. Formation constants at infinite dilution and different temperatures of the CH3Hg+/Dop− species. Table 7. Formation constants at infinite dilution and different temperatures of the CH3Hg+/Dop−specie C
−0.62 ± 0.02
0.49 ± 0.04
−0.49 ± 0.03
−0.24 ± 0.04
T = 318.15 K
a I/mol dm−3; b refers to the general equilibrium: pMn+ + qLz−+ rH+ + Cl−= MpLqHrCl(np−zq+r−1); c ±Std. Dev.; d parameter for the
dependence of logβpqr on I/mol dm−3. Biomolecules 2021, 11, 1312 14 of 28 Table 8. Formation constants at infinite dilution and different temperatures of the Mg2+/Dop−species. c/m = d0 + a1c +a2c2 3.3.3. Sn2+/Dop−System The c/m ratio can be
determined by the following formula: c/m = d0 + a1c +a2c2
(17) (17) Biomolecules 2021, 11, 1312 15 of 28 where d0 is the water density at T = 273.15 K, d0 = 0.99987 g/cm3. The dependence of
water density on temperature can be modeled by the following equation: d(T/K) = [(d0 + p1·T/K −p2·(T/K)2)/(p3·(T/K) + 1))]·10−3
(18) (18) where d(T/K) is the water density at the desired temperature; p1 = 8.119, p2 = 7.8735·10−3
and p3 = 8.0574·10−3 are empirical parameters obtained by modeling the data pair, pure
water density (g/cm3) derived from literature data [40] vs. T/K. The knowledge of the
water density at the desired temperature and the molarity and molality of the electrolytic
solution (NaCl in our case) at a given saline concentration, allows the calculation of the c/m
value. Using Equation (17), the following parameters have been calculated: a1 = −0.017765,
a2 = −6.525·10−4. The range of validity for conversion in NaCl is: 0 ≤I (NaCl)/mol kg−1
≤6. The stability constants converted in the molal concentration scale, by using the above
approach, are reported in Tables S3–S6 of the Supplementary Material. From the ionic strength and formation constants expressed in the molal concentra-
tion scale, the application of the SIT model allows the determination of the specific ion
interaction coefficients of all the ion pairs formed by the interactions of the cations with
dopamine. These calculations also require the use of the specific ion interaction coefficients of the
other ionic species present in the solution, namely: ε(H+,Cl−) = 0.12 [41]; ε(Dop−,Na+) =
−0.228 [10]; ε(Ca2+,Cl−) = 0.14 [29]; ε(Mg2+,Cl−) = 0.209 [29] and ε(Sn2+,Cl−) = 0.032 [35]. For the CH3Hg+/Dop−system, this calculation is not possible, because the SIT co-
efficient for the interaction of methylmercury with chloride is unknown. In this case, the
determination of ∆ε instead of classical SIT coefficients is particularly indicated, since
the system of equations related to various equilibria results undetermined, hampering
the calculation of other single ε. However, this approach was also applied to the other
metal/dopamine systems here investigated. The specific ion interaction coefficients (ε and ∆ε) of the ionic species and the Setschenow
coefficients for the neutral ones are reported in Table 10. Table 10. Specific ion interaction parameters and Setschenow coefficients for the species of the metal/dopamine systems. 3.3.3. Sn2+/Dop−System Species
logTβpqr a
C b
∆H c
T∆S c
CH3Hg+/Dop−
ML
10.62 ± 0.03 d
0.27 ± 0.06 d
−76 ± 5 d
−15 ± 5 d
MLH
19.64 ± 0.05
0.01 ± 0.10
−8 ± 5
104 ± 5
MLCl
11.41 ± 0.02
0.08 ± 0.05
−34 ± 2
31 ± 2
MLOH
2.00 ± 0.04
−0.04 ± 0.05
2 ± 3
13 ± 3
Ca2+/Dop−
ML
5.30 ± 0.07 d
−0.88 ± 0.12 d
-
MLH
13.80 ± 0.06
0.33 ± 0.15
−58 ± 25 d,e
20 ± 25 d,e
MLOH
−6.18 ± 0.06
−0.06 ± 0.09
99 ± 43
64 ± 43
Mg2+/Dop−
ML
3.560 ± 0.036 d
0.07 ± 0.04 d
−72 ± 2
52 ± 2 d
MLOH
−5.366 ± 0.108
−1.03 ± 0.03
24 ± 3
−7 ± 3
Sn2+/Dop−
M2L2
36.40 ± 0.29 d
−2.90 ± 0.50 d
−211 ± 28 d,e
−4 ± 28 d,e
ML2
25.56 ± 0.20
−3.33 ± 0.36
−286 ± 35
−141 ± 35
MLOH
10.43 ± 0.19
−1.75 ± 0.33
−84 ± 20
−24 ± 21
M2L(OH)
16.11 ± 0.04
0.30 ± 0.06
−42 ± 21
50 ± 21
a Refers to the general equilibrium: pMn+ + qLz−+ rH+ + Cl−= MpLqClHr(np−zq+r−1); b parameter for the dependence on I/mol dm−3; c
enthalpy and entropy change values of formation in kJ/mol; d ±Std. Dev.; e calculated at I = 0.15 mol dm−3 and T = 298.15 K. ion constants at infinite dilution and T = 298.15 K for the Mn+/Dop−species and enthalpy and entropy Table 9. Formation constants at infinite dilution and T = 298.15 K for the Mn+/Dop−species and enthalpy and entropy
change values. Table 9. Formation constants at infinite dilution and T = 298.15 K for the Mn+/Dop−species and
change values. From a comparison between the formation constants at the same experimental conditions,
it is evident that Sn2+ forms complexes with dopamine featured by a higher stability, with
respect to the other cations, and that it is the only metal that forms polynuclear complexes. Concerning the second model used to study the dependence of the stability constants
on the ionic strength, namely the SIT approach, both the concentrations and the stabil-
ity constants were converted from the molar to the molal scale. 3.3.3. Sn2+/Dop−System CH3Hg+
∆εML a
∆εMLH a
∆εMLCl a
∆εMLOH a
T = 288.15 K
1.53 ± 0.02 b
−0.88 ± 0.06
1.61 ± 0.02
−0.15 ± 0.03
T = 298.15 K
0.38 ± 0.02
2.43 ± 0.15
0.48 ± 0.04
0.99 ± 0.15
T = 310.15 K
−0.64 ± 0.02
0.31 ± 0.04
−0.52 ± 0.02
−0.24 ± 0.03
Ca2+
T = 298.15 K
−1.11 ± 0.07
0.19 ± 0.09
-
−0.16 ± 0.06
Mg2+
T = 288.15 K
0.26 ± 0.06
-
-
−1.22 ± 0.04
T = 298.15 K
0.06 ± 0.07
-
-
−1.02 ± 0.06
T = 310.15 K
0.11 ± 0.06
-
-
−1.63 ± 0.02
Sn2+
∆εM2L2 a
∆εML2 a
∆εMLOH a
∆εM2LOH a
T = 298.15 K
−2.88 ± 0.19 b
−3.29 ± 0.14
−1.72 ± 0.13
0.30 ± 0.02
Species
Ca2+
Mg2+
Sn2+
T = 298.15 K
ε(ML+,Cl−)
c
1.03 ± 0.07 b
−0.20 ± 0.07
-
ε(MLH2+,Cl−)
c
−0.16 ± 0.09
-
-
kMLOH c,d
−0.06 ± 0.01
0.99 ± 0.05
1.39 ± 0.13
ε(M2L22+,Cl−)
c
-
-
2.49 ± 0.19
kML2 c,d
-
-
2.90 ± 0.14
ε(M2LOH2+,Cl−)
c
-
-
−0.60 ± 0.02
a Calculated by means of Equation (10); b ±Std. Dev.; c Calculated by means of Equations (12)–(15); d Setschenow coefficient for the neutral
species calculated by means of Equation (11). action parameters and Setschenow coefficients for the species of the metal/dopamine systems. Table 10. Specific ion interaction parameters and Setschenow coefficients for the species of the metal/dop a Calculated by means of Equation (10); b ±Std. Dev.; c Calculated by means of Equations (12)–(15); d Setschenow coefficient for the neutral
species calculated by means of Equation (11). Biomolecules 2021, 11, 1312 16 of 28 16 of 28 3.5. Sequestering Ability of Dopamine towards Cations and Effect of pH, Ionic Strength,
and Temperature 3.5. Sequestering Ability of Dopamine towards Cations and Effect of pH, Ionic Strength,
and Temperature In studies concerning problems involving natural or biological fluids, it is often es-
sential to have a parameter that allows estimating the ability of one or more ligands to
sequester a given component, such as a metal ion. This estimation is particularly difficult
when comparing two different systems or the same system studied at different temper-
atures, ionic strengths, and ionic media. 3.3.3. Sn2+/Dop−System When the systems to be compared also have
different speciation and only a few (or none) are the common species among them, under-
standing which systems have higher or lower sequestering ability is fairly impossible. This
problem is even more complex if the system of interest is contained in a multicomponent
solution, where the possible simultaneous presence of several metal and/or organic and
inorganic components makes this study more complex. For example, if we want to calculate
the sequestering ability of a given Mn+ metal with an organic ligand Lz−, the metal may
undergo hydrolysis reactions and form complexes with the anions of the ionic medium or
with other competing ligands. The ligand Lz−in turn can be present in its different protonate or deprotonate forms
and reacts with the cation of the ionic medium or with other metals. Therefore, it is essential
to use a parameter that can allow the calculation of the sequestering ability of a ligand
towards a metal ion at different experimental conditions. Many years ago, this research group introduced the pL0.5 parameter, which, once the
conditions of pH, ionic strength, ion medium, and temperature are established, gives an
objective representation of the sequestering ability towards one or more metals [23,24]. It is
determined by applying a Boltzmann type equation to the couple of data: the sum of the
mole fraction of all the formed metal–ligand species vs. the logarithm-changed sign of the
ligand analytical concentration (pL).The proposed equation is: x = 1/(1 + 10(pL−pL0.5))
(19) (19) where x is the total fraction of complexed metal plotted versus pL, (pL = −log [L]tot and
[L]tot is the analytical concentration of ligand). This function is like a sigmoidal curve with
an asymptote of 1 for pL→−∞and 0 for pL→+∞. pL0.5 is a quantitative parameter and
represents the concentration of ligand required for the sequestration of 50% of the metal
cation. It is important to remember that this property varies as experimental conditions
change, but is independent of the analytical concentration of metal when it is present in
traces (~10−10 mol dm−3 or less). In the calculation of pL0.5, all the parallel reactions (metal
hydrolysis, ligand protonation, reactions with other components, etc.) are considered in
the speciation model, but are successively excluded in the calculation of pL0.5. 3.3.3. Sn2+/Dop−System p
y
p
Using this approach, the dopamine sequestering ability against the metal cations here
investigated was determined at different experimental conditions (I/mol dm−3, pH, T/K),
and the pL0.5 values are reported in Tables 11–13. g
p
nd the pL0.5 values are reported in Tables 11–13. p
p
For a better visualization of the results, some sequestering diagrams have been drawn
(Figures 8–11). From the sequestering diagrams here reported, it is possible to observe that the
sequestering ability of dopamine towards the metals is dependent both on the ionic strength
values, on the temperature, and pH. The increase of the pL0.5 with pH (Figure 7) is related
to the deprotonation of the ligand and an indication of the electrostatic nature of the
interactions. At the same pH, the dependence of dopamine sequestering ability on the ionic
strength (Figure 8) is due both to the metal acid-base property, the strength of interaction
between the components, the tendency of the metal to form hydrolytic species and stable
weak complexes with the anion of the ionic medium, which contribute to reduce the
amount of free metal ion to interact with the ligand. Biomolecules 2021, 11, 1312 17 of 28 17 of 28 Table 11. Sequestering ability of dopamine towards the metal ions at I = 0.15 mol dm−3, pH = 7.4 and
different temperatures. Metal
T/K
pL0.5
288.15
2.36
CH3Hg+
298.15
2.63
310.15
3.74
318.15
4.50
Mg2+
288.15
2.23 a)
298.15
2.27 a)
310.15
2.94 a)
288.15
0.81
Ca2+
298.15
1.15
310.15
1.86
288.15
3.74
Sn2+
298.15
3.46
310.15
3.75
a) Owing to the low values at pH = 7.4, for Mg2+ the pL0.5 were calculated at pH = 9.5. 288.15
0.81
Ca2+
298.15
1.15
310.15
1.86
288.15
3.74
Sn2+
298.15
3.46
310.15
3.75
Owing to the low values at pH = 7.4, for Mg2+ the pL0.5 were calculated at pH = 9.5. For a better visualization of the results, some sequestering diagrams have been
drawn (Figures 8–11). From the sequestering diagrams here reported, it is possible to observe that the se-
questering ability of dopamine towards the metals is dependent both on the ionic strength
values, on the temperature, and pH. The increase of the pL0.5 with pH (Figure 7) is related
o the deprotonation of the ligand and an indication of the electrostatic nature of the in-
eractions. 3.3.3. Sn2+/Dop−System 19 of 30 3
4
5
6
7
0.0
0.2
0.4
0.6
0.8
1.0
pH = 2.0,
3.0, 4.0
pH = 9.0
pH = 8.0
pH = 7.4
pH = 6.0
pH = 5.0
pL
(a) 3
4
5
6
7
0.0
0.2
0.4
0.6
0.8
1.0
pH = 2.0,
3.0, 4.0
pH = 9.0
pH = 8.0
pH = 7.4
pH = 6.0
pH = 5.0
pL
(a)
1
2
3
4
5
6
0
20
40
60
80
100
I = 1.0 mol dm
-3
I = 0.75 mol dm
-3
I = 0.5 mol dm
-3
I = 0.15 mol dm
-3
x
pL
(b)
Figure 9. Sequestering diagram of dopamine towards Sn2+. (a) T = 298.15 K, I = 0.15 mol dm−3 and
different pHs. (b) T = 298.15 K, pH = 7.4 and different ionic strengths. pL0.5 values: (a) 1.30 (pH = 5.0)
< 2.33 (pH = 6.0) < 3.74 (pH = 7.4) < 4.31 (pH = 8.0) < 5.07 (pH = 9.0). (b) 3.74 (I = 0.15 mol dm−3) < 3.70
(I = 0.50 mol dm−3) < 3.12 (I = 0.75 mol dm−3) < 2.60 (I = 1.0 mol dm−3). 1.0
Figure 9. Sequestering diagram of dopamine towards Sn2+. (a) T = 298.15 K, I = 0.15 mol dm−3 and
different pHs. (b) T = 298.15 K, pH = 7.4 and different ionic strengths. pL0.5 values: (a) 1.30 (pH = 5.0) <
2.33 (pH = 6.0) < 3.74 (pH = 7.4) < 4.31 (pH = 8.0) < 5.07 (pH = 9.0). (b) 3.74 (I = 0.15 mol dm−3) < 3.70
(I = 0.50 mol dm−3) < 3.12 (I = 0.75 mol dm−3) < 2.60 (I = 1.0 mol dm−3). 1
2
3
4
5
6
0
20
40
60
80
100
I = 1.0 mol dm
-3
I = 0.75 mol dm
-3
I = 0.5 mol dm
-3
I = 0.15 mol dm
-3
x
pL
(b) Figure 9. Sequestering diagram of dopamine towards Sn2+. (a) T = 298.15 K, I = 0.15 mol dm−3 and
different pHs. (b) T = 298.15 K, pH = 7.4 and different ionic strengths. pL0.5 values: (a) 1.30 (pH = 5.0)
< 2.33 (pH = 6.0) < 3.74 (pH = 7.4) < 4.31 (pH = 8.0) < 5.07 (pH = 9.0). 3.3.3. Sn2+/Dop−System For a better visualization of the results, some sequestering diagrams have been
drawn (Figures 8–11). From the sequestering diagrams here reported, it is possible to observe that the se-
questering ability of dopamine towards the metals is dependent both on the ionic strength
values, on the temperature, and pH. The increase of the pL0.5 with pH (Figure 7) is related
o the deprotonation of the ligand and an indication of the electrostatic nature of the in-
eractions. At the same pH, the dependence of dopamine sequestering ability on the ionic
trength (Figure 8) is due both to the metal acid-base property, the strength of interaction
etween the components, the tendency of the metal to form hydrolytic species and stable
weak complexes with the anion of the ionic medium, which contribute to reduce the
mount of free metal ion to interact with the ligand. Table 11. Sequestering ability of dopamine towards the metal ions at I = 0.15 mol dm−3, pH = 7.4 and
different temperatures. Ca2+
298.15
1.15
310.15
1.86 1
2
3
4
5
0.0
0.2
0.4
0.6
0.8
1.0
I = 0.15 mol dm-3
I = 0.50 mol dm-3
I = 0.75 mol dm-3
I = 1.00 mol dm-3
pL
(a) 1
2
3
4
5
0.0
0.2
0.4
0.6
0.8
1.0
T = 310.15 K
T = 298.15 K
T = 288.15 K
pL
(b) gure 8. Sequestering diagram of dopamine towards Ca2+. (a) T = 298.15 K, pH = 7.4 and different
nic strengths; (b) I = 0.15 mol dm−3, pH = 7.4 and different temperatures. pL0.5 values: (a) 1.15 (I =
15 mol dm−3) < 1.19 (I = 0.50 mol dm−3) < 1.68 (I = 0.75 mol dm−3) < 2.58 (I= 1.0 mol dm−3). (b) 0.81 (T
288.15 K) < 1.15 (T = 298.15 K) < 1.86 (T = 318.15 K). Figure 8. Sequestering diagram of dopamine towards Ca2+. (a) T = 298.15 K, pH = 7.4 and different ionic
strengths; (b) I = 0.15 mol dm−3, pH = 7.4 and different temperatures. pL0.5 values: (a) 1.15 (I = 0.15 mol
dm−3) < 1.19 (I = 0.50 mol dm−3) < 1.68 (I = 0.75 mol dm−3) < 2.58 (I= 1.0 mol dm−3). (b) 0.81 (T = 288.15
K) < 1.15 (T = 298.15 K) < 1.86 (T = 318.15 K). 3.3.3. Sn2+/Dop−System At the same pH, the dependence of dopamine sequestering ability on the ionic
trength (Figure 8) is due both to the metal acid-base property, the strength of interaction
between the components, the tendency of the metal to form hydrolytic species and stable
weak complexes with the anion of the ionic medium, which contribute to reduce the
amount of free metal ion to interact with the ligand. 1
2
3
4
5
0.0
0.2
0.4
0.6
0.8
1.0
I = 0.15 mol dm-3
I = 0.50 mol dm-3
I = 0.75 mol dm-3
I = 1.00 mol dm-3
pL
(a)
1
2
3
4
5
0.0
0.2
0.4
0.6
0.8
1.0
T = 310.15 K
T = 298.15 K
T = 288.15 K
pL
(b)
Figure 8. Sequestering diagram of dopamine towards Ca2+. (a) T = 298.15 K, pH = 7.4 and different
onic strengths; (b) I = 0.15 mol dm−3, pH = 7.4 and different temperatures. pL0.5 values: (a) 1.15 (I =
.15 mol dm−3) < 1.19 (I = 0.50 mol dm−3) < 1.68 (I = 0.75 mol dm−3) < 2.58 (I= 1.0 mol dm−3). (b) 0.81 (T
288.15 K) < 1.15 (T = 298.15 K) < 1.86 (T = 318.15 K). Figure 8. Sequestering diagram of dopamine towards Ca2+. (a) T = 298.15 K, pH = 7.4 and different ionic
strengths; (b) I = 0.15 mol dm−3, pH = 7.4 and different temperatures. pL0.5 values: (a) 1.15 (I = 0.15 mol
dm−3) < 1.19 (I = 0.50 mol dm−3) < 1.68 (I = 0.75 mol dm−3) < 2.58 (I= 1.0 mol dm−3). (b) 0.81 (T = 288.15
K) < 1.15 (T = 298.15 K) < 1.86 (T = 318.15 K). 19 of 30 Table 11. Sequestering ability of dopamine towards the metal ions at I = 0.15 mol dm−3, pH = 7.4 and
different temperatures. Metal
T/K
pL0.5
288.15
2.36
CH3Hg+
298.15
2.63
310.15
3.74
318.15
4.50
Mg2+
288.15
2.23 a)
298.15
2.27 a)
310.15
2.94 a)
288.15
0.81
Ca2+
298.15
1.15
310.15
1.86
288.15
3.74
Sn2+
298.15
3.46
310.15
3.75
a) Owing to the low values at pH = 7.4, for Mg2+ the pL0.5 were calculated at pH = 9.5. Ca2+
298.15
1.15
310.15
1.86
288.15
3.74
Sn2+
298.15
3.46
310.15
3.75
Owing to the low values at pH = 7.4, for Mg2+ the pL0.5 were calculated at pH = 9.5. 3.3.3. Sn2+/Dop−System (b) 3.74 (I = 0.15 mol dm−3) < 3.70
(I = 0.50 mol dm−3) < 3.12 (I = 0.75 mol dm−3) < 2.60 (I = 1.0 mol dm−3). Figure 9. Sequestering diagram of dopamine towards Sn2+. (a) T = 298.15 K, I = 0.15 mol dm−3 and
different pHs. (b) T = 298.15 K, pH = 7.4 and different ionic strengths. pL0.5 values: (a) 1.30 (pH = 5.0) <
2.33 (pH = 6.0) < 3.74 (pH = 7.4) < 4.31 (pH = 8.0) < 5.07 (pH = 9.0). (b) 3.74 (I = 0.15 mol dm−3) < 3.70
(I = 0.50 mol dm−3) < 3.12 (I = 0.75 mol dm−3) < 2.60 (I = 1.0 mol dm−3). 18 of 28
m and
H = 5.0)
)
3 70
and
= 5.0)
3 70 Biomolecules 2021, 11, 1312 2
3
4
5
6
7
0.0
0.2
0.4
0.6
0.8
1.0
Sn
2+
CH3Hg
+
Ca
2+
Mg
2+
x
pL
Figure 10. Sequestering diagram of dopamine towards the metal ions here investigated, at I = 0.15
mol L−1, T = 298.15 K, and pH = 9.0. Figure 10. Sequestering diagram of dopamine towards the metal ions here investigated, at
I = 0.15 mol L−1, T = 298.15 K, and pH = 9.0. 2
3
4
5
6
7
0.0
0.2
0.4
0.6
0.8
1.0
Sn
2+
CH3Hg
+
Ca
2+
Mg
2+
x
pL
igure 10. Sequestering diagram of dopamine towards the metal ions here investigated, at I = 0.15
mol L−1, T = 298.15 K, and pH = 9.0. 2
3
4
5
6
7
0.0
0.2
0.4
0.6
0.8
1.0
Sn
2+
CH3Hg
+
Ca
2+
Mg
2+
x
pL
2
3
4
5
6
7
0.0
0.2
0.4
0.6
0.8
1.0
Sn
2+
CH3Hg
+
Ca
2+
Mg
2+
x
pL igure 10. Sequestering diagram of dopamine towards the metal ions here investigated, at I = 0.15
mol L−1, T = 298.15 K, and pH = 9.0. Figure 10. Sequestering diagram of dopamine towards the metal ions here investigated, at
I = 0.15 mol L−1, T = 298.15 K, and pH = 9.0. gure 10. Sequestering diagram of dopamine towards the metal ions here investigated, at I = 0.15
ol L−1, T = 298.15 K, and pH = 9.0. 3.3.3. Sn2+/Dop−System 0.2
0.4
0.6
0.8
1.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
I/mol dm
-3
(a)
285
290
295
300
305
310
315
320
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
T/K
(b)
Figure 11. Dependence of pL0.5 on: (a) I/mol dm−3 and (b) T/K. (a) pL0.5 values calculated at T = 298.15
K, pH = 7.4 and different ionic strengths; (b) pL0.5 values calculated at I = 0.15 mol dm−3, pH = 7.4
and different temperatures. 0.2
0.4
0.6
0.8
1.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
I/mol dm
-3
(a)
285
290
295
300
305
310
315
320
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
T/K
(b)
igure 11. Dependence of pL0.5 on: (a) I/mol dm−3 and (b) T/K. (a) pL0.5 values calculated at T = 298.15
, pH = 7.4 and different ionic strengths; (b) pL0.5 values calculated at I = 0.15 mol dm−3, pH = 7.4
nd different temperatures. Figure 11. Dependence of pL0.5 on: (a) I/mol dm−3 and (b) T/K. (a) pL0.5 values calculated at
T = 298.15 K, pH = 7.4 and different ionic strengths; (b) pL0.5 values calculated at I = 0.15 mol dm−3,
pH = 7.4 and different temperatures. 0.2
0.4
0.6
0.8
1.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
I/mol dm
-3
(a)
0.2
0.4
0.6
0.8
1.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
I/mol dm
-3
(a) 285
290
295
300
305
310
315
320
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
T/K
(b)
285
290
295
300
305
310
315
320
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
CH3Hg
+
Mg
2+
Ca
2+
Sn
2+
pL0.5
T/K
(b) T/K
T/K Figure 11. Dependence of pL0.5 on: (a) I/mol dm−3 and (b) T/K. (a) pL0.5 values calculated at T = 298.15
K, pH = 7.4 and different ionic strengths; (b) pL0.5 values calculated at I = 0.15 mol dm−3, pH = 7.4
and different temperatures. gure 11. In Figure 9, the effect of pH and saline concentration on the pL0.5 values of the
Sn2+/Dop−system is reported. Concerning the ionic strength influence, (Figure 9b)
∆pL0.5 values of ~1.14 units from I = 0.15 to 1.0 mol dm−3 was obtained. With respect to 3.3.3. Sn2+/Dop−System p
g
g
Similarly, temperature gradients also affect the ligand sequestering ability towards metals. Table 13 compares the pL0.5 values of the different systems, at different temperatures. Table 13. Sequestering ability of dopamine towards metal ions at I = 0.15 mol dm−3, T = 298.15 K and different pHs. pL0.5
pH
CH3Hg+
CH3Hg+ a
Ca2+
Mg2+
Sn2+
5
-
-
-
-
1.30
6
-
-
-
-
2.33
7.4
2.63
3.74
1.15
0.08
3.74
8.2
3.46
4.40
2.00
1.06
4.31
9
4.51
5.18
3.03
1.31
5.07
9.5
5.23
5.75
3.65
2.27
-
10.5
6.16
6.16
4.72
3.97
-
a Calculated at T = 310.15 K. Sequestering ability of dopamine towards metal ions at I = 0.15 mol dm−3, T = 298.15 K and different pHs. f dopamine towards metal ions at I = 0.15 mol dm−3, T = 298.15 K and different pHs. In this case, independent of the system and pH range investigated, an increasing trend
of the pL0.5 values with increasing the temperature is observed. In order to evaluate the sequestering ability of dopamine towards the metal ions under
study, the pL0.5 values were calculated at the same experimental conditions, i.e., T = 298.15
K, I = 0.15 mol dm−3 and pH = 9.0 (but similar results can be obtained from a comparison
between all the data reported in Tables 13–15). From the pL0.5 values reported in Figure 10,
the following sequestering ability trend of dopamine towards the investigated metals can
be observed: pL0.5: Mg2+ < Ca2+ < CH3Hg+ < Sn2+ pL0.5: Mg2+ < Ca2+ < CH3Hg+ < Sn2+ The effect of the variables, T/K, I/mol dm−3 and pH on the sequestering ability of
dopamine towards the metal ions can be also observed in Figures 11 and 12. pL0.5: Mg2+ = 1.31 < Ca2+ = 2.00 < CH3Hg+ = 4.58 < Sn2+ = 5.08. Table 14. Empirical parameters for the modeling of pL0.5 values of the metal/dopamine systems with respect to T/K, I/mol
dm−3 and pH. Metal
p1
p2
p3
p4
Std. Dev. b
Mean Dev. 3.3.3. Sn2+/Dop−System Dependence of pL0.5 on: (a) I/mol dm−3 and (b) T/K. (a) pL0.5 values calculated at T = 298.15
pH = 7.4 and different ionic strengths; (b) pL0.5 values calculated at I = 0.15 mol dm−3, pH = 7.4
d different temperatures. Figure 11. Dependence of pL0.5 on: (a) I/mol dm−3 and (b) T/K. (a) pL0.5 values calculated at
T = 298.15 K, pH = 7.4 and different ionic strengths; (b) pL0.5 values calculated at I = 0.15 mol dm−3,
pH = 7.4 and different temperatures. Table 12. Sequestering ability of dopamine towards metal ions at T = 298.15 K, pH = 7.4 and different
ionic strengths. Table 12. Sequestering ability of dopamine towards metal ions at T = 298.15 K, pH = 7.4 and different
ionic strengths. Table 12. Sequestering ability of dopamine towards metal ions at T = 298.15 K, pH = 7.4 and different
ionic strengths. Table 12. Sequestering ability of dopamine towards metal ions at T = 298.15 K, pH = 7.4 and different
ionic strengths. Metal
I/mol dm−3
pL0.5
0.15
2.63
CH3Hg+
0.50
2.90
0.75
3.02
1.00
3.12
0.15
2.27 a
Mg2+
0.50
2.11 a
0.75
2.04 a
1.00
2.10 a
0.15
1.15
Ca2+
0.50
1.19
0.75
1.68
1.00
2.58
0.15
3.74
Sn2+
0.50
3.70
0.75
3.12
1.00
2.60
a Calculated at pH = 9.5. In Figure 9, the effect of pH and saline concentration on the pL0.5 values of the
Sn2+/Dop−system is reported. Concerning the ionic strength influence, (Figure 9b)
∆pL0.5 values of ~1.14 units from I = 0.15 to 1.0 mol dm−3 was obtained. With respect to Biomolecules 2021, 11, 1312 19 of 28 19 of 28 Ca2+/Dop−system, we can observe an opposite trend, namely the increase of the ionic
strength (i.e., NaCl concentration) produces a lowering of pL0.5. This effect can be explained
considering that increasing the Cl−concentration, the formation of the SnCli (i = 1–3) and
SnOHCl species is favorite, contributing to a lowering of the Sn2+ free concentration. Similarly, temperature gradients also affect the ligand sequestering ability towards metals. T bl 13
th
L
l
f th diff
t
t
t diff
t t
t g
g
,
i (
)
SnOHCl species is favorite, contributing to a lowering of the Sn2+ free concentration. Similarly, temperature gradients also affect the ligand sequestering ability towards metals. Table 13 compares the pL0.5 values of the different systems, at different temperatures. 3.3.3. Sn2+/Dop−System (a) pL0.5 values calculated at T = 298.15 K and I = 0.15 mol
dm-3; (b) pL0.5 values of CH3Hg+/Dop−system calculated at I = 0.15 mol dm−3, pH = 7.4, and T =
298.15 and 310.15 K. Considering the amount of available data the dependence of pL
on the variables Table 15. Thermal decomposition steps for the Ca2+/Dop−precipitates. Scheme. Percentages by Weight at the Beginning
of the Process
Percentage Loss of Weight during the
Decomposition Process
Ca-Dop(A)
1◦process
99.8%
9.45%
2◦process
90.31%
34.49%
3◦process
55.82%
17.96%
4◦process
37.82%
3.10%
5◦process
34.77%
10.56%
Stoichiometry (M:L)
1:1
Ca-Dop(B)
1◦process
99.76%
14.77%
2◦process
84.98%
57.02%
3◦process
27.97%
15.22%
4◦process
12.75%
3.89%
Stoichiometry (M:L)
1:3
Ca-Dop(C)
1◦process
99.86%
10.01%
2◦process
89.85%
67.93%
3◦process
21.92%
11.79%
4◦process
10.12%
3.48%
Stoichiometry (M:L)
1:5
Biomolecules 2021, 11, x
21 of 30
pL0.5: Mg2+ < Ca2+ < CH3Hg+ < Sn2+
The effect of the variables, T/K, I/mol dm−3 and pH on the sequestering ability of do-
pamine towards the metal ions can be also observed in Figures 11 and 12. pL0.5: Mg2+ = 1.31 < Ca2+ = 2.00 < CH3Hg+ = 4.58 < Sn2+ = 5.08. Table 14. Empirical parameters for the modeling of pL0.5 values of the metal/dopamine systems with respect to T/K, I/mol
dm−3 and pH. Metal
p1
p2
p3
p4
Std. Dev. b
Mean Dev. b
CH3Hg+
−19.0 ± 2.6 a
0.050 ± 0.007
−0.08 ± 0.20
0.97 ± 0.03
0.14
0.27
Mg2+
−18.8 ± 4.0
0.035 ± 0.013
−0.37 ± 0.19
1.12 ± 0.09
0.12
0.24
Ca2+
−22.4 ± 2.6
0.050 ± 0.009
1.33 ± 0.12
1.16 ± 0.02
0.05
0.09
Sn2+
−3.8 ± 2.0
0.001 ± 0.007
−1.04 ± 0.08
0.95 ± 0.03
0.07
0.10
a ±Std. Dev.; b standard and mean deviation on the fit of Equation (20)
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
-1
0
1
2
3
4
5
6
7
8
9
5
6
7
8
9
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Sn
2+
pL0.5
pH
CH3Hg
+
Ca
2+
Mg
2+
pL0.5
pH
(a)
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
T = 298.15 K
T = 310.15 K
pL0.5
pH
(b)
Figure 12. Dependence of pL0.5 on pH. 3.3.3. Sn2+/Dop−System b
CH3Hg+
−19.0 ± 2.6 a
0.050 ± 0.007
−0.08 ± 0.20
0.97 ± 0.03
0.14
0.27
Mg2+
−18.8 ± 4.0
0.035 ± 0.013
−0.37 ± 0.19
1.12 ± 0.09
0.12
0.24
Ca2+
−22.4 ± 2.6
0.050 ± 0.009
1.33 ± 0.12
1.16 ± 0.02
0.05
0.09
Sn2+
−3.8 ± 2.0
0.001 ± 0.007
−1.04 ± 0.08
0.95 ± 0.03
0.07
0.10
a ±Std. Dev.; b standard and mean deviation on the fit of Equation (20). Table 14. Empirical parameters for the modeling of pL0.5 values of the metal/dopamine systems with respect to T/K, I/mol
dm−3 and pH. parameters for the modeling of pL0.5 values of the metal/dopamine systems with respect to T/K, I/mol Figure 11a, shows the variation of the pL0.5 values with respect to ionic strength;
for Mg2+ and CH3Hg+, a linear trend, even if opposite, is observed increasing the ionic
strength. For Ca2+ and Sn2+, a non-linear variation is reported, and also in this case with
an opposite trend. The net increase of pL0.5 with the ionic strength is an indication of a stabilizing effect
of the ion coming from the ionic media on the stability of the complexes. In particular, for
CH3Hg+, the trend can be explained observing the distribution diagram reported in Figure 2,
where increasing the ionic strength, there is an increase of the formation percentage of the
MLCl species. Biomolecules 2021, 11, 1312 20 of 28 20 of 28 The opposite effect was observed for Sn2+ (see also Figure 6). In this case, the decrease of
the pL0.5 values can be explained considering that increasing the ionic strength, a lowering
of the formation percentage of the species can be observed, especially at pH > 7, where the
percentage of formation of the ML2 decreases from ~22% at I = 0.15 mol dm−3 to ~5% at
I = 1.0 mol dm−3. Figure 11b reports the effect of the temperature on pL0.5, obtaining an
increasing trend. In Figure 12a,b, the effect of the variation of the pH on the sequestering ability
of dopamine towards the metals is highlighted. For CH3Hg+ (Figure 12b), the effect of the
temperature is less evident, and increasing the pH up to ~10, the differences are fairly nil. p
,
g
p
p
,
y
Table 15. Thermal decomposition steps for the Ca2+/Dop−precipitates. Scheme. 3.3.3. Sn2+/Dop−System Percentages by Weight at the Beginning
of the Process
Percentage Loss of Weight during the
Decomposition Process
Ca-Dop(A)
1◦process
99.8%
9.45%
2◦process
90.31%
34.49%
3◦process
55.82%
17.96%
4◦process
37.82%
3.10%
5◦process
34.77%
10.56%
Stoichiometry (M:L)
1:1
Ca-Dop(B)
1◦process
99.76%
14.77%
2◦process
84.98%
57.02%
3◦process
27.97%
15.22%
4◦process
12.75%
3.89%
Stoichiometry (M:L)
1:3
Ca-Dop(C)
1◦process
99.86%
10.01%
2◦process
89.85%
67.93%
3◦process
21.92%
11.79%
4◦process
10.12%
3.48%
Stoichiometry (M:L)
1:5
iomolecules 2021, 11, x
21 of 30
pL0.5: Mg2+ < Ca2+ < CH3Hg+ < Sn2+
The effect of the variables, T/K, I/mol dm−3 and pH on the sequestering ability of do-
pamine towards the metal ions can be also observed in Figures 11 and 12. pL0.5: Mg2+ = 1.31 < Ca2+ = 2.00 < CH3Hg+ = 4.58 < Sn2+ = 5.08. Table 14. Empirical parameters for the modeling of pL0.5 values of the metal/dopamine systems with respect to T/K, I/mol
dm−3 and pH. Metal
p1
p2
p3
p4
Std. Dev. b
Mean Dev. b
CH3Hg+
−19.0 ± 2.6 a
0.050 ± 0.007
−0.08 ± 0.20
0.97 ± 0.03
0.14
0.27
Mg2+
−18.8 ± 4.0
0.035 ± 0.013
−0.37 ± 0.19
1.12 ± 0.09
0.12
0.24
Ca2+
−22.4 ± 2.6
0.050 ± 0.009
1.33 ± 0.12
1.16 ± 0.02
0.05
0.09
Sn2+
−3.8 ± 2.0
0.001 ± 0.007
−1.04 ± 0.08
0.95 ± 0.03
0.07
0.10
a ±Std. Dev.; b standard and mean deviation on the fit of Equation (20)
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
-1
0
1
2
3
4
5
6
7
8
9
5
6
7
8
9
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Sn
2+
pL0.5
pH
CH3Hg
+
Ca
2+
Mg
2+
pL0.5
pH
(a)
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
T = 298.15 K
T = 310.15 K
pL0.5
pH
(b)
Figure 12. Dependence of pL0.5 on pH. (a) pL0.5 values calculated at T = 298.15 K and I = 0.15 mol
dm-3; (b) pL0.5 values of CH3Hg+/Dop− system calculated at I = 0.15 mol dm−3, pH = 7.4, and T =
298.15 and 310.15 K. Fi u e 11a
ho
the
a iatio
of the
L
alue
ith e
e t to io i
t e
th fo
Figure 12. Dependence of pL0.5 on pH. 3.3.3. Sn2+/Dop−System (a) pL0.5 values calculated at T = 298.15 K and I = 0.15 mol
dm-3; (b) pL0.5 values of CH3Hg+/Dop− system calculated at I = 0.15 mol dm−3, pH = 7.4, and T =
298.15 and 310.15 K. Figure 12. Dependence of pL0.5 on pH. (a) pL0.5 values calculated at T = 298.15 K and I = 0.15 mol
dm-3; (b) pL0.5 values of CH3Hg+/Dop−system calculated at I = 0.15 mol dm−3, pH = 7.4, and T =
298.15 and 310.15 K. Table 15. Thermal decomposition steps for the Ca2+/Dop−precipitates. 7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
T = 298.15 K
T = 310.15 K
pL0.5
pH
(b) 7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
-1
0
1
2
3
4
5
6
7
8
9
5
6
7
8
9
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Sn
2+
pL0.5
pH
CH3Hg
+
Ca
2+
Mg
2+
pL0.5
pH
(a) ure 12. Dependence of pL0.5 on pH. (a) pL0.5 values calculated at T = 298.15 K and I = 0.15 mol
-3; (b) pL0.5 values of CH3Hg+/Dop− system calculated at I = 0.15 mol dm−3, pH = 7.4, and T =
.15 and 310.15 K. Figure 12. Dependence of pL0.5 on pH. (a) pL0.5 values calculated at T = 298.15 K and I = 0.15 mol
dm-3; (b) pL0.5 values of CH3Hg+/Dop−system calculated at I = 0.15 mol dm−3, pH = 7.4, and T =
298.15 and 310.15 K. Figure 11a, shows the variation of the pL0.5 values with respect to ionic strength; for
g2+ and CH3Hg+, a linear trend, even if opposite, is observed increasing the ionic
rength. For Ca2+ and Sn2+, a non-linear variation is reported, and also in this case with an
pposite trend. Considering the amount of available data, the dependence of pL0.5 on the variables
(I/mol dm−3, T/K, and pH) was modeled by means of the LIANA computer program. For each metal/dopamine system, the best results in term of statistical parameters, were
obtained when the following simple empirical equation was used: of pL0.5 with the ionic strength is an indication of a stabilizing effect
m the ionic media on the stability of the complexes. 3.6. Thermogravimetric Analysis Some thermogravimetric analyses were performed, by using a PerkinElmer thermo-
gravimetric balance model Pyris Diamond (PerkinElmer, Milano, Italy), in oxidant purging
atmosphere (air flow), in the temperature range T = 293.15–1073.15 K. These investigations
were carried out on solid samples obtained at the end of potentiometry titrations of the
Ca2+/Dop−system and from solutions prepared at different metal to ligand molar ratios. Before the TGA analysis, the samples, once collected, were filtered on 0.45 µm cellulose
filt
d
h d
ith
ll
t
f
lt
t
O
h d th
i it t Some thermogravimetric analyses were performed, by using a PerkinElmer thermo-
gravimetric balance model Pyris Diamond (PerkinElmer, Milano, Italy), in oxidant purging
atmosphere (air flow), in the temperature range T = 293.15–1073.15 K. These investigations
were carried out on solid samples obtained at the end of potentiometry titrations of the
Ca2+/Dop−system and from solutions prepared at different metal to ligand molar ratios Before the TGA analysis, the samples, once collected, were filtered on 0.45 µm cellulose
filters, and washed with small amounts of ultrapure water. Once washed, the precipitates
were treated with small amounts of acetone and vacuum dried. Figures 13 and 14 report the thermogravimetric diagrams for three different Ca2+/Dop−
precipitates; it is possible to observe that the curves have different decomposition profiles with
residual values at the end of analysis, where the metal oxide is present, clearly different. 23 of 30 Figure 13. Thermogravimetric curve for the Ca2+/Dop− precipitates obtained at different metal:lig-
and molar ratios and I = 0.15 mol dm−3. Component concentration in the solution: cCa2+ = 1.5 mmol
dm−3 and cDop- = 1.5–7.5 mmol dm−3. Atmosphere of air (gaseous mixture of nitrogen and oxygen
with 80% and 20%, v/v, respectively) at a flow rate of 100 mL min−1 and a scanning rate of 10 K min−1. Figure 13. Thermogravimetric curve for the Ca2+/Dop−precipitates obtained at different
metal:ligand molar ratios and I = 0.15 mol dm−3. Component concentration in the solution: cCa2+ =
1.5 mmol dm−3 and cDop−= 1.5–7.5 mmol dm−3. Atmosphere of air (gaseous mixture of nitrogen
and oxygen with 80% and 20%, v/v, respectively) at a flow rate of 100 mL min−1 and a scanning rate
of 10 K min−1. Figure 13. Thermogravimetric curve for the Ca2+/Dop− precipitates obtained at different metal:lig-
and molar ratios and I = 0.15 mol dm−3. where pi = 1–4 are the empirical parameters. where pi = 1–4 are the empirical parameters. The values of the pi parameters and the corresponding standard and mean deviation
on the fit are reported in Table 14. 3.3.3. Sn2+/Dop−System In particular, for
pL0.5 = p1 + p2·T/K + p3·(I/mol dm−3)2 + p4·pH
(20) f pL0.5 with the ionic strength is an indication of a stabilizing effect
m the ionic media on the stability of the complexes. In particular, for
b
l
d b
h d
b
d
d
pL0.5 = p1 + p2·T/K + p3·(I/mol dm−3)2 + p4·pH
(20) ct
or (20) Biomolecules 2021, 11, 1312 21 of 28 21 of 28 where pi = 1–4 are the empirical parameters. Table 15 reports the
4. Literature Data Table 15 reports the percentage weight loss for each Ca /Dop system, calculated
the thermal decomposition of the precipitates. Considering these experimental data,
oichiometry of the precipitates was calculated, assuming that the residual of the de-
osition process is CaO. As it can be observed, the stoichiometry of the precipitate
ge between a metal:ligand molar ratios of 1:3 to 1:5, in dependence on the initial con-
ation of the components in the vessel. erature Data
Literature reports different papers where the interaction of dopamine towards metal
has been investigated. However, to our knowledge, no thermodynamic studies on
i
i h h
i
d
i
i
i
h
b
d
f
Literature reports different papers where the interaction of dopamine towards metal
ions has been investigated. However, to our knowledge, no thermodynamic studies on the
interaction with the cations under investigation have ever been reported, except for Mg2+. The stability constants for the systems published in the literature are listed in Table 16. The
main difficulty for a direct comparison of these data with the results here obtained is the
different approaches used for the determination of the acid-base properties of dopamine. In some cases, the ligand was considered as a triprotic ligand (L2−), by the calculation of
the third protonation constant having a log KH value of about 13–14, out of the pH range of
physiological interest. In other cases, only the protonation constants of one phenolic group
and of the amine group of the alkyl chain were considered. nteraction with the cations under investigation have ever been reported, except for
The stability constants for the systems published in the literature are listed in Table
he main difficulty for a direct comparison of these data with the results here obtained
different approaches used for the determination of the acid-base properties of do-
The literature stability constants reported in Table 16, refer to low ionic strength values
(I ≤0.2 mol dm−3), and this aspect does not allow a comparison with the stability constants
here reported up to I = 1.0 mol dm−3, as well as for the dependence on the ionic strength
and temperature. ne. 3.6. Thermogravimetric Analysis Component concentration in the solution: cCa2+ = 1.5 mmol
dm−3 and cDop- = 1.5–7.5 mmol dm−3. Atmosphere of air (gaseous mixture of nitrogen and oxygen
with 80% and 20%, v/v, respectively) at a flow rate of 100 mL min−1 and a scanning rate of 10 K min−1. Figure 13. Thermogravimetric curve for the Ca2+/Dop−precipitates obtained at different
metal:ligand molar ratios and I = 0.15 mol dm−3. Component concentration in the solution: cCa2+ =
1.5 mmol dm−3 and cDop−= 1.5–7.5 mmol dm−3. Atmosphere of air (gaseous mixture of nitrogen
and oxygen with 80% and 20%, v/v, respectively) at a flow rate of 100 mL min−1 and a scanning rate
of 10 K min−1. Table 15 reports the percentage weight loss for each Ca2+/Dop−system, calculated
from the thermal decomposition of the precipitates. Considering these experimental data,
the stoichiometry of the precipitates was calculated, assuming that the residual of the
decomposition process is CaO. As it can be observed, the stoichiometry of the precipitate
change between a metal:ligand molar ratios of 1:3 to 1:5, in dependence on the initial
concentration of the components in the vessel. 22 of 28
ol
n Biomolecules 2021, 11, 1312 e 14. Thermogravimetric profile of the percentage weight loss for Ca2+/Dop− B. Figure 14. Thermogravimetric profile of the percentage weight loss for Ca2+/Dop−B. 14. Thermogravimetric profile of the percentage weight loss for Ca2+/Dop− B. Figure 14. Thermogravimetric profile of the percentage weight loss for Ca2+/Dop−B. Table 15 reports the
4. Literature Data In some cases, the ligand was considered as a triprotic ligand (L2−), by the calcu-
n of the third protonation constant having a log KH value of about 13–14, out of the
The only attempt of comparison that can be done between our and literature data
regards the speciation models. They are mainly formed by simple mononuclear species,
such as: ML, ML2, and some ternary protonated ones. For the systems reported in the
literature, the formation of metal polynuclear species, such as M2L2 and M2LOH, obtained
for Sn2+, is not observed. A fairly good agreement between our and the literature data there is for Mg2+, logβML
= 4.57–4.49 at I = 0.2 mol dm−3 in NaCl aqueous solution and different temperatures (from
T = 288.15 to 308.15 K) [42]. As an example, from our investigation we obtained at I = 0.15 mol dm−3 and T = 298.15
K, logβML = 3.034 ± 0.044. The difference with respect the literature value can be explained
considering that our speciation model also includes the MLOH species, which becomes
significant at pH values higher than 8.5, and cannot be neglected in the speciation of the
system. The formation constant of the ternary MLOH hydrolytic species is: logβMLOH =
−6.111 ± 0.016 at I = 0.15 mol dm−3 and T = 298.15 K. Another attempt of comparison can be made for the CH3Hg+/Dop−complexes here
determined and those already reported in a previous investigation for the CH3Hg+/Eph−
(adrenaline) interactions [18]. A similar speciation model has been obtained; if we compare,
as a pure example, the stability of the complexes at I = 0.15 mol dm−3 and T = 298.15 K
in NaCl aqueous solution, for adrenaline we have the following values: logβML = 8.56
(10.43 for Dop); logβMLH = 17.33 (19.43 for Dop); logβMLOH = −0.79 (1.99 for Dop) and
logβMLCl = 9.17 (11.21 for Dop). It is possible to observe that dopamine forms more stable Biomolecules 2021, 11, 1312 23 of 28 complexes with CH3Hg+ with respect to adrenaline, with a difference of about of two
orders of magnitude. complexes with CH3Hg
with respect to adrenaline, with a difference of about of two
orders of magnitude. Table 16. Literature data for the complexation of dopamine towards metal ions. I/mol dm−3
T/K
Metal
Medium
logβML
logβMLH
logβML2
logβM(LH)2
logβML2H
Ref. Table 15 reports the
4. Literature Data 0.1
298.15
Cu2+
HClO4
20.56
26.91
48.26
[43]
0.2
298.15
Y3+
KCl
7.95
14.84
[44]
La3+
6.35
11.7
[45]
0.2
288.15
Mg2+
NaCl
4.57
[42]
298.15
4.50
308.15
4.49
0.2
288.15
Zn2+
NaCl
52.94
[46]
298.15
52.14
308.15
51.22
0.15
310.15
Cu2+
NaClO4
15.44
22.07
23.25
[47]
Ni2+
9.66
18.12
26.07
Zn2+
11.43
18.89
0.2
288.15
MoO42−
NaCl
51.48
[48]
298.15
51.08
308.15
50.66
0.2
298.15
Cu2+
KCl
16.60
24.22
24.78
45.83
35.66
[49]
Ni2+
9.42
19.38
14.81
35.66
25.61
Zn2+
20.21
18.05
38.93
28.67
0.37
293.15
Ni2+
NaNO3
18.373
13.86
34.05
[50]
Cu2+
16.01
23.29
23.47
44.26
Zn2+
19.33
Cd2+
17.991
Pb2+
22.23 Table 16. Literature data for the complexation of dopamine towards metal ions. However, the difficulty to carry out an accurate comparison between our and literature
systems, in terms of stability of the species, is also due to the apparent neglection from
the different authors of the hydrolysis of the metals investigated. In fact, in those papers,
authors report and discuss the acid-base properties of dopamine, but no information on the
hydrolysis of the metal and their hydrolytic constants is reported. From our investigations,
we have evidenced that the metal hydrolytic species coexist in some cases, especially in
excess of metals, with the metal/dopamine complexes, as observed for Sn2+, where the
Sn(OH)+ and Sn3(OH)42+ species reach about 5% of formation in pH range values, where
the formation of the Sn2+/Dop−complexes occur, whilst the SnOHCl—about 20%. The use of the pL0.5 values can be useful to estimate the different sequestering ability
of dopamine towards the metal ions. In this light, we selected the systems listed in Table 16,
which reports the stability of the complexes determined at similar experimental conditions. By using the protonation constants proposed in those literature papers [44,45,48–53] and
adding to the speciation models, the hydrolytic constants of the metals, taken from Brown
and Edberg [54], the pL0.5 values were calculated at T = 298.15K, I = 0.2 mol dm−3 and pH
= 7.4. The obtained values reported in Figure 15, can be compared with the values here
presented for our systems. p
y
From Figure 15, the following pL0.5 values have been obtained: pL0.5: Cd2+ = 0.23; Ni2+ = 1.77; Zn2+ = 2.61; Cu2+ = 6.60. Table 15 reports the
4. Literature Data For La3+, a negligible amount of metal was sequestered by dopamine; for this reason,
the pL0.5 value was calculated at pH = 9.5, resulting to be pL0.5 = 2.85. As a pure comparison
on the effect of the pH on the sequestration of Cd2+ by dopamine, also in this case the
pL0.5 was calculated at pH = 7.4 and 9.5; the value obtained for Cd2+ are pL0.5 = 0.23 and
3.80, respectively. The very low pL0.5 value calculated for Cd2+ with respect to Zn2+ or
Cu2+, can be explained considering that in chloride media, it forms quite stable CdCli (i For La3+, a negligible amount of metal was sequestered by dopamine; for this reason,
the pL0.5 value was calculated at pH = 9.5, resulting to be pL0.5 = 2.85. As a pure comparison
on the effect of the pH on the sequestration of Cd2+ by dopamine, also in this case the
pL0.5 was calculated at pH = 7.4 and 9.5; the value obtained for Cd2+ are pL0.5 = 0.23 and
3.80, respectively. The very low pL0.5 value calculated for Cd2+ with respect to Zn2+ or
Cu2+, can be explained considering that in chloride media, it forms quite stable CdCli (i Biomolecules 2021, 11, 1312 24 of 28
the pL0.5
and 3 80, 24 of 28
the pL0.5
and 3 80 = 1–3) complexes and the CdOHCl one, that reduce the free metal concentration for the
interaction with the dopamine. be explained considering that in chloride media, it forms quite stable CdCli (i 1 3) com
plexes and the CdOHCl one, that reduce the free metal concentration for the interaction
with the dopamine. = 1–3) complexes and the CdOHCl one, that reduce the free metal concentration for the
interaction with the dopamine. be explained considering that in chloride media, it forms quite stable CdCli (i 1 3) com
plexes and the CdOHCl one, that reduce the free metal concentration for the interaction
with the dopamine. Table 15 reports the
4. Literature Data Mg2+ (0.08) < Cd2+ (0.23) < Ca2+ (1.15) < Ni2+ (1.77) < Zn2+ (2.61) < CH3Hg+ (2.63) < Sn2+ (3.74) < Cu2+ (6.60). Mg2+ (0.08) < Cd2+ (0.23) < Ca2+ (1.15) < Ni2+ (1.77) < Zn2+ (2.61) < CH3Hg+ (2.63) < Sn2+ (3.74)
Mg2+ (0.08) < Cd2+ (0.23) < Ca2+ (1.15) < Ni2+ (1.77) < Zn2+ (2.61) < CH3Hg+ (2.63) < Sn2+ (3.74) < Some simple correlations can be obtained from our and literature data; considering the
pL0.5 values here calculated at T = 298.15 K and I~0.15–0.20 mol dm−3 (pH = 7.4), a simple
fairly linear relationship is obtained with the first hydrolytic constant (M(OH) species) of
the M2+ metal ions reported in Figure 16. The linear relationship is reported in Figure 16,
where we observe a correlation coefficient r = 0.95. Some simple correlations can be obtained from our and literature data; considering
the pL0.5 values here calculated at T = 298.15 K and I~0.15–0.20 mol dm−3 (pH = 7.4), a
simple fairly linear relationship is obtained with the first hydrolytic constant (M(OH) spe-
cies) of the M2+ metal ions reported in Figure 16. The linear relationship is reported in
Figure 16, where we observe a correlation coefficient r = 0.95. 0
1
2
3
4
5
6
7
-12
-11
-10
-9
-8
-7
-6
-5
Cu
2+
Zn
2+
Ni
2+
Mn
2+
Cd
2+
log KMOH
r = 0.95
pL0.5
Mg
2+
Figure 16. Relationship between the value log KMOH value of some M2+ metal ions vs. pL0.5 calculated
at I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4. Similar results can be obtained from the comparison of the pL0.5 values vs. the for-
mation constants of the ML species of the M2+ metal/dopamine species, determined at I =
0.15 mol dm−3 and T = 298.15 K. Data for Mn2+ and (CH3CH2)2Sn2+ (DET) reported in Figure
17 are unpublished data from this research group. Moreover, for Figure 17, a fairly linear
relationship (r = 0.90) was obtained. Figure 16. Relationship between the value log KMOH value of some M2+ metal ions vs. pL0.5
calculated at I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4. Similar results can be obtained from the comparison of the pL0.5 values vs. Table 15 reports the
4. Literature Data 2
4
6
8
0.0
0.2
0.4
0.6
0.8
1.0
Cd
2+
Cu
2+
Zn
2+
Ni
2+
La
3+ pH = 9.5
Cd
2+ pH = 9.5
x
pL 2
4
6
8
0.0
0.2
0.4
0.6
0.8
1.0
Cd
2+
Cu
2+
Zn
2+
Ni
2+
La
3+ pH = 9.5
Cd
2+ pH = 9.5
Data: PL_CdpH95
Model: Boltzman
Chi^2 = 6.064E-6
A1
100
±0
A2
0
±0
x0
3.79713
±0.00004
dx
0.43431
±0.00003
x
pL
Figure 15. Sequestering diagram for some metal/dopamine systems, at I = 0.15 mol dm−3, T = 298.15
K and pH = 7.4.pL0.5: Cd2+ = 0.23; Ni2+ = 1.77; Zn2+ = 2.61; Cu2+ = 6.60 (pH = 9.5: La3+ = 1.85; Cd2+ = 3.80). These literature data allow a merge with those here experimentally obtained at I =
Figure 15. Sequestering diagram for some metal/dopamine systems, at I = 0.15 mol dm−3, T = 298.15
K and pH = 7.4.pL0.5: Cd2+ = 0.23; Ni2+ = 1.77; Zn2+ = 2.61; Cu2+ = 6.60 (pH = 9.5: La3+ = 1.85; Cd2+
= 3.80). Figure 15. Sequestering diagram for some metal/dopamine systems, at I = 0.15 mol dm−3, T = 298.15
K and pH = 7.4.pL0.5: Cd2+ = 0.23; Ni2+ = 1.77; Zn2+ = 2.61; Cu2+ = 6.60 (pH = 9.5: La3+ = 1.85; Cd2+ = 3.80). The e lite atu e data allo
a
e
e
ith tho e he e e
e i
e tally obtai ed at I
Figure 15. Sequestering diagram for some metal/dopamine systems, at I = 0.15 mol dm−3, T = 298.15
K and pH = 7.4.pL0.5: Cd2+ = 0.23; Ni2+ = 1.77; Zn2+ = 2.61; Cu2+ = 6.60 (pH = 9.5: La3+ = 1.85; Cd2+
= 3.80). These literature data allow a merge with those here experimentally obtained at I
0.15 mol dm−3 in NaCl aqueous solutions, T = 298.15 K and pH = 7.4; the following seques-
tering trend of dopamine towards the metals was obtained:
These literature data allow a merge with those here experimentally obtained at I = 0.15
mol dm−3 in NaCl aqueous solutions, T = 298.15 K and pH = 7.4; the following sequestering
trend of dopamine towards the metals was obtained:
26 of 30 Mg2+ (0.08) < Cd2+ (0.23) < Ca2+ (1.15) < Ni2+ (1.77) < Zn2+ (2.61) < CH3Hg+ (2.63) < Sn2+ (3.74) < Cu2+ (6.60). Table 15 reports the
4. Literature Data 0
1
2
3
4
5
6
7
2
4
6
8
10
12
14
16
18
Cu
2+
DET
2+
Ni
2+
Mn
2+
Ca
2+
log KML
pL0.5
r = 0.90
Mg
2+ Figure 17. Relationship between the value log KML value of some M2+ metal/dopamine system vs. pL0.5. Data refer to I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4 (for pL0.5 values). Figure 17. Relationship between the value log KML value of some M2+ metal/dopamine system vs. pL0.5. Data refer to I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4 (for pL0.5 values). Obviously, these correlations should be considered only as simple approximations
since the pL0.5 also depends on many other variables, such as the other hydrolytic constants
of the metal and the conditions where the pL0.5 is calculated, for example, pH, temperature,
ionic strength, etc. Table 15 reports the
4. Literature Data the
formation constants of the ML species of the M2+ metal/dopamine species, determined at
I = 0.15 mol dm−3 and T = 298.15 K. Data for Mn2+ and (CH3CH2)2Sn2+ (DET) reported in
Figure 17 are unpublished data from this research group. Moreover, for Figure 17, a fairly
linear relationship (r = 0.90) was obtained. 0
1
2
3
4
5
6
7
-12
-11
-10
-9
-8
-7
-6
-5
Cu
2+
Zn
2+
Ni
2+
Mn
2+
Cd
2+
log KMOH
r = 0.95
pL0.5
Mg
2+ Figure 16. Relationship between the value log KMOH value of some M2+ metal ions vs. pL0.5 calculated
at I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4. Figure 16. Relationship between the value log KMOH value of some M2+ metal ions vs. pL0.5
calculated at I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4. Similar results can be obtained from the comparison of the pL0.5 values vs. the for-
mation constants of the ML species of the M2+ metal/dopamine species, determined at I =
0.15 mol dm−3 and T = 298.15 K. Data for Mn2+ and (CH3CH2)2Sn2+ (DET) reported in Figure
17 are unpublished data from this research group. Moreover, for Figure 17, a fairly linear
relationship (r = 0.90) was obtained. Similar results can be obtained from the comparison of the pL0.5 values vs. the
formation constants of the ML species of the M2+ metal/dopamine species, determined at
I = 0.15 mol dm−3 and T = 298.15 K. Data for Mn2+ and (CH3CH2)2Sn2+ (DET) reported in
Figure 17 are unpublished data from this research group. Moreover, for Figure 17, a fairly
linear relationship (r = 0.90) was obtained. 25 of 28
ly linear Biomolecules 2021, 11, 1312 0
1
2
3
4
5
6
7
2
4
6
8
10
12
14
16
18
Cu
2+
DET
2+
Ni
2+
Mn
2+
Ca
2+
log KML
pL0.5
r = 0.90
Mg
2+
Figure 17. Relationship between the value log KML value of some M2+ metal/dopamine system vs. pL0.5. Data refer to I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4 (for pL0.5 values). Figure 17. Relationship between the value log KML value of some M2+ metal/dopamine system vs. pL0.5. Data refer to I = 0.15 mol dm−3, T = 298.15 K and pH = 7.4 (for pL0.5 values). 5. Discussion The speciation studies carried out on dopamine interactions towards monovalent (CH3Hg+)
and bivalent (Mg2+, Ca2+, and Sn2+) metal cations lead to the following conclusions: 1. Interactions with Ca2+ allow the formation of a simple speciation model with species
of low stability. 2. Similar results were obtained for the Mg2+/Dop−system, even if, in this case, the
speciation model contains only the ML and MLOH species. The formation of the
MLH species was checked at each experimental condition, but was always rejected by
the computer program. The different speciation model with respect to Ca2+ can be
attributed to the different acid-base properties of Mg2+ with respect to calcium. In
fact, a comparison between the distribution diagrams of these two systems, highlight
for Mg2+ a “complexation” with dopamine at higher pH values (pH~8.5) with respect
to Ca2+ (pH~7.5), avoiding the formation of MLH. p
g
3. The behavior of CH3Hg+ in NaCl aqueous solutions is also particular; in this case, the
CH3HgCl species influences the speciation of the CH3Hg+/Dop−system. In fact, the
significant stability of the CH3HgCl species and the weakness of the complexes with
dopamine lead to the formation of a mixed ternary complex (MLCl), which forms in a
wide pH interval and at each investigated experimental condition. p
g
p
4. Only for the Sn2+/Dop−system, the processing of the experimental data highlights
the formation of both mononuclear and binuclear complexes of medium-high stability,
with variation of logβpqr values dependent on the ionic strength and temperature
investigated. The strength of the polynuclear complexes, also at low concentrations, avoid the
formation of the simple ML ones, but allows the formation of the binary Sn2L2 species. This complex, together with the M2LOH ones, reaches high formation percentage along a
wide pH interval. 5. The effect of the supporting electrolyte (NaCl) on the speciation of the metal/ligand
systems can be observed for CH3Hg+ and Sn2+, since they tend to form stable complexes
with chloride, influencing both the stability and distribution of the Mn+/dopamine species
and preventing the hydrolysis of the metal ions. Biomolecules 2021, 11, 1312 26 of 28 6. Measurements carried out at different ionic strengths and temperatures allowed to
observe that the stability of the complexes is influenced by variation of these two
variables, in particular that the entropy is generally the driving force of the formation
of the metal/dopamine species. 6. 5. Discussion Measurements carried out at different ionic strengths and temperatures allowed to
observe that the stability of the complexes is influenced by variation of these two
variables, in particular that the entropy is generally the driving force of the formation
of the metal/dopamine species. 7. The dependence of the stability constants on the ionic strength and temperature was
modeled by means of different approaches (see Section 3.4), allowing the calculation
of the Debye–Hückel parameters for the dependence on I/mol dm−3 and the specific
ion interaction parameters (SIT) of the ion-pairs (and of the Setschenow coefficient for
the neutral species) formed by the interaction of the metals with dopamine. p
y
p
8. By using the pL0.5 parameter, it was possible to quantify, at different pH values,
ionic strengths, and temperatures, the effective sequestering ability of dopamine
towards the metals. This approach is very important, since the simple comparison of
the stability constants values can lead to incorrect considerations, especially when
comparing systems with different speciation and in different experimental conditions
of temperatures, ionic strengths, pH values, or in different ionic media. The results
here obtained highlight that the sequestering ability tends to vary as the temperature
and ionic strength change. The sequestering ability of dopamine towards the metals
follows the trend: pL0.5: Sn2+ > CH3Hg+ > Ca2+> Mg2+. p
g
g
9. The precipitates collected at the end of potentiometric titrations were analyzed by
thermogravimetric analysis (TGA) under a constant airflow (10 K/min). The analysis
of the thermogravimetric profile of the curves allowed observing different stoichiom-
etry of the solids, dependent on the different metal:ligand ratios, and concentrations
of the solutions containing these components. p
g
g
9. The precipitates collected at the end of potentiometric titrations were analyzed by
thermogravimetric analysis (TGA) under a constant airflow (10 K/min). The analysis
of the thermogravimetric profile of the curves allowed observing different stoichiom-
etry of the solids, dependent on the different metal:ligand ratios, and concentrations
of the solutions containing these components. 10. g
p
10. An analysis of the literature data allowed observing different speciation models
and stability of the metal/dopamine complexes. The different sequestering ability
of dopamine towards the metal ions was quantified by using the pL0.5 parameter,
observing fairly linear relationships between this parameter and the first hydrolytic
constants of the metals (log KMOH) or with the log KML. 10. References Cataldo, S.; Crea, F.; Gianguzza, A.; Pettignano, A.; Piazzese, D. Solubility and acid-base properties an
chitosan in different ionic media and at different ionic strengths, at T = 25 ◦C. J. Mol. Liq. 2009, 148, 120 14. Cigala, R.M.; Crea, F.; De Stefano, C.; Lando, G.; Milea, D.; Sammartano, S. Modeling the acid–base properties of glutathione in
different ionic media, with particular reference to natural waters and biological fluids. Amino Acids 2012, 43, 629–648. [CrossRef]
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self-medium and in NaClaq at T = 298.15 K. J. Chem. Thermodyn. 2010, 42, 1393–1399. [CrossRef] 16. Crea, F.; Cucinotta, D.; De Stefano, C.; Milea, D.; Sammartano, S.; Vianelli, G. Modeling solubility, acid–base properties and activity
coefficients of amoxicillin, ampicillin and (+)6-aminopenicillanic acid, in NaCl(aq) at different ionic strengths and temperatures. Eur. J. Pharm. Sci. 2012, 47, 661–677. [CrossRef] 17. Bretti, C.; Cigala, R.M.; Crea, F.; De Stefano, C.; Vianelli, G. Solubility and modeling acid–base properties of adrenaline in NaCl
aqueous solutions at different ionic strengths and temperatures. Eur. J. Pharm. Sci. 2015, 78, 37–46. [CrossRef] 18. Crea, F.; De Stefano, C.; Irto, A.; Lando, G.; Materazzi, S.; Milea, D.; Pettignano, A.; Sammartano, S. Understanding the Solution
Behavior of Epinephrine in the Presence of Toxic Cations: A Thermodynamic Investigation in Different Experimental Conditions. Molecules 2020, 25, 511. [CrossRef] [PubMed] 19. Materazzi, S.; Foti, C.; Crea, F.; Risoluti, R.; Finamore, J. Biomimetic complexes of divalent cobalt and zinc with N-heterocyclic
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and stability of the metal/dopamine complexes. The different sequestering ability
of dopamine towards the metal ions was quantified by using the pL0.5 parameter,
observing fairly linear relationships between this parameter and the first hydrolytic
constants of the metals (log KMOH) or with the log KML. Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/biom11091312/s1, Table S1. Literature protonation constants of dopamine at different
ionic strengths and temperatures in NaCl aqueous solutions; Table S2. Literature hydrolytic and
chloride constants of CH3Hg+, Mg2+, Ca2+ and Sn2+ ions in NaCl aqueous solutions at different
ionic strengths and temperatures; Table S3. Formation constants of the CH3Hg+/Dop−complexes at
different ionic strengths and temperatures in molal concentration scale; Table S4. Formation constants
of the Ca2+/Dop−complexes at different ionic strengths and temperatures in molal concentration
scale; Table S5. Formation constants of the Mg2+/Dop−complexes at different ionic strengths
and temperatures in molal concentration scale; Table S6. Formation constants of the Sn2+/Dop−
complexes at different ionic strengths and temperatures in molal concentration scale; Figure S1. Distribution diagram for the Mg2+/Dop−species at I = 0.15 mol dm−3 and T = 310.15 K; Figure S2. Derivative curves for the different Ca2+/Dop−precipitates; Figure S3. Thermogravimetric profile of
the percentage weight loss for Ca2+/Dop−A; Figure S4. Thermogravimetric profile of the percentage
weight loss for Ca2+/Dop−C. Author Contributions: Conceptualization, A.G., F.C., A.I. and D.M.; methodology, F.C., C.D.S. and
R.M.C.; software F.C. and D.M.; validation, F.C., D.M., A.I. and C.D.S.; formal analysis, F.C., A.I. and
S.M.; investigation, F.C., A.G. and A.I.; data curation, F.C., A.I., R.M.C., M.R.F. and A.P.; writing—
original draft preparation, F.C. and C.D.S.; writing—review and editing, F.C., A.G., M.R.F. and D.M.;
supervision, C.D.S. and F.C.; project administration, F.C., C.D.S.; funding acquisition, C.D.S. All
authors have read and agreed to the published version of the manuscript. Funding: The authors A.G., F.C., D.M. C.D.S., R.M.C., and A.I. thank the University of Messina for
research and mobility 2017 funds (ARCADIA project). Funding: The authors A.G., F.C., D.M. C.D.S., R.M.C., and A.I. thank the University of Messina for
research and mobility 2017 funds (ARCADIA project). Institutional Review Board Statement: Not applicable Institutional Review Board Statement: Not applicable Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. 27 of 28 Biomolecules 2021, 11, 1312 Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ure, A.M.; Davidson, C.M. (Eds.) Chemical Speciation in the Environment, 2nd ed.; Wiley-Blackwell: 2. Bernhard, M.; Bernhard, M.; Brinckman, F.E.; Sadler, P.J. The Importance of Chemical “Speciation” in Environmental Processes; Springer
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2017, 62, 1018–1027. [CrossRef] 34. Baes, C.F.; Mesmer, R.E. The Hydrolysis of Cations; John Wyley & Sons: New York, NY, USA, 1976. 34. Baes, C.F.; Mesmer, R.E. The Hydrolysis of Cations; John Wyley & Sons: New York, NY, USA, 1976. 35. Cigala, R.M.; Crea, F.; De Stefano, C.; Lando, G.; Milea, D.; Sammartano, S. The inorganic speciation of tin(II) in aqueous solution. Geochim. Cosmochim. Acta 2012, 87, 1–20. [CrossRef] 36. Bretti, C.; Cigala, R.M.; Crea, F.; Lando, G.; Sammartano, S. Thermodynamics of proton binding and weak (Cl−, Na+ and K+)
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54. Brown, P.L.; Ekberg, C. Hydrolysis of Metal Ions; Wiley & Sons: Weinheim, Germany, 2016; Available online: https://onlinelibrary. wiley.com/doi/book/10.1002/9783527656189 (accessed on 1 April 2016).
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Attention Matters: Pitch vs. Pattern Processing in Adolescence
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Frontiers in psychology
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Keywords: attention, auditory, adolescents, mismatch negativity, pitch, pattern processing INTRODUCTION
“My experience is what I agree to attend to.” (James, 1890,
p. 402). The current study took a slightly different focus on selective
listening, in which selection was initiated by altering task per-
formance with the same set of sounds. There was one stream of
sounds that was fully attended to perform different tasks. The
“object” was determined by the task requirements that put differ-
ent attributes of the same sequence into focus, not by competing
bottom-up inputs that could be ignored. Thus, competition was
derived fully by the task demand and not by ignoring different
parts of the input. More sensory information reaches us that we can possibly deal
with. One of the goals of attention is to limit the amount of infor-
mation we perceive. When we choose to attend to certain parts of
the sensory input it changes our experience of the environment. If I now ask you to direct your attention to your shoes, you can
feel the pressure of them against your feet. Though, you probably
didn’t notice your shoes before I asked you to attend to them even
though the sensory information was always available to you.Active
selection initiates an interaction between the input (bottom-up
processes) and task goals (top-down influences) (Treue,2001;Beck
andKastner,2009;ShapiroandMiller,2011;MillerandBuschman,
2012), modulating the flow of information. This effectively alters
our experience of the environment by putting into focus what we
choose (or select) to attend to. Attention is adaptive. Adolescence is a time of considerable growth in both cognitive
and brain functions (Crone,2009). The ability to selectively attend
to the environment has been shown to improve during develop-
ment (Stuss,1992). Changes in executive function during this time
indicate an increased ability to switch between tasks (Crone et al.,
2006), to monitor actions (Ladouceur et al., 2010), and to hold
items in memory (Crone et al., 2006; Bunge and Wright, 2007). Concurrently, the brain is undergoing continued developmental
changes (Gogtay et al., 2004; Blakemore and Choudhury, 2006;
Casey et al., 2008), such as a decrease in gray matter consequential
to an increase in synaptic pruning (Sowell et al.,2001; Gogtay et al.,
2004). Consistent with continued brain maturation through ado-
lescence, the cortically generated obligatory event-related brain
potentials (ERPs) that are measured at the scalp have not yet
reached the maturity level of adulthood (Ponton et al.,2000; Gilley
et al., 2005; Wunderlich and Cone-Wesson, 2006; Sussman et al.,
2008). ORIGINAL RESEARCH ARTICLE
bli h d 10 J
2013 Elyse S. Sussman* Elyse S. Sussman* Departments of Neuroscience and Otorhinolaryngology-HNS, Albert Einstein College of Medicine, Bronx, NY, USA Edited by:
Nicole Wetzel, University of Leipzig,
Germany
Reviewed by:
Marie Gomot, Institut National de la
Santé et de la Recherche Médicale,
France
Anna Shestakova, St. Petersburg
State University, Russia
*Correspondence:
Elyse S. Sussman, Departments of
Neuroscience and
Otorhinolaryngology-HNS, Albert
Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY
10461, USA
e-mail: elyse.sussman@
einstein.yu.edu Keywords: attention, auditory, adolescents, mismatch negativity, pitch, pattern processing Edited by: From the moment we wake up, we are flooded with more sensory inputs than we can
possibly process. Selective attention mechanisms serve to limit the sensory onslaught,
while facilitating the ability to perform everyday tasks. However, not much is known about
the typical development of selective attention mechanisms during childhood even though
impairments of attention are commonly noted in neurodevelopmental disorders. The cur-
rent study focuses on a transitional time in child development, adolescence, to determine
in what way specific auditory tasks have a modulatory effect on underlying brain activity to
facilitate behavioral goals. Neural mechanisms of selective attention were tested through
auditory pitch and pattern perception, using a measure of event-related brain potentials
(ERPs) called the mismatch negativity (MMN). Sounds with a regular five-tone pattern
were presented in three conditions. The conditions differed only in how participants were
instructed to listen to the sounds. Focus was either on the pitch of the sounds, the pattern
of the sounds, or on a close-captioned movie. Even though the sound input was identical in
all conditions, task-specific modifications were manifest in the MMN evoked by the deviant
sounds embedded in the test sequences. The results demonstrate that in adolescence,
as in adults, selective attention alters neural activity specific to performance goals, thus
indicating specific neural adaptation modulated by behavior. Edited by:
Nicole Wetzel, University of Leipzig,
Germany Nicole Wetzel, University of Leipzig,
Germany Reviewed by: e
e
ed by
Marie Gomot, Institut National de la
Santé et de la Recherche Médicale,
France Marie Gomot, Institut National de la
Santé et de la Recherche Médicale,
France *Correspondence:
Elyse S. Sussman, Departments of
Neuroscience and
Otorhinolaryngology-HNS, Albert
Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY
10461, USA e-mail: elyse.sussman@
einstein.yu.edu Keywords: attention, auditory, adolescents, mismatch negativity, pitch, pattern processing Thus, this developmental time period is a unique age group
for investigation, with little known about the link between brain
responses and higher level cognitive skills. It is well known that selecting among inputs can bias underly-
ing neural activity associated with it (Hubel et al., 1959; Desimone
and Duncan, 1995; Shomstein and Yantis, 2004). In the biased-
competition model of visual selective attention (Desimone and
Duncan, 1995), for example, when there are multiple objects in
a visual scene, the attended object “wins” the competition for
neural representation in favor of the unattended objects. Atten-
tion mediates the neural responses, enhancing the activity of the
selected object. The indication of such results is that attention acts
to facilitate behavioral goals. June 2013 | Volume 4 | Article 333 | 1 www.frontiersin.org Attention matters Sussman is particularly useful for addressing the question because (1)
MMN provides an index of deviance detection regardless of the
direction of attention (Näätänen, 1992; Sussman et al., 2003b;
Winkler et al., 2005). Therefore, it could be elicited in all con-
ditions of attention; and (2) MMN is highly context dependent
(Sussman and Steinschneider, 2006; Rahne et al., 2007; Sussman,
2007; Rahne and Sussman, 2009), and thus can index how sounds
are organized in memory. MMN elicitation is based on tones
that are detected as deviant in comparison with tones detected
as standard (Näätänen, 1992; Näätänen et al., 2001; Sussman,
2007). Based on our previous studies in adults, we expected
that selectively listening to the pitches of the sounds vs. the
patterns of the sounds would modulate the neurophysiological
response to the B tones in accordance with the task being per-
formed (Sussman et al., 1998, 2002; Sussman and Gumenyuk,
2005). The goal of the current study, in adolescents, was to test task-
specific modulation of neural activity resulting from selectively
attending to either the pitch or the pattern of a single stream
of sounds to determine how selective attention modulates neural
activity associated with task goals. That is, the listener is not
selecting a subset of the whole information, but rather refocus-
ing attention to different aspects of the sounds by combining or
separating elements of a single sound stream to perform a task. Keywords: attention, auditory, adolescents, mismatch negativity, pitch, pattern processing To test this, we presented a patterned sequence of sounds and
instructed participants to focus on the pitches of the sounds
(Attend-Pitch condition), the pattern of the sounds (Attend-
Pattern condition), or away from the sounds (Attend-Video con-
dition). The tone pattern in the sequence was determined by
two tones occurring in a regularly repeating five-tone pattern
(AAAABAAAAB . . ., where“A” represents a tone of one frequency
and B represents a tone of a slightly higher frequency, Figure 1). A third tone (slightly lower in frequency) was presented ran-
domly, and rarely, in the sequence. This tone served as the target
so that participants would never be pressing the response key
to the B tones that provided the dependent measure. Listening
to the pitches required focus on the individual sounds to com-
pare among them, whereas listening to the pattern of sounds
involved temporally connecting them to perceive the repeating
sequence. We predicted that if the B tone was always detected as an infre-
quently occurring tone of a different frequency than the A tone
(focus on pitch), then MMN would be elicited by the B tone in
all conditions. In contrast, if the five-tone repeating pattern of the
sequence was detected in all conditions (focus on pattern), then
no MMN would be elicited by the B tone because it would be held
in memory as part of the standard repeating pattern, and not as
a deviant. Thus, task modulation of the neural activity to support
performance goals would be observed by MMN evoked by B tones
only when the pitches of the tones were used to perform the task,
but not when the pattern was relevant for the task. We measured components of the ERPs associated with deviance
and target detection. In particular, the mismatch negativity
(MMN) component was used to index deviance detection. MMN FIGURE 1 | Schematic of the stimulus paradigm. The frequency (in Hz) of
the tones is indicated on the ordinate and time is represented on the
abscissa. The 880 Hz tones, represented by filled black squares, occurred
80%, and the 988 Hz tones, represented by the open squares, occurred 18%. The sound sequence containing the five-tone repeating pattern was
presented in all three conditions: Attend-Video (top row), Attend-Pitch (middle
row), and Attend-Pattern (bottom row). Target tones are represented by the
open square with a “T” inside. DATA REDUCTION AND ANALYSIS ERPs evoked by each stimulus type was separately averaged
together. Responses evoked by the A tone immediately follow-
ing the B tone were excluded from analysis. Approximately 10% of
the overall epochs were rejected due to artifact. The MMN com-
ponent was statistically measured using a 40 ms window centered
on the peak obtained at the mastoid electrodes in the grand-mean
difference waveforms. The inversion at the mastoid that is typi-
cally observed for the MMN component was used to obtain a peak
measurement because of overlap at frontal electrode sites (e.g., Fz)
with the N2 component in the sound task conditions. Fz was used
in the Attend-Video condition. The peak of 152 ms was used for
the Attend-Pitch and Attend-Pattern conditions for all stimulus
types. In the Attend-Video conditions, the peak was 174 ms for all
stimulus types. The N2 component was measured using a 40 ms
window centered on the peak in the grand-mean difference wave-
form peak at the Cz electrode (greatest S/N ratio), with a peak
latency of 218 ms in the Attend-Pattern condition, and 202 ms in
the Attend-Pitch condition. The P3b component was measured
using a 50 ms window centered on the peak of the grand-mean
difference waveform at the Pz electrode (greatest S/N ratio), with
a peak latency of 364 ms in the Attend-Pattern condition, and
354 ms in the Attend-Pitch condition. No attention-related com-
ponents were elicited in the Attend-Video condition because the
sounds were ignored. PARTICIPANTS Ten adolescents (six males), ranging in age from 13 to 17 years
(M = 15, SD = 1) participated in the study. All procedures were
approved by the Internal Review Board of the Albert Einstein Col-
lege of Medicine. Parents were reimbursed for their travel expenses
and adolescents were given a gift certificate for their participation. After the experimental protocol was explained to them, parents
gave informed consent and children gave written assent. All of the
children were in their age-appropriate grade in school, passed a
hearing screening test (20 dB HL or better from 500 to 4000 Hz),
and had no reported history of neurological disorders. ELECTRODE PLACEMENT AND ELECTROENCEPHALOGRAM RECORDING
Electroencephalogram recordings were obtained using a 32-
channel electrode cap that incorporates a subset of the Interna-
tional 10–20 system (Jasper, 1958). Additionally, electrodes were
placed over the left and right mastoids (LM and RM, respectively). The tip of the nose was used as the reference electrode during
recordings. F7 and F8 electrode sites were used in a bipolar con-
figuration to monitor the horizontal electro-oculogram (HEOG). FP1 and an electrode placed below the left eye were used in a
bipolar configuration to monitor the vertical electro-oculogram
(VEOG). All impedances were maintained below 5 kΩ. The EEG
and EOG were digitized (Neuroscan SynAmps amplifier, Com-
pumedics Corp., El Paso, TX, USA) at a sampling rate of 500 Hz
(0.05–100 Hz bandpass). EEG was then filtered offline (Butter-
worth, zero phase shift) with a lowpass designation of 30 Hz. Artifact rejection was set to exclude activity exceeding 100 µV
after EEG epochs were baseline corrected. Epochs were 600 ms in
duration, starting 100 ms pre-stimulus onset and ending 500 ms
post-stimulus onset. STIMULI Three pure tone stimuli, 50 ms duration (5 ms rise/fall time, cali-
brated to 79 dB peak-to-peak equivalent using a Brüel and Kjaer
2209 sound level meter), were presented with a 575 ms onset-to-
onset pace binaurally through insert earphones. The three stimuli
differed only in tone frequency (784, 880, and 988 Hz). Two of
the tones were presented in a continuously repeating five-tone
pattern (AAAABAAAAB . . ., where “A” denotes the 880 Hz tone
and “B” denotes 988 Hz). Thus, the B tone was presented as every
fifth tone in the sequence (p = 0.20). The 784 Hz tone randomly
replaced the A tones (p = 0.02), and was the target tone when a
task was performed with the sounds (Figure 1). Keywords: attention, auditory, adolescents, mismatch negativity, pitch, pattern processing The position of the target shown in each
condition denotes that the target occurred randomly (2%), whereas the other
tones were presented in a fixed order. The dashed square outlines the
smallest tonal element needed to perform the task, thus denoting the
standard used in the MMN deviance detection process. Frontiers in Psychology | Developmental Psychology
June 2013 | Volume 4 | Article 333 | 2 row), and Attend-Pattern (bottom row). Target tones are represented by the
open square with a “T” inside. The position of the target shown in each
condition denotes that the target occurred randomly (2%), whereas the other
tones were presented in a fixed order. The dashed square outlines the
smallest tonal element needed to perform the task, thus denoting the
standard used in the MMN deviance detection process. FIGURE 1 | Schematic of the stimulus paradigm. The frequency (in Hz) of
the tones is indicated on the ordinate and time is represented on the
abscissa. The 880 Hz tones, represented by filled black squares, occurred
80%, and the 988 Hz tones, represented by the open squares, occurred 18%. The sound sequence containing the five-tone repeating pattern was
presented in all three conditions: Attend-Video (top row), Attend-Pitch (middle FIGURE 1 | Schematic of the stimulus paradigm. The frequency (in Hz) of
the tones is indicated on the ordinate and time is represented on the
abscissa. The 880 Hz tones, represented by filled black squares, occurred
80%, and the 988 Hz tones, represented by the open squares, occurred 18%. The sound sequence containing the five-tone repeating pattern was
presented in all three conditions: Attend-Video (top row), Attend-Pitch (middle Frontiers in Psychology | Developmental Psychology Frontiers in Psychology | Developmental Psychology June 2013 | Volume 4 | Article 333 | 2 Attention matters Sussman MATERIALS AND METHODS
PARTICIPANTS seated but took a moment to shift position. One longer break
(10–15 min) was provided at roughly the mid-point, in which par-
ticipants were disconnected from the recording system and given
time to walk around and have a snack break. PROCEDURES The patterned tone sequences were presented in three conditions
of attention: Attend-Pitch, Attend-Pattern, and Attend-Video. In
the Attend-Pitch condition, participants were instructed to listen
to the three different pitches of the tones and press the response
key when they heard the rarely occurring, lowest-pitched tone. In the Attend-Pattern condition, participants were instructed that
there was a five-tone repeating pattern of tones in the sequence
and to press the response key when they detected a different pat-
tern that occurred rarely. Consequently, in both task conditions,
the target tone was the same rarely occurring lower-pitched tone
and only the instruction of when to press the response key differed
(i.e., for a pitch change or a pattern change). In the Attend-Video
condition, participants were told that they would hear sounds in
their ears, to ignore the sounds and watch the captioned video of
their choosing. Thus, the patterned sequences were played in all
three conditions and only the instructions of how to attend to the
stimuli differed (attend to the pitch, to the pattern, or ignore the
sounds). Participants sat in a comfortable chair in an electrically shielded
and sound-attenuated booth (IAC, Bronx, NY, USA). Five blocks
of 300 stimuli were presented for each of the attend conditions
(∼3 min per block) and three blocks of 500 stimuli (∼5 min per
block) for the ignore condition. Attend condition blocks were
shorter than the Ignore condition blocks to reduce sustained atten-
tion effects or fatigue that may occur during longer focused task
demands. However, the overall presentation amount was the same
in all conditions. About 1155 A tones, 300 B tones, and 45 target
tones were obtained in each condition. Total session time was 1.5–
2 h,which included time for electrode placement and breaks. Short
breaks (1–3 min) were provided in which participants remained To statistically verify the presence of the MMN, N2, and P3b
components, one-sample t-tests were conducted to determine
whether the mean amplitude at the electrode of greatest signal-to-
noiseratioforeachcomponent(FzforMMN,Fzfortarget-N2,and
Pz for P3b) was significantly greater than zero. To compare ampli-
tudes of the components across conditions, repeated-measures
analysis of variance (ANOVA) was calculated and Huynh–Feldt June 2013 | Volume 4 | Article 333 | 3 www.frontiersin.org www.frontiersin.org Attention matters Sussman ERP RESULTS
d corrections were reported as appropriate. Fisher’s least signifi-
cant difference (LSD) test was performed for all post hoc cal-
culations. Hits, misses, false alarms, and correct rejections were
calculated to determine accuracy of behavioral responses to the
target tones. Button press responses were considered correct if
they occurred between 100 and 900 ms from target stimulus onset. Student’s t-test for dependent measures was used to determine
whether the hit rate (HR), false alarm rate (FAR), and reac-
tion time (RT) differed between target types (Attend-Pitch vs. Attend-Pattern). Figure 2 displays the ERPs elicited by the standard stimuli,overlay-
ing the responses from each condition of attention. In adolescence,
the P1 (peak ∼68 ms),N1 (peak ∼90 ms),P2 (peak ∼144 ms),and
the obligatory-N2 (peak ∼276 ms) were observed in the standard
waveforms. Observed peak latencies and amplitudes of the obliga-
tory components are consistent with their age (Ponton et al., 2000;
Sussman et al., 2008). Figure 3 displays the ERPs evoked by the A and B tones overlain
fortheAttend-Pattern,Attend-Pitch,andAttend-Videoconditions
at the midline (Fz, Cz, and Pz) electrodes. Table 1 provides the
mean amplitudes of the difference (response to B-minus-response
to A) waveforms for the ERP components, and statistical presence
of MMN, target-N2, and P3b components. Figure 3 displays the ERPs evoked by the A and B tones overlain
fortheAttend-Pattern,Attend-Pitch,andAttend-Videoconditions
at the midline (Fz, Cz, and Pz) electrodes. Table 1 provides the
mean amplitudes of the difference (response to B-minus-response
to A) waveforms for the ERP components, and statistical presence
of MMN, target-N2, and P3b components. BEHAVIORAL RESULTS Hit rate to targets was high in both the Attend-Pattern (M = 0.93,
SD = 0.07) and Attend-Pitch (M = 0.95, SD = 0.04) conditions,
with no significant difference between them (t 9 = 1.03, p = 0.33). FAR was low in both conditions (M = 0.001, SD < 0.01) and also
did not significantly differ by task (t 9 < 1, p = 0.80). RT to tar-
gets in the Attend-Pattern (M = 463, SD = 84) and Attend-Pitch
(M = 463, SD = 86) conditions also did not significantly differ by
task (t 9 < 1, p = 0.96). Overall, there were no task effects on HR,
RT, or FAR. Figure 4 displays the difference waveforms (ERP response to
the B tone-minus-ERP response to the A tone) and voltage maps. MMNs were elicited by the target tones in all conditions (Table 1). MMNs were elicited by the B tones (the final tone of the standard
repeating pattern) in the Attend-Pitch and in the Attend-Video
conditions, but not in the Attend-Pattern condition (Table 1). The
amplitudes of the MMNs elicited by the target tones were larger
than the MMN elicited by the B tones (F1,9 = 32.13, p < 0.001). FIGURE 2 | Standard obligatory responses for all conditions. The ERP
responses to the frequently occurring tone (880 Hz) are overlain at the midline
(Fz, Cz, Pz) and lateral (T7, T8) electrodes for the Attend-Video (short dashed
line), Attend-Pattern (solid line), and Attend-Pitch (long dashed line) conditions. Obligatory ERP components are labeled at the electrode of greatest
signal-to-noise ratio for the midline (P1, N1, P2, and N2) and the t-complex
(Na, Ta, and Tb). x-axis is displayed in milliseconds. y-axis is the amplitude in
microvolts. Obligatory ERP components are labeled at the electrode of greatest
signal-to-noise ratio for the midline (P1, N1, P2, and N2) and the t-complex
(Na, Ta, and Tb). x-axis is displayed in milliseconds. y-axis is the amplitude in
microvolts. FIGURE 2 | Standard obligatory responses for all conditions. The ERP
responses to the frequently occurring tone (880 Hz) are overlain at the midline
(Fz, Cz, Pz) and lateral (T7, T8) electrodes for the Attend-Video (short dashed
line), Attend-Pattern (solid line), and Attend-Pitch (long dashed line) conditions. BEHAVIORAL RESULTS Frontiers in Psychology | Developmental Psychology June 2013 | Volume 4 | Article 333 | 4 Frontiers in Psychology | Developmental Psychology | 4 Attention matters Sussman FIGURE 3 | ERPs evoked by the A and B tones. The ERP responses evoked
by the A tones (solid red line) and the B tones (solid blue line) are overlain and
displayed for the Attend-Pattern (top row), Attend-Pitch (middle row), and
Attend-Video (bottom row) conditions. Midline electrodes are in columns (Fz,
left; Cz, middle; and Pz, right). Ordinate is the amplitude in microvolts, and the
abscissa shows the timeline in milliseconds. Attend-Video (bottom row) conditions. Midline electrodes are in columns (Fz,
left; Cz, middle; and Pz, right). Ordinate is the amplitude in microvolts, and the
abscissa shows the timeline in milliseconds. FIGURE 3 | ERPs evoked by the A and B tones. The ERP responses evoked
by the A tones (solid red line) and the B tones (solid blue line) are overlain and
displayed for the Attend-Pattern (top row), Attend-Pitch (middle row), and To determine whether there was an effect of task on MMN, a two-
way repeated-measures ANOVA was conducted on the difference
waveforms with factors of Attention (Pattern/Pitch/Video) and
Electrode (Fz, F3, F4). There was no effect of attention on MMN
amplitude for the target tones (F2,18 < 1, p = 0.42), nor was there
a topographic difference (no main effect of electrode: F2,18 = 1,
i = 0.38), and no interaction. In contrast, there was a main effect
of attention on B tones (F2,18 = 4.96, ε = 0.86, p = 0.019). Post hoc
calculation showed that MMN amplitude in the Attend-Pitch con-
dition (−1.47 µV) was larger than that in the Attend-Video condi-
tion (−0.80 µV), and both MMNs were larger than the amplitude
evoked in the Attend-Pattern condition (−0.26 µV), where there
was no significant MMN. no difference in amplitude according to task (no main effect of
attention, F1,9 < 1, p = 0.92), and no main effect of electrode after
correction (F5,45 = 2.83, ε = 0.52, p = 0.067), indicating that the
target-N2 had a fronto-central distribution in adolescence. For the
target-P3b, a two-way repeated-measures ANOVA was conducted
on the difference waveforms with factors of attention (pattern
vs. pitch) and electrode (Pz, P3, P4) to assess task effects. BEHAVIORAL RESULTS Sim-
ilarly, task had no effect on the target-P3b (no main effect of
attention, F1,9 < 1, p = 0.70). However, there was a main effect
of electrode (F2,18 = 7.15, ε = 0.99, p = 0.005). Post hoc calcula-
tions showed that the amplitude was largest at the Pz electrode,
which is a distribution that is similar to adults. DISCUSSION Target-N2 and P3b components were elicited only to target
stimuli (Table 1; Figure 4) when either the pattern or the pitch of
the sounds was attended (Attend-Pattern and Attend-Pitch con-
ditions), but not when the sounds were ignored (Attend-Video
condition), as would be expected. For the target-N2, two-way
repeated-measures ANOVA were conducted on the difference
waveforms with factors of attention (pattern vs. pitch) and elec-
trode (Fz, F3, F4, Cz, C3, C4) to assess task effects. There was The goal of the study was to assess, in adolescence, task-specific
modulation of neural activity resulting from selectively attending
to either the pitch or the pattern of sounds in a sequence. In all con-
ditions, the sound sequences were presented in a fixed temporal
order (AAAABAAAAB . . .), and only the task instructions dif-
fered. The main finding was that the task performance modulated
the brain’s response to the sound. This was demonstrated by the June 2013 | Volume 4 | Article 333 | 5 www.frontiersin.org Attention matters Sussman Table 1 | ERP component mean amplitudes. Attend/stimulus
Mean
SD
t-value
*≤0.05; **≤0.01;
ns, not sig. MMN (Fz)
Pattern/Target
−4.85
3.66
−4.19
**
Pattern/B
−0.23
1.45
−0.51
ns
Pitch/Target
−4.25
1.93
−6.96
**
Pitch/B
−1.43
1.07
−4.24
**
Video/Target
−3.51
4.30
−2.58
*
Video/B
−0.87
1.32
−2.08
*
N2 (Fz)
Pattern/Target
−7.63
5.12
−4.71
**
Pattern/B
0.53
1.44
1.16
ns
Pitch/Target
−7.15
4.42
−5.11
**
Pitch/B
−0.47
1.31
−1.13
ns
P3b (Pz)
Pattern/Target
16.02
10.46
4.84
**
Pattern/B
0.44
1.00
1.38
ns
Pitch/Target
14.98
9.90
4.79
**
Pitch/B
0.71
1.31
1.71
ns Table 1 | ERP component mean amplitudes. tones were deviants in the Attend-Pitch condition, on the basis
of probability of occurrence (Sussman et al., 2003a), and MMN
was elicited by them. The absence of MMN to the B tones in
the Attend-Pattern condition can be explained by their function
in the sequence as a part of the repeating standard pattern: they
were not deviant. Thus, the way in which the sounds were used
to perform the task altered the repeating standard that was main-
tained in memory, which modulated the change-detection process
(Sussman, 2007). When participants had no task with the sounds (Attend-Video
condition), MMNs were elicited by both the B tones and the
target (784 Hz) tones. This suggests that the regularity of the
sequence was not automatically detected, which may be explained
solely by the stimulus rate. DISCUSSION The 575 ms onset-to-onset pace may
have been too slow for automatic detection of the pattern while
attending the video (Sussman and Gumenyuk, 2005; Wang et al.,
2009). An alternative explanation, in conjunction with the slow
presentation rate, is that MMN elicitation to B tones occurred
because the patterning of the sequence was irrelevant to the task
(watching a video), whether or not the B tones were detected as
occurring regularly. This would suggest that MMN was elicited
strictly on the basis of the ratio of tone frequencies within the
sound sequence (Sussman et al., 2002; Sussman and Gumenyuk,
2005), with the A tones occurring frequently (standard) and the
B tones and target tones both infrequently (deviants) (Sussman
et al., 2003a). MMN response to the B tones under the different task conditions. When the pitches of the sounds were relevant to performing the
task, MMN was elicited by the infrequently occurring B tones. In
contrast, when the pattern of sounds was relevant to perform the
task, no MMN was elicited by the B tones, as they were an integral
part of the repeating standard pattern. That is, the same regularly
occurring B tones evoked different brain responses depending on
their relevance in performing the task. These results thus demon-
strate that selective attention alters neural activity, adapting the
neural response to the pertinent elements of the input required
for performing a task: in this case, adapting to a five-tone standard
in one condition and to single tone standard in the other. Further, these results are consistent with previous studies using
similar paradigms in adult participants (Sussman et al., 1998,
2002; Sussman and Gumenyuk, 2005), indicating that top-down
processes in adolescence have similar modulatory effects in facil-
itating task goals. Even though the obligatory cortical ERPs in
adolescents are not yet adult-like, they differ both in morphology
and scalp distribution compared to adults (Sussman et al., 2008),
task effects evoking the MMN component appear to be similar. This suggests that the MMN component, which is driven by the
internal state of the individual, reflects aspects of auditory cogni-
tion not strictly bound to cortical maturation, which is consistent
with findings of MMN elicitation in infants and toddlers (Shafer
et al., 2000; Huotilainen et al., 2003). DISCUSSION The target 784 Hz tone was a deviant in both conditions,
even though in the Attend-Pattern condition the target tone was
a “pattern deviant” and in the Attend-Pitch condition it was a
“pitch deviant.” MMN, target-N2, and P3b components were
elicited by the targets. In the current study, in the adolescents,
the target-detection N2 component had a more fronto-central
scalp distribution than the more centro-parietal distribution gen-
erally found in adults. This could be due to overlap of multiple
N2 components in adolescents, if, for example, anterior cin-
gulate cortex were more involved in performing the task. This
would be consistent with continued changes occurring in pre-
frontal cortex through adolescence (Blakemore and Choudhury,
2006). The target-P3b component, on the other hand, had a sim-
ilar topography to that found in adults, with its maximal peak
at the Pz electrode (Friedman and Simpson, 1994; Fabiani et al.,
1998). Finally, the ability to selectively attend to sounds and ignore
irrelevant sounds in the environment has important implications
for clinical populations. The paradigm of the current study may
be useful for testing various clinical populations. This is because
changes in the neurophysiologic responses can be attributed to
attentional control and not to differences in the physical charac-
teristics of the stimuli or to the stimulus presentation rate, since
the physical auditory input was the same in all conditions. This
protocol may thus provide a unique way to assess attention deficits
in various neurodevelopmental disorders. Frontiers in Psychology | Developmental Psychology frontal lobe function. Psychophysiol-
ogy 35, 698–708. doi:10.1111/1469-
8986.3560698 SUMMARY (C) Stimulus sequence (bottom row) is displayed as a
reminder of the stimulus-eliciting tones. “T” denotes the target tones when
sounds were attended and “B” denotes the fifth tone of the pattern. FIGURE 4 | Difference waveforms and scalp voltage maps for all FIGURE 4 | Difference waveforms and scalp voltage maps for all
conditions. (A) Difference waveforms (top row) are displayed for the target
tones (thick lines) and B tones (thin lines) at Fz (solid line) and the left mastoid
(LM, dashed lines) in the Attend-Pattern (left column), Attend-Pitch (middle
column), and Attend-Video (right column). (B) Scalp voltage maps (middle
row) are displayed at the peak of the ERP components (peak latency above adjusting the time scale of adaptation dependent upon task
goals. current study, even though the stimulus-driven input was biased
toward the patterned sequence, attention mediated the neural
activity to support performance goals. Task-dependent facili-
tation was demonstrated in that the neural response adapted
either to the single tone standard or to the five-tone pattern
standard inherent in the sequence. Overall, the results show,
in adolescence as in adults, that attention plays an important
role in modulating neural activity to facilitate performance, SUMMARY The same tone evoked different change-detection responses spe-
cific to the task, not the stimulus input. This is consistent with
studies demonstrating task-specific effects in the visual system
(Beck and Kastner, 2009), and consistent with idea that atten-
tion is an “emergent property” of competitive interactions with
stimulus-driven processes (Desimone and Duncan, 1995). In the In contrast, the B tones had a functionally different role in
the two conditions depending on whether the listener was detect-
ing separate pitches, or the sequential five-tone pattern. The B
tones were used to perform the task but were not target tones, and
required no button presses in any of the three conditions. The B Frontiers in Psychology | Developmental Psychology June 2013 | Volume 4 | Article 333 | 6 Attention matters Sussman FIGURE 4 | Difference waveforms and scalp voltage maps for all
conditions. (A) Difference waveforms (top row) are displayed for the target
tones (thick lines) and B tones (thin lines) at Fz (solid line) and the left mastoid
(LM, dashed lines) in the Attend-Pattern (left column), Attend-Pitch (middle
column), and Attend-Video (right column). (B) Scalp voltage maps (middle
row) are displayed at the peak of the ERP components (peak latency above the head shots, and the ERP components labeled below). No MMN was
elicited by the B tone in the Attend-Pattern condition. Target-N2 and P3b
components were not elicited in the Attend-Video condition, in which the
sounds were ignored. (C) Stimulus sequence (bottom row) is displayed as a
reminder of the stimulus-eliciting tones. “T” denotes the target tones when
sounds were attended and “B” denotes the fifth tone of the pattern. E 4 | Difference waveforms and scalp voltage maps for all the head shots, and the ERP components labeled below). No MMN was
elicited by the B tone in the Attend-Pattern condition. Target-N2 and P3b
components were not elicited in the Attend-Video condition, in which the
sounds were ignored. (C) Stimulus sequence (bottom row) is displayed as a
reminder of the stimulus-eliciting tones. “T” denotes the target tones when
sounds were attended and “B” denotes the fifth tone of the pattern. the head shots, and the ERP components labeled below). No MMN was
elicited by the B tone in the Attend-Pattern condition. Target-N2 and P3b
components were not elicited in the Attend-Video condition, in which the
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adolescence. Front. Psychol. 4:333. doi:
10.3389/fpsyg.2013.00333 Frontiers in Psychology | Developmental Psychology ACKNOWLEDGMENTS This is an
open-access article distributed under the
terms of the Creative Commons Attribu-
tion License, which permits use, distrib-
ution and reproduction in other forums,
provided the original authors and source
are credited and subject to any copy-
right notices concerning any third-party
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Citation for published version (Harvard):
Rolfe, S, Garnham, L, Godwin, J, Anderson, I, Seaman, P & Donaldson, C 2020, 'Housing as a social
determinant of health and wellbeing: developing an empirically-informed realist theoretical framework', BMC
Public Health, vol. 20, 1138. https://doi.org/10.1186/s12889-020-09224-0 Housing as a social determinant of health and wellbeing: developing an empirically-
informed realist theoretical framework
Rolfe, Steve; Garnham, Lisa; Godwin, Jon; Anderson, Isabel; Seaman, Pete; Donaldson,
Cam
Published in:
BMC Public Health
DOI:
10.1186/s12889-020-09224-0
Publication date:
2020
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Publisher's PDF, also known as Version of record
Link to publication in ResearchOnline
Citation for published version (Harvard):
Rolfe, S, Garnham, L, Godwin, J, Anderson, I, Seaman, P & Donaldson, C 2020, 'Housing as a social
determinant of health and wellbeing: developing an empirically-informed realist theoretical framework', BMC
Public Health, vol. 20, 1138. https://doi.org/10.1186/s12889-020-09224-0 Document Version
Publisher's PDF, also known as Version of record Link to publication in ResearchOnline Citation for published version (Harvard):
Rolfe, S, Garnham, L, Godwin, J, Anderson, I, Seaman, P & Donaldson, C 2020, 'Housing as a social
determinant of health and wellbeing: developing an empirically-informed realist theoretical framework', BMC
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of how to contact us. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Rolfe et al. BMC Public Health (2020) 20:1138
https://doi.org/10.1186/s12889-020-09224-0 Open Access Abstract Background: The role of housing as a social determinant of health is well-established, but the causal pathways are
poorly understood beyond the direct effects of physical housing defects. For low-income, vulnerable households
there are particular challenges in creating a sense of home in a new tenancy which may have substantial effects on
health and wellbeing. This study examines the role of these less tangible aspects of the housing experience for
tenants in the social and private rented sectors in west central Scotland. Methods: The paper analyses quantitative data from a mixed methods, longitudinal study of tenants from three
housing organisations, collected across the first year of their tenancy. The paper postulates causal hypotheses on
the basis of staff interviews and then uses a Realist Research approach to test and refine these into a theoretical
framework for the connections between tenants’ broader experience of housing and their health and wellbeing. Results: Housing service provision, tenants’ experience of property quality and aspects of neighbourhood are all
demonstrated to be significantly correlated with measures of of health and wellbeing. Analysis of contextual factors
provides additional detail within the theoretical framework, offering a basis for further empirical work. Conclusions: The findings provide an empirically-informed realist theoretical framework for causal pathways
connecting less tangible aspects of the housing experience to health and wellbeing. Applying this within housing
policy and practice would facilitate a focus on housing as a public health intervention, with potential for significant
impacts on the lives of low-income and vulnerable tenants. The framework also offers a basis for further research to
refine our understanding of housing as a social determinant of health. Keywords: Housing, Health, Social determinants, Causal mechanisms, Realist evaluation empirically understood. This paper aims to develop an
empirically-informed theoretical framework to elucidate
some of the possible causal pathways between less
tangible aspects of housing experience and health and
wellbeing, for low-income households in rented accom-
modation. It is concerned with the subjective experience
of housing, from the perspective of low income house-
hold in rented accommodation. Housing as a social determinant of health
and wellbeing: developing an empirically-
informed realist theoretical framework Steve Rolfe1*
, Lisa Garnham2
, Jon Godwin3
, Isobel Anderson1
, Pete Seaman2
and Ca 2
and Cam Donaldson4 Background Housing is often cited as an important social determin-
ant of health, recognising the range of ways in which a
lack of housing, or poor quality housing, can negatively
affect health and wellbeing [1–4]. However, the causal
pathways from housing to health are inherently complex,
as with all the social determinants of health [5], so many
of these pathways are neither fully conceptualised, nor The causal relationships between tangible physical
housing defects and poor health outcomes are widely ac-
cepted [6, 7], with clear evidence of negative physical * Correspondence: steve.rolfe1@stir.ac.uk
1Faculty of Social Sciences, University of Stirling, Stirling FK9 4LA, UK
Full list of author information is available at the end of the article * Correspondence: steve.rolfe1@stir.ac.uk
1Faculty of Social Sciences, University of Stirling, Stirling FK9 4LA, UK
Full list of author information is available at the end of the article * Correspondence: steve.rolfe1@stir.ac.uk
1Faculty of Social Sciences, University of Stirling, Stirling FK9 4LA, UK
Full list of author information is available at the end of the article Rolfe et al. BMC Public Health (2020) 20:1138 Page 2 of 19 Rolfe et al. BMC Public Health (2020) 20:1138 This paper attempts to make progress in this area, by
developing an empirically-informed, realist theoretical
framework for causal pathways linking less tangible as-
pects of housing as experienced by tenants to health and
wellbeing outcomes. Utilising quantitative data from a
mixed
methods,
longitudinal
study
of
tenants
in
Scotland, the paper employs realist analysis [31, 32] to
test and refine four hypotheses relating to: relationships
between tenants and their housing provider; tenants’ ex-
perience of property quality; affordability; and aspects of
neighbourhood and social support. Firstly, we focus on
whether the hypotheses are supported by the evidence. Secondly, we consider the contextual factors which play
a role in determining who is most affected and in what
circumstances [31]. To undertake this analysis, the study
focuses on new tenants, conceptualising the change of
tenancy and the related changes in housing experience
and housing service as a complex intervention in the
lives of participating tenants [33]. The paper is inher-
ently exploratory, attempting to develop and refine a
realist understanding of the causal pathways which may
link less tangible aspects of the housing experience to
health and wellbeing, on the basis of empirical data, to
provide a framework for further analysis and research. Further analysis of the qualitative data from the study, to
elaborate the precise nature of the causal mechanisms
involved will be the subject of a later paper. health effects of toxins within the home, damp and
mould, cold indoor temperatures, overcrowding and
safety factors [4, 6–8], and also of negative mental health
effects arising from cold indoor temperatures, over-
crowding/lack of personal space, and damp and mould
[4, 6, 7, 9]. Moreover, analysis of the impact of housing
improvement interventions provides evidence for causal
direction and pathways [10, 11]. Beyond these impacts of physical aspects of housing,
the literature regarding health impacts of less tangible
aspects of the housing experience is relatively sparse, al-
though the literature around the notion of ‘home’ pro-
vides some theoretical and empirical starting points. The
social, psychological and cultural value of home as
something more than the material object of housing has
long been recognised [12], indicating the range of ways
in which dwellings offer sites of control, autonomy and
socialisation, and a basis for social identity and status
[13, 14]. Thus from the perspective of ‘ontological secur-
ity’ [15, 16], the home is seen as providing a secure base
from which people can develop confidence in self and
social identity [17, 18]. Moreover, research across differ-
ent housing sectors [19, 20] and examining the specific
experiences of different social groups [21–23] suggests
that the subjective experience of housing and housing
services can be important in creating a sense of home
and underpinning ontological security. Taking this a stage further, the work of Kearns and
colleagues examining the ‘psychosocial benefits of home’
[24–26], has gone some way to develop theoretical and
empirical connections between housing, home and well-
being. Their findings suggest that aspects of control, au-
tonomy,
status
and
empowerment
are
related
to
measures of wellbeing, with some variance in relative
importance between households with different charac-
teristics, but the use of cross-sectional data restricts the
examination of causality. The evidence from longitudinal
panel datasets reviewed by Clapham et al. [27] provides
more evidence for the causal effect of physical housing
quality and tenure on measures of subjective wellbeing,
but these studies face difficulty in identifying mediators,
many of which appear to relate to the psychosocial as-
pects of home, including autonomy, security and status. Methods This study treats the entire housing experience as the
intervention from which health and wellbeing impacts
result. It seeks to understand the mechanisms through
which that subjective experience generates impacts, the
different contexts in which those mechanisms operate
and impacts vary, and why. It focuses, in particular, on
the less tangible aspects of that housing experience,
owing to a lack of empirical evidence in the literature. It
uses realist methodology in order to achieve this. In this
section we provide an introduction to realist evaluation
(RE) for readers unfamiliar with this methodology,
highlighting key characteristics which are of particular
relevance to this study. We explain why we selected this
methodology and how we employed it in practice. RE is an established methodology within the school of
theory-based evaluation (TBE) approaches. These meth-
odologies as a whole attempt to move away from before-
and-after evaluation designs, using theory to attempt to
uncover and understand the causal processes and mech-
anisms at play within any policy or programme [34, 35]. Examining health and wellbeing impacts of aspects of
housing beyond bricks and mortar is particularly import-
ant in the context of declining rates of home ownership
and consequent increases in renting across much of
Europe and the US, following the Global Financial Crisis
[28–30]. Aside from the obvious relevance of tenure se-
curity for this growing group of tenants, the role of
landlords and housing organisations may be salient, par-
ticularly in terms of the constraints that may be placed
on tenants’ agency in generating their own sense of
home. Understanding causality within realist evaluation
As Gates & Dyson [36] argue, there is a ‘growing ac-
knowledgement that there are multiple ways to think
about causal relationships’ and therefore a diversity of Understanding causality within realist evaluation Rolfe et al. BMC Public Health (2020) 20:1138 Page 3 of 19 Page 3 of 19 outcomes [39]. Initial, tentative theories in the form of
hypothesised CMOCs are developed from existing evi-
dence in the literature and stakeholder engagement,
employing ‘abductive reasoning’, which Jagosh et al. [40]
define as “inference to the best explanation”. Mixed
methods data collection is then utilised to examine the
‘outcome
regularities’
which
relate
to
these
initial
CMOCs, in two phases. Firstly, quantitative data is
employed to examine the patterns of outcomes across
different contexts in order to test and refine the initial
CMOCs. The causal theories represented by these
CMOCs are then refined further by using qualitative
data to elucidate the underlying mechanisms through
exploring and triangulating stakeholders’ understand-
ings. These refined CMOCs provide a more nuanced
picture of what works, for whom in different circum-
stances, which can be utilised in practice and also pro-
vides the starting point for a further iteration of the
realist research process. ways to make causal claims in social science. RE starts
from a realist view of causality, which has two important
features. Firstly, causality is conceptualised as generative
rather than successionist. Generative causation empha-
sises that it is the latent ‘powers and liabilities’ within
things which generates effects in particular contexts
[37]. Whilst methodologies based on successionist caus-
ality, such as RCTs, attempt to control for contextual in-
fluences,
RE
deliberately
incorporates
context
to
examine how it influences the operation of causal mech-
anisms. Thus, the RE position is that research needs to
focus not on whether programmes work in a general
sense, but on ‘what works, for whom, in what circum-
stances’ [31], often now extended to include questions of
‘how and why?’ This is particularly valuable in situations
where ‘interventions’ and contexts are interconnected in
complex ways, such as social situations where the inter-
vention is shaped by the agency of the beneficiary. Secondly, building on the realist notion of a ‘stratified
reality’ [31, 38], RE recognises that most mechanisms
will be hidden. Whilst many elements of social reality,
such as human behaviour or the existence and activities
of institutions are directly observable, the mechanisms
which generate social outcomes are often hidden within
individual reasoning or complex organisational interac-
tions
and
hence
are
not
necessarily
tangible. Understanding causality within realist evaluation As
Westhorp [37] argues, the invisible nature of mecha-
nisms arises because they (often) operate at different
levels of the system than the outcome, they operate at
different timescales to the outcome, and they depend on
relationships and interactions, some of which cannot be
observed. However, the practical impossibility of directly
observing such causal mechanisms does not preclude
understanding, it merely highlights the need for theory
to provide an explanation. In a sense, this perspective
provides an additional angle to the truism that ‘correl-
ation does not imply causation’, since the implication is
that correlation requires a theory of mechanisms to
identify the likelihood of a causal relationship. Importantly, RE attempts to develop theoretical under-
standing at different levels. On the one hand, the aim is
to develop ‘middle-range theory’ (MRT) [41] regarding
causal mechanisms. MRT lies between the (impossible)
grand, unified theory of social behaviour, organisation
and change, and the very specific understandings of par-
ticular contexts [31, 42]. On the other hand, individual
RE studies aim to improve the specification of CMOCs,
thereby “learning more and more about less and less”
[31] in order to enhance our understanding of the par-
ticular contexts within which causal mechanisms work
for particular groups of people. The process of know-
ledge cumulation within RE involves traversing repeat-
edly between abstraction and specification to refine the
MRT and examine how it applies in particular contexts
[31]. This paper focuses only on the first of these two el-
ements, examining the outcome regularities exhibited
within the quantitative data in order to develop a
middle-range theoretical framework. The second stage
of this analysis will be presented elsewhere. Housing Association • Community-based Housing Association, providing social rented hous-
ing and operating a subsidiary regeneration organisation which fo-
cuses on employment and community development. Aims to provide
affordable housing in the social rented sector (SRS) to low income
households with a variety of needs, as well as contributing to commu-
nity sustainability and regeneration through non-housing activities. Owns and manages around 5500 properties. Letting Agency • Social enterprise letting agency which manages property for private
rented sector (PRS) landlords. Combined with investment arm which
purchases its own property and rents it, through the letting agency
arm. Social mission to provide high quality housing in the PRS to
vulnerable households. Provides tenancy support service, funded from
service charge income. Manages around 250 properties on behalf of
private landlords and owns a further 200. Secondly, given that tenants are active agents within
their housing experience, the notion of generative caus-
ality is likely to be valuable in exploring the pathways
between aspects of housing experience and health and
wellbeing outcomes. Moreover, whilst health and well-
being can be measured at static points in time, there is a
sense in which health and (especially) wellbeing are
emergent properties which are constantly in flux. Hence,
understanding the causal relationships is likely to require
an exploration of multiple, inter-related mechanisms
which operate on different timescales [37]. Rent Deposit Schemes • Voluntary sector organisation running two Rent Deposit Schemes
(RDS), which facilitate access to the PRS for households at risk of
homelessness. Provides deposit guarantee to enable vulnerable
households without savings to access PRS tenancies, as well as a level
of tenancy support over the first year of the tenancy. Tenants are
expected to save up their deposit over the first year of their tenancy
instead of being asked to provide it up-front, before their tenancy be-
gins. Combined, the two schemes support around 100 people into ten-
ancies each year. organisation’s approach to working with tenants. Table 2
provides
an
overview
of
the
interviewees
in
each
organisation. We therefore employed RE methodology in order to
examine the complex, contingent and emergent nature
of the less tangible aspects of housing. We aimed to
examine the causal impact of the subjective housing ex-
perience, and to clarify the nature of the mechanisms in-
volved and the contexts influencing their operation. The data from these interviews provided the implicit
causal understandings of practitioners, which was then
combined with existing evidence from the literature to
examine the plausibility of the suggested mechanisms
and contextual factors, and to develop the initial, tenta-
tive CMOCs, as laid out in Table 3. There were some
differences between the organisations regarding the spe-
cific contextual factors that might be relevant, but across
the interviews the same four mechanisms were seen as
likely to have a notable impact on health and wellbeing. Project design In order to explore a range of possible mechanisms and
contexts, we worked with three quite different housing
organisations, described in Table 1. The organisations
operate across the social and private rented sectors, but
with a similar client group of low-income tenants. Low-
income households are most likely to be at risk from
poor health and wellbeing and more likely to experience
poor housing. They therefore represent the portion of
the population for whom it is more important to under-
stand the relationship between housing and health and
wellbeing, if we are to effectively address and reduce
health inequalities. Table 2 Overview of scoping study interviewees
Organisation
Interviewees
A
Housing Association
• Assistant Director of Housing Services
• Housing Manager
• Housing Officer
• Concierge
• Regeneration Manager
• Community Support/Development Officers × 2
• Cultural Officer
• Development Officer (money advice service)
B
Letting Agency
• Director
• Assistant Director
• Tenancy Support Officer
• Property Inspection Officer
C
Rent Deposit Schemes
• Service Manager
• Team Leader × 2
• Senior Development Officer
• Development Officer/Support Worker × 4
• Admin Worker × 2 Table 2 Overview of scoping study interviewees
Organisation
Interviewees Rationale for using RE in this study Building on these understandings of causality and the
nature of mechanisms, RE departs from experimental
methodologies that attempt to control contextual influ-
ences to isolate the effect of particular interventions or
mechanisms. Rather,
it
explicitly
recognises
that
mechanisms operate differently in different contexts and
therefore develops causal hypotheses in the form of ‘Con-
text-Mechanism-Outcome
Configurations’
(CMOCs),
attempting to identify how causal mechanisms may oper-
ate to generate outcomes within particular contexts. This research attempts to examine possible causal path-
ways between the less tangible aspects of the housing ex-
perience and health and wellbeing outcomes. More
specifically, we set out to investigate these impacts by
studying tenants entering a new tenancy, conceptualising
this change as a complex intervention in the lives of
these tenants. A number of aspects of this area of study
suggested the value of using RE. Firstly, the the intervention being studied is both
multi-faceted and complex, in the sense that there are
multiple, interacting components, including the agency
of tenants and housing staff [43]. Where interventions as
well as outcomes are emergent, and where context is In practical terms, RE proceeds in a cyclical fashion to
iteratively refine our understanding of mechanisms and
the contexts within which they operate to generate Page 4 of 19 Rolfe et al. BMC Public Health (2020) 20:1138 Rolfe et al. BMC Public Health (2020) 20:1138 likely to be important, an evaluation approach is needed
which can operate at the level of the system [44]. RE ex-
plicitly attempts to grapple with the fact that society and
human behaviour are in a permanent state of self-
transformation, by recognising the mutating nature of
social programmes and the role of agency in generative
causation [32, 45]. Whilst we can attempt to understand
the mechanisms which may generate health and well-
being outcomes, it is neither possible nor practically use-
ful to attempt to separate ‘intervention’ elements from
the complex open system within which they take place. Rather, we used RE in order to examine the ‘interven-
tional systems’ [46] within which health and wellbeing
change may be generated for and by tenants. Phase 2 – data collection Data was collected from a cohort of new tenants, over
the period 2016–2018. All new tenants were invited to
participate in the study, being given initial information
by housing organisation staff prior to a more detailed
conversation and opt-in consent process with the re-
search team. Participation was voluntary, with around
50% of new tenants agreeing to take part in the study. Data was collected through structured interviews carried
out at three time points: the start of the tenancy (Wave
1), collecting background data on tenants’ prior housing
situation; 2–4 months into the tenancy (Wave 2); and 9–
12 months into the tenancy (Wave 3). At each wave,
quantitative data was collected on satisfaction with vari-
ous aspects of the housing service, community and social
networks, health and wellbeing, financial circumstances
and demographics. At Waves 2 and 3, these elements
were also explored qualitatively through face-to-face in-
terviews conducted in the tenant’s home, although this
data is not presented here. Table 4 sets out the numbers
of tenants involved at each Wave and Table 5 provides a
demographic overview of the sample, based on those
completing
at
least
the
first
two
Waves
of
data
collection. g
For the purposes of this analysis, the definition of
health
aligns
with
that
of
the
World
Health
Organization: “a state of complete physical, mental and
social wellbeing, and not merely the absence of infirm-
ity” [47]. The definition of wellbeing itself is complex,
but we use it to mean a combination of positive psycho-
logical state and a functional balance between individual
resources and challenges [48]. Crucially, these concep-
tions of health and wellbeing overlap considerably,
reflecting the growing evidence base indicating that psy-
chological wellbeing is a significant determinant of phys-
ical health, particularly over the life course [49] and
that measures of wellbeing are highly correlated with
measures of health [50, 51]. As both a close analogue
to and a determinant of health, we suggest that Table 4 Numbers of participating tenants at each Wave
Organisation
Wave 1
Wave 2
Wave 3
Housing Association
56
33
23
Letting Agency
50
34
17
Rent Deposit Schemes
15
8
5
Total
121
75
45 The drop-out rates between the waves are largely due
to two factors. Phase 1 – developing the initial hypotheses B In the first phase of the research, individual semi-
structured interviews were carried out with 23 staff
across the three organisations, in order to uncover the
program theories underlying their practice, with specific
reference to potential health and wellbeing impacts. In-
terviewees were selected in order to provide a cross-
section of staff, encompassing different aspects of each Rolfe et al. BMC Public Health (2020) 20:1138 Page 5 of 19 Table 3 CMO-Cs through which housing situation may affect health and wellbeing and potential contextual influences
CMO-C
Contextual factors
Mechanism
Outcome
1
• Security of tenure
• Tenancy support
• Responsiveness of landlord to problems
• Expectations, situation and capacity
of tenant
Positive tenancy experience reduces stress and provides
tenants with autonomy and control
Improved health
and wellbeing
2
• Level of investment in property prior
to tenancy
Quality housing provides tenants with a comfortable space
in which to relax and a sense of status
Improved health
and wellbeing
3
• Rent levels
• Income levels
• Benefits system (especially changes)
• Landlord responses to financial issues
Affordable housing reduces financial stress and frees up
income for other expenditure
Improved health
and wellbeing
4
• Community development activities of landlord
• Opportunities for choice of neighbourhood
• Existing networks of tenants
• Tenancy support
Good neighbourhood environment and supportive social/
community networks around housing location reduce stress
and increase opportunities for socialisation
Improved health
and wellbeing Good neighbourhood environment and supportive social/
community networks around housing location reduce stress
and increase opportunities for socialisation • Tenancy support length. The attrition at Wave 3 was exacerbated by the
timescale of the project – some Wave 3 interviews could
not be scheduled before data collection had to be com-
pleted. These patterns were relatively consistent across
the three organisations and the number of tenants mov-
ing on or losing their tenancy was very small (< 5%). We
also compared demographic data for the participant
groups at each wave with each other and with the wider
population of new tenants within each organisation. This
analysis showed only minor differences, suggesting a
limited degree of selection bias. Phase 1 – developing the initial hypotheses Given the similar conceptions of potential mechanisms
across the organisations, we therefore set out to collect
data from tenants which would enable us to test and re-
fine these CMOCs, treating the housing organisation as
just one contextual factor amongst many that might im-
pact upon the tenants’ housing experience. Phase 2 – data collection At Wave 1, data was collected through a
short telephone interview (around 15 min), whereas
Waves 2 and 3 involved more onerous face-to-face inter-
views in the tenants’ home of around 30–60 min in Rolfe et al. BMC Public Health (2020) 20:1138 Page 6 of 19 Table 5 Demographic overview of participating tenants (Wave 2 completers)
Housing Assoc. Letting Agency
Rent Deposit Schemes
Total
Full sample
33
34
8
75
Gender
Female
17
18
5
40
Male
16
16
3
35
Age
Younger (< 35)
12
20
2
34
Older (= > 35)
21
14
6
41
Disability
Disabled
14
5
3
22
Non-disabled
19
29
5
53
Employment
Employed
8
23
0
31
Not employed
25
11
8
44
Household type
Household without children
21
26
5
52
Household with children
12
8
3
23
Household income
< 50% median
30
21
8
59
50–60% median
1
4
0
5
60–100% median
2
7
0
9
> 100% median
0
2
0
2
Housing Benefit
Full or partial Housing Benefit
25
7
8
40
No Housing Benefit
8
27
0
35
Previous housing situation
Social housing
9
2
2
13
Private rented sector
8
21
2
31
Homeless
10
5
4
19
Other
6
6
0
12 Table 5 Demographic overview of participating tenants (Wave 2 completers) not expect to be substantially affected by the housing ex-
perience within a year. Moreover, this pilot work indi-
cated that broader conceptions of health and wellbeing
overlap considerably in the public mind, reflecting the
connections demonstrated in the literature. We there-
fore included an additional question on self-rated change
in overall health and wellbeing and deliberately situated
it alongside qualitative exploration to create a focus on
the broader conception of health and wellbeing as a
combination
of
physical
state,
mental
state
and
functioning. wellbeing is an important outcome to consider in
housing research [27]. On the basis of these definitions and given the low
likelihood of significant impacts on clinical health indi-
cators arising from social determinants within a single
year, we used three self-report questions to measure
health and wellbeing at each wave. Whilst self-rated
health status has clear limitations, there is good evidence
to suggest that it provides a reliable indicator of object-
ively measured health [52]. We employed the World
Health Organization’s 5-point wellbeing scale (WHO5)
as an internationally-validated measure of wellbeing [51]. Phase 3 – analysis to test and refine the theories The analysis of the quantitative data was undertaken in
two stages. Firstly, the data was used to examine the out-
come regularities and thereby test whether the hypothe-
sised mechanisms appeared to be operating to generate
impacts on health and wellbeing. Bivariate tests were
carried out using the full sample (using Spearman’s Rho
for non-parametric data) to examine correlations be-
tween the independent variables related to each hypoth-
esis and two health and wellbeing outcome variables. Outcome regularities evidenced by correlations do not
in themselves provide clear evidence of causality, but
provide a basis for further investigation of the underlying
mechanisms and the contextual factors which may be af-
fecting their operation. g
g
Secondly, further tests (again using Spearman’s Rho)
were conducted for sub-populations within the full sam-
ple, in order to examine potential contextual factors
which may be influencing the operation of mechanisms
within each hypothesised CMO-C, including demo-
graphic
characteristics,
socio-economic
status
and
household type, as well as differences between the orga-
nisations. This analysis was carried out using the Wave
2 data, in order to provide a sufficient sample size at
sub-population levels. All of the sub-populations defined
in Table 5 above were tested in this analysis, but house-
hold type was simplified into households with children
and those without, whilst the analysis based on house-
hold income categories is not presented here, as the
small numbers of households outside the lowest income
category makes comparison between income groups im-
possible. Most variables have very few missing values
and analysis suggests that they are missing completely at
random (with one exception highlighted in the findings),
so pairwise exclusion was used. The Rent Deposit Importantly, the two health and wellbeing variables
serve different purposes within the analysis. The WHO5
scale provides a validated, internationally-recognised
measure [51], focused primarily on the positive psycho-
logical state aspect of wellbeing. Due to challenges in
contacting tenants prior to their move, 42% of partici-
pants did not complete the Wave 1 WHO5 question-
naire until more than 2 weeks after their move-in date. As such, Wave 1 WHO5 does not reliably describe pre-
move wellbeing for all tenants and therefore cannot be
used to assess improvement pre- and post-move across
the whole sample. Phase 2 – data collection As a general measure of health, we used a self-rated
health status question drawn from the Scottish House-
hold Survey. Unsurprisingly, the data from this question
showed no significant change between waves, although it
was still useful in demonstrating that our sample was
somewhat more unhealthy than the general population
(17% ‘bad’ or ‘very bad’ health in the sample, compared
to 9% in SHS 2017 data), as would be expected for this
group of vulnerable and low-income households. A
focus group and pilot interviews with tenants prior to
the main data collection phase had indicated that most
people were likely to interpret this question relatively
narrowly as relating to physical disease, which we did Independent variables were selected from existing
questions in national surveys (Scottish Household Sur-
vey and Scottish Social Housing Charter indicators) to
measure aspects of the housing experience which might
plausibly trigger each of the mechanisms in Table 3. Given the RE conception of mechanisms as hidden,
these variables do not attempt to measure the mecha-
nisms directly, but to provide an indication of the poten-
tial that the theorised mechanism has been triggered,
when combined with the outcome data. Additional ques-
tions relating to contextual aspects were also asked, in-
cluding demographics and previous housing situations. The key outcome and independent variables are set
out in Table 6. More information on these variables Page 7 of 19 Rolfe et al. BMC Public Health (2020) 20:1138 Page 7 of 19 and their related questions available in the supple-
mentary material. aspects of housing service, housing quality, financial
coping and neighbourhood. The self-rating of health and
wellbeing collected at Waves 2 and 3, provides a direct
measure of tenants’ perspectives on what has changed
for them since the start of their new tenancy and is
therefore used within the analysis to examine potential
correlations across Waves, testing for ‘change’ effects of
the same aspects of housing situation. Since the question
specifically asks for self-rated change since the start of
the tenancy, this variable provides a direct indicator of
tenants’ perceptions of the impact of their change in
housing situation on their health and wellbeing. Phase 3 – analysis to test and refine the theories Hence it is primarily used within the
analysis to examine potential correlations within each
Wave, testing for ‘static’ health and wellbeing effects of Table 6 Key variables used to explore hypothesised causal pathways
Hypothesis
Variable
Type of data
Independent variables
1
Overall satisfaction with housing organisation
5-point Likert-style scale
1
Comparison of current and previous experience of renting
5-point rating from ‘A lot better’ to ‘A lot worse’
2
Rating of property quality
5-point rating from ‘Very good’ to ‘Very poor’
2
Satisfaction with maintenance service
5-point Likert-style scale
3
Rating of ability to cope financially over the last few months
5-point rating from ‘All of the time’ to ‘Never’
3
Rating of ability to cope with paying rent over the last few months
5-point rating from ‘All of the time’ to ‘Never’
4
Rating of neighbourhood quality
4-point rating from ‘Very good’ to ‘Very poor’
4
Index created from four social support questionsa
Index (5-point Likert-style scale for each question)
Dependent variables
All hypotheses
World Health Organization 5-point Wellbeing Scale (WHO5) –
score created from five statements of wellbeing over the preceding
two weeks
6-point rating from ‘All of the time’ to ‘At no time’
All hypotheses
Self-rated change in health and wellbeing since moving into new
property (self-rated H&WB change)
5-point rating from ‘A lot better’ to ‘A lot worse’
a These questions were included to create an index, drawing on their use in other social surveys, such as the Scottish Household Survey. Cronbach’s alpha is >
0.75 at all three Waves Table 6 Key variables used to explore hypothesised causal pathways
H
th
i
V i bl Rolfe et al. BMC Public Health (2020) 20:1138 Page 8 of 19 Fig. 1 Health and wellbeing change from start of tenancy WHO5 index as the dependent variable, whilst those la-
belled ‘Wave 1–2′ and ‘Wave 1–3′ provide the results
for tests using the variable for self-rated health and well-
being change since the start of the tenancy. Schemes (RDS) are also excluded from the organisa-
tional breakdown analysis because the low numbers of
participating tenants make it impossible to perform
meaningful tests. RDS tenants are included in the other
sub-population tests. These results provide patterns of ‘outcome regularities’
in the language of RE, which suggest a number of poten-
tial refinements to the CMOCs. 1‘Significant’ change in the WHO-5 scale is considered to be 10 points
when the raw score is scaled to create an index of 0–100 (Topp et al.,
2015). Phase 3 – analysis to test and refine the theories For each hypothesised
causal pathway, the presence or absence of significant
correlations provides evidence as to whether the relevant
mechanism may be operating, bearing in mind that the
subsequent stage of the analysis may qualify these find-
ings by considering the contextual factors involved. Examining outcome regularities across full sample Note:This data relates only to those tenants who completed their Wave 1 WHO5
questionnaire prior to moving in to their new tenancy Fig. 2 Change in WHO5 wellbeing score from start of tenancy. Note:This data relates only to those tenants who completed their Wave 1 WHO5
questionnaire prior to moving in to their new tenancy Thirdly, in terms of neighbourhood and social support,
there is support for Hypothesis 4, since there are signifi-
cant correlations between neighbourhood quality and
the social support index, and health and wellbeing. and, if so, which contextual factors influence their
operation. Clearly care must also be taken in drawing conclusions
about differences in correlations between the ‘static’ and
‘change’ tests, since the dependent variables measure
somewhat different aspects of health and wellbeing. Nevertheless, variations in the significance level of the
correlations across hypotheses suggests that it is the Whilst correlation does not imply causation, these pat-
terns in the data provide indications of potential rela-
tionships which merit further investigation in order to
understand whether causal mechanisms are involved Table 7 Summary of hypothesis tests on full sample Table 7 Summary of hypothesis tests on full sample Table 7 Summary of hypothesis tests on full sample
Hypothesis
Independent variable
Wave 2
(WHO5)
Wave 3
(WHO5)
Wave 1–2
(HWB change)
Wave 1–3
(HWB
change)
Rho
Sig. Rho
Sig. Rho
Sig. Rho
Sig. Examining outcome regularities across full sample The data for both outcome variables indicates improve-
ment in health and wellbeing across the first year of the
tenancies. Figure 1 shows the data for tenants’ self-rated
health and wellbeing change at Waves 2 and 3, showing
a clear improvement at both time points. Firstly, in terms of the experience of the property and
housing service, there appears to be some support for
Hypotheses 1 and 2. All the variables relating to these
aspects of housing experience, with the exception of
maintenance service satisfaction at Wave 2, show a sig-
nificant correlation with health and wellbeing outcomes. In particular, the strength of the correlations in relation
to change in health and wellbeing from Wave 1 to Wave
2 suggests the possibility of a causal connection which
merits further investigation. Figure 2 provides the data for change in tenants’
WHO5 score from the start of their tenancy to the
Wave 2 and 3 time points.1 Again, this data suggests that
tenants’ wellbeing is improving over time in their new
tenancy. A similar pattern can be seen for the tenants of
each participant organisation when analysed separately. This overall picture of improving health and wellbeing
by comparison with tenants’ prior situations suggests
that there may be aspects of their new housing experi-
ence which are generating this change. The first stage of
the data analysis examines the possible role of the
hypothesised CMO-Cs by testing for correlations with
related independent variables. A summary of this ana-
lysis is provided in Table 7. The columns labelled ‘Wave
2′ and ‘Wave 3′ set out the results of the tests using the Secondly, the data relating to tenants’ self-rated finan-
cial coping shows that whilst overall financial coping is
strongly correlated with wellbeing at Wave 2, this effect
does not appear in between-wave analyses. Alongside
this, there is no significant correlation between tenants’
self-rated ability to cope with paying their rent and their
health and wellbeing, either within or across the Waves. This combination suggests that Hypothesis 3 is not sup-
ported by this data, since neither rent nor a change in
housing situation affects health and wellbeing as mea-
sured in this study. Rolfe et al. BMC Public Health (2020) 20:1138 Page 9 of 19 Fig. 2 Change in WHO5 wellbeing score from start of tenancy. 2Additional chi-square tests were conducted to check against these
correlations. Again, the results need to be interpreted tentatively given
the sample size, but these tests suggest a similar pattern. Hypothesis 2 – property quality Table 9 summarises the analysis by sub-population for
the two key variables relating to property quality: tenant
rating of the overall condition of the property and satis-
faction with the maintenance service. Looking firstly at property quality, there are notable
differences between sub-populations in terms of correla-
tions between changes in property quality and changes
in health and wellbeing. The data suggests that partici-
pants who are tenants of the Letting Agency, female,
young, non-disabled, employed, not receiving Housing
Benefit, with no children in the household and coming
from the PRS are more likely to exhibit a correlation be-
tween change in property quality and change in their
health and wellbeing. Some of these may be intercon-
nected, inasmuch as the sub-populations are connected. For example, Letting Agency tenants are more likely to
be employed, non-disabled and coming from the PRS
than
others
in
the
sample. However,
other Hypothesis 1 – positive tenancy experience Hypothesis 1 – positive tenancy experience
Table 8 summarises the analysis by sub-population for
the two key variables relating to overall tenancy experi-
ence: satisfaction with the housing organisation, and
overall
rental
experience
compared
to
previous
experiences. The key finding here is that the strongly significant
correlations for the whole population of participants are
largely reflected in the vast majority of sub-populations,
for both variables. Whilst there are differences in the
level of significance between sub-populations and some
sub-populations which do not show a significant correl-
ation on the overall renting experience variable, the dif-
ferences in p values are small and may plausibly be
explained by the smaller size of some of the sub-
samples.2 Hence tenancy experience, at least insofar as it
is captured by satisfaction with the housing organisation
and comparison with previous renting experiences, has a
universal relationship with health and wellbeing across
the different sub-populations of tenants in this study. Examining outcome regularities across full sample 1 Experience of secure, stable tenancy reduces
stress and provides tenants with a secure base
from which to exercise autonomy
Overall satisfaction with
housing organisation
0.27
0.02*
0.28
0.06
0.43
0.001*** 0.26
0.09
Comparison of current
and previous experience
of rentinga
NA
NA
NA
NA
0.38
0.002**
0.23
0.1
2 Quality housing provides tenants which a
comfortable space in which to relax and a
sense of status
Rating of property quality
0.28
0.02*
0.30
0.05*
0.31
0.007**
0.29
0.05*
Satisfaction with maintenance
service
0.18
0.2
0.095 0.6
0.46
0.009**
−0.033 0.9
3 Affordable housing reduces financial stress
and frees up income for other expenditure
Rating of ability to cope
financially over the last few
months
0.32
0.005**
0.57
0.001*** 0.14
0.2
0.18
0.2
Rating of ability to cope with
paying rent over the last few
months
0.047 0.7
0.081 0.6
0.030 0.8
0.17
0.3
4 Good neighbourhood environment and
supportive social/community networks
around housing location reduce stress and
increased opportunities for socialisation
Rating of neighbourhood
quality
0.46
0.001*** 0.44
0.003**
0.25
0.04*
0.20
0.2
Index created from four social
support questions
0.33
0.005**
0.30
0.04*
0.28
0.02*
−0.001 1.0
*p < 0.05, **p < 0.01, ***p < 0.001. N = 75 at Wave 2, N = 45 at Wave 3
aThis variable is explicitly about change in rental experience and therefore was not tested against the ‘static’ WHO5 measure Page 10 of 19 Page 10 of 19 Page 10 of 19 Rolfe et al. BMC Public Health (2020) 20:1138 of the housing experience. The outcome variable for all
of the subsequent tables is therefore self-rated change in
health and wellbeing. The one exception to this is overall
satisfaction with the housing organisation, since this
variable is not available at Wave 1. Many tenants did not
have a ‘housing organisation’ at Wave 1, because they
were either homeless or living with friends or family. Hence, for this test the ‘static’ variable of housing satis-
faction at Wave 2 was used. of the housing experience. The outcome variable for all
of the subsequent tables is therefore self-rated change in
health and wellbeing. The one exception to this is overall
satisfaction with the housing organisation, since this
variable is not available at Wave 1. Many tenants did not
have a ‘housing organisation’ at Wave 1, because they
were either homeless or living with friends or family. Examining outcome regularities across full sample Hence, for this test the ‘static’ variable of housing satis-
faction at Wave 2 was used. experience of the housing and the housing service rela-
tive to a tenant’s previous experiences, and not the
current experience of the housing or service in itself,
that may predict improvements in health and wellbeing. Conversely, the effects of neighbourhood quality and the
availability of local social support appear to show the op-
posite pattern, in that current experience may predict
changes in health and wellbeing outcomes, regardless of
tenants’ previous housing situation(s). The overall pattern of correlations at Wave 3 is very
similar to that at Wave 2, although there appears to be
some drop-off in terms of the strength of the relation-
ships. This may be due to the smaller sample size at
Wave 3 (N = 45), which inevitably limits the strength of
possible correlations by comparison with Wave 2 (N =
75). Furthermore, given these relatively small sample
sizes, there is a possibility that selection effects may be
influencing the apparent differences between Wave 2
and Wave 3. To examine this possibility, the analysis
was repeated across the two waves using data from only
those tenants who had completed all three Waves. This
analysis showed a very similar pattern of within-Wave
correlations at Wave 2 to the analysis on the full sample. Moreover, tests for difference between the group of ten-
ants who dropped out after Wave 2 and those who con-
tinued to Wave 3 on the independent and dependent
variables, as well as demographic characteristics, show
no significant difference between these two groups ex-
cept for one – rating of neighbourhood quality at Wave
2 (2p = 0.04). Hence selection bias does not appear to
affect the analysis outlined above to any great degree. Alongside this, the between-wave analysis in Table 7 ap-
pears to show an ‘adaptation effect’, whereby the impact
of housing changes on health and wellbeing diminishes
over time. However, the sub-sample analysis suggests
that this apparent ‘adaptation effect’ may simply be an
artefact of the smaller sample size at Wave 3. Within the
sub-sample there are very limited changes in significance
of correlations, suggesting that the data does not dem-
onstrate an adaptation effect. Exploring contextual factors In order to explore the potential role of contextual fac-
tors in influencing the operation of the hypothesised
mechanisms, the same bivariate tests were carried out
on sub-populations, in order to identify potential differ-
ences in impact based on tenant characteristics and
housing organisation. The analysis focuses on the change
between Wave 1 and Wave 2, specifically to examine the
short-term difference that a new tenancy and home with
a new housing organisation makes to tenants’ health and
wellbeing. Thus, the tests look for correlations between
self-rated changes in health and wellbeing since the start
of the tenancy and self-rated changes in various aspects Rolfe et al. BMC Public Health (2020) 20:1138 Page 11 of 19 Table 8 Sub-population analysis related to Hypothesis 1 Table 8 Sub-population analysis related to Hypothesis 1
Correlation with Wave 1–2 health and wellbeing change
Satisfaction with organisation
Renting experience (comparison to previous)
n
Rho
Sig. Rho
Sig. Total
Total
75
0.43***
0.001
0.38**
0.002
Organisationa
Housing Assoc. 33
0.51**
0.003
0.50*
0.01
Letting Agency
34
0.38*
0.03
0.45*
0.01
Gender
Female
40
0.49**
0.001
0.33
0.05
Male
35
0.35*
0.04
0.42*
0.03
Age
Young
34
0.55**
0.001
0.35
0.08
Old
41
0.34*
0.03
0.41*
0.01
Disability
Disabled
22
0.51*
0.02
0.38
0.09
Not disabled
53
0.38**
0.005
0.43**
0.005
Employment
Employed
31
0.56**
0.001
0.47*
0.02
Not employed
44
0.35*
0.02
0.35*
0.03
Housing Benefit
Full or partial HB
40
0.46**
0.003
0.42*
0.01
No HB
35
0.40*
0.02
0.37
0.06
Household Type
Children
23
0.42*
0.05
0.30
0.2
No children
52
0.46**
0.001
0.42**
0.005
Previous housing situation
Homeless
19
0.54*
0.02
0.70**
0.005
Social housing
13
0.63*
0.02
−0.18
0.6
PRS
31
0.43*
0.02
0.38*
0.04
Other
12
−0.053
0.9
0.58
0.2
a As noted in the methodology, the RDS tenants are excluded from the analysis by organisation (in this and subsequent tables), as the numbers of participating
tenants are too small to perform meaningful tests a As noted in the methodology, the RDS tenants are excluded from the analysis by organisation (in this and subsequent tables), as the numbers of participating
tenants are too small to perform meaningful tests should be noted, however, that the data for this variable
is more limited, since some tenants (e.g. Exploring contextual factors those coming
from the family home) did not have a maintenance ser-
vice to rate at Wave 1 and others had no experience of
the maintenance service in their new tenancy by Wave
2. Missing value analysis suggests that this particular
variable may be somewhat biased as a result. Moreover,
it could be argued that this variable is less closely related
to this hypothesis than tenants’ rating of property qual-
ity, since the experience of maintenance services could
also be connected to Hypothesis 1 as an element of the
overall tenancy experience. Thus, any conclusions relat-
ing to the maintenance variable need to be particularly
tentative. characteristics, such as gender, are evenly distributed
across the organisations. Perhaps most interestingly, the data on rating of prop-
erty quality at Wave 2 relative to Wave 1 does not show
a significant difference between the organisations (p =
0.44 using Mann-Whitney U test), so it does not appear
that these patterns are an artefact of differences in prop-
erty standards between the housing providers in this
study. That is, it appears that tenants of the Letting
Agency are not more (or less) likely to be satisfied by
the quality of their properties, but a change in the qual-
ity of their property is more likely to be accompanied by
a change in their health and wellbeing. Further qualita-
tive analysis is required to understand whether there is a
causal link here and why this might be the case. Hypothesis 3 – affordability The patterns relating to satisfaction with maintenance
are somewhat different, but again exhibit notable differ-
ences between sub-populations. Thus, participants who
are tenants of the Housing Association, female, older,
non-disabled, in receipt of Housing Benefit, with no chil-
dren and coming from the PRS are more likely to show
a correlation between change in their maintenance ser-
vice satisfaction and change in their health and well-
being. Again, some of these are likely to be related,
thanks to overlaps between the sub-populations. It Hypothesis 3 – affordability
As outlined earlier, the tests for the full sample show no
significant correlations between changes in health and
wellbeing and changes in either self-rated ability to cope
with paying rent or to cope financially. Looking at the
descriptive data for these variables, the lack of relation-
ship between health and wellbeing and rent coping is
perhaps unsurprising, given that more than 70% of par-
ticipants show no change in their ability to cope with
paying their rent. This likely reflects the number of Rolfe et al. BMC Public Health (2020) 20:1138 Page 12 of 19 Table 9 Sub-population analysis related to Hypothesis 2 Table 9 Sub-population analysis related to Hypothesis 2
Correlation with Wave 1–2 health and wellbeing change
Tenant rating of property quality
Satisfaction with maintenance service
n
Rho
Sig. n
Rho
Sig. Total
Total
73
0.31**
0.007
32
0.46**
0.009
Organisation
Housing Assoc. 31
0.081
0.7
12
0.67*
0.02
Letting Agency
33
0.56**
0.001
15
0.50
0.06
Gender
Female
38
0.44**
0.006
20
0.52*
0.02
Male
35
0.16
0.4
12
0.33
0.3
Age
Young
33
0.50**
0.003
12
0.19
0.6
Old
40
0.16
0.3
20
0.54*
0.01
Disability
Disabled
22
0.056
0.8
11
0.51
0.1
Not disabled
51
0.57***
0.001
21
0.45*
0.04
Employment
Employed
31
0.46**
0.009
12
0.41
0.2
Not employed
42
0.25
0.1
20
0.42
0.06
Housing Benefit
Full or partial HB
38
0.17
0.3
19
0.50*
0.03
No HB
35
0.51**
0.002
13
0.35
0.2
Household Type
Children
23
0.25
0.3
11
0.086
0.8
No children
50
0.34*
0.01
21
0.59**
0.005
Previous housing situation
Homeless
17
0.32
0.2
4
0.33
0.7
Social housing
13
0.18
0.6
7
0.35
0.4
PRS
31
0.51**
0.003
21
0.49*
0.02
Other
12
0.45
0.1
0
NA
NA tenants receiving full Housing Benefit, with 39% of the
sample having their rent entirely covered. Housing Association tenants. Hypothesis 3 – affordability These in turn seem to be
reflected by the correlations in sub-populations split by
disability, employment and Housing Benefit receipt, all
of which are distributed unevenly across the organisa-
tions, as shown in Table 5. Perhaps more interestingly,
there are also differences between the sub-populations
coming from different prior housing situations, which
do not appear to reflect the differences between the or-
ganisations. Previously homeless tenants (who are more
likely to be Housing Association tenants) show a signifi-
cant correlation with neighbourhood quality whilst ten-
ants coming from the PRS (who are more likely to be
Letting Agency tenants) show a significant correlation
with social support. For both variables there is a signifi-
cant correlation for households without children, but
not for those with children, which is perhaps counter-
intuitive. It is somewhat difficult to hypothesise under-
lying reasons for these patterns of correlations from the
quantitative data alone. The lack of correlation with change in self-rated health
and wellbeing for the whole sample is mirrored in the
sub-populations, with no correlations for change in rent
coping for any sub-population except households with
children (at the 5% level), and only the groups of
employed or non-HB recipients showing a correlation
with financial coping (also at the 5% level), as shown in
Table 10. This analysis therefore suggests that the over-
all picture of no significant relation between change in
rent or financial coping and health and wellbeing is also
present across the various sub-populations, with only
very minor indications of variation between groups. Hypothesis 4 – Neighbourhood and support networks
Table 11 summarises the analysis for the two key vari-
ables relating to neighbourhood quality and social sup-
port networks: tenant rating of the neighbourhood as a
place to live and the index of social support indicators. Whilst both variables are significantly correlated with
a change in self-rated health and wellbeing change for
the sample as a whole, the sub-population analysis re-
veals some differences. The correlations with neighbour-
hood quality are significant for Letting Agency tenants,
whereas those with social support are significant for Hypothesis 4 – Neighbourhood and support networks Hypothesis 4 – Neighbourhood and support networks
Table 11 summarises the analysis for the two key vari-
ables relating to neighbourhood quality and social sup-
port networks: tenant rating of the neighbourhood as a
place to live and the index of social support indicators. However, this analysis may be somewhat advanced by
turning to more objective measures of neighbourhood
quality. Data on the deprivation level of the areas that
tenants have moved from (at Wave 1) and to (at Wave
2/3) shows that tenants’ rating of neighbourhood quality
is not significantly correlated with the neighbourhood’s
Scottish Index of Multiple Deprivation (SIMD) decile Rolfe et al. BMC Public Health (2020) 20:1138 Page 13 of 19 Table 10 Sub-population analysis related to Hypothesis 3a
Correlation with Wave 1–2 health and wellbeing change
Tenant rating of ability to cope with paying rent
Tenant rating of ability to cope financially
n
Rho
Sig. n
Rho
Sig. Total
Total
55
0.030
0.8
75
0.14
0.2
Organisation
Housing Assoc. 23
−0.13
0.6
32
0.19
0.3
Letting Agency
24
0.24
0.3
34
0.26
0.1
Gender
Female
29
0.31
0.1
40
0.095
0.6
Male
26
−0.37
0.06
35
0.17
0.3
Age
Young
21
0.057
0.8
34
0.089
0.6
Old
34
−0.054
0.8
41
0.16
0.3
Disability
Disabled
21
−0.065
0.8
22
0.12
0.6
Not disabled
34
0.065
0.7
53
0.22
0.1
Employment
Employed
22
0.22
0.3
31
0.38
0.03*
Not employed
33
−0.15
0.4
44
0.018
0.9
Housing Benefit
Full or partial HB
31
0.10
0.6
40
0.086
0.6
No HB
24
−0.15
0.5
35
0.37
0.03*
Household Type
Children
17
0.51
0.04*
23
0.33
0.1
No children
38
−0.21
0.21
52
0.074
0.6
Previous housing situation
Homeless
8
−0.23
0.6
19
0.21
0.4
Social housing
13
0.17
0.6
13
0.47
0.1
PRS
31
−0.22
0.2
31
−0.22
0.2
Other
3
NA
NA
12
0.48
0.1
a Note that sample sizes for rent coping are smaller, due to the number of participants who did not answer this question at Wave 1, either because they were
homeless, or living with family Table 10 Sub-population analysis related to Hypothesis 3a a Note that sample sizes for rent coping are smaller, due to the number of participants who did not answer this question at Wave 1, either because they were
homeless, or living with family (2p = 0.6 using Spearman’s Rho). Hypothesis 4 – Neighbourhood and support networks There is, however, a
strongly significant correlation (2p = 0.001) between the
Wave 1–2 change in tenants’ rating of neighbourhood
quality and the change in SIMD decile. That is, tenants
moving between areas with different levels of deprivation
are more likely to describe a significant change in the
quality of their neighbourhood than those who are mov-
ing between deprived areas. Thus, rating of neighbour-
hood quality appears to be a relative concept for tenants,
likely based on a complex mixture of their neighbour-
hood history and expectations. In this context, it is
worth noting the relatively limited degree of neighbour-
hood choice available to tenants in this study, given their
predominantly low incomes. At Wave 1, 59% of tenants
in this study were living in the most deprived SIMD
quintile, whilst at Wave 2, 81% of tenants were in the
most deprived quintile. important for tenants, particularly given the probable
interaction with previous housing experiences. Discussion This analysis demonstrates that the health and wellbeing
of participants in this study does appear to be affected
by the change in subjective housing experience resulting
from a move into a new tenancy. Moreover, where their
new housing experience is positive, they are likely to de-
scribe improvements in health and wellbeing that are
sustained, or even increased, over the first year. This evi-
dence casts some doubt on notion of adaptation in the
relationship between housing and wellbeing [27], al-
though we acknowledge that longer-term data may show
a different pattern. Our analysis presents statistically significant relation-
ships between a variety of aspects of the housing experi-
ence
from
tenants’
perspectives,
and
health
and
wellbeing outcomes. These enable us to identify which
causal pathways are likely to be important in shaping
health and wellbeing for different groups of low-income
tenants in different contexts. By refining the CMO-Cs in
this way, the analysis delineates a theoretical framework
for ways in which less tangible aspects of housing ex-
perience can act as a social determinant of health and Picking apart these relationships between neighbour-
hood, social support and health and wellbeing clearly re-
quires further investigation, since the patterns in the
quantitative data are difficult to make sense of alone. Moreover, it seems plausible to suggest that these vari-
ables are particularly limited in terms of capturing the
underlying mechanisms, since there are so many aspects
of neighbourhood and social support which may be Rolfe et al. BMC Public Health (2020) 20:1138 Page 14 of 19 Table 11 Sub-population analysis related to Hypothesis 4
Correlation with Wave 1–2 health and wellbeing change
Tenant rating of neighbourhood quality
Social support network index
n
Rho
Sig. Rho
Sig. Total
Total
75
0.25*
0.04
0.28*
0.02
Organisation
Housing Assoc. Discussion 33
0.25
0.2
0.42*
0.02
Letting Agency
34
0.34*
0.05
0.20
0.3
Gender
Female
40
0.16
0.3
0.30
0.07
Male
35
0.31
0.08
0.26
0.1
Age
Young
34
0.26
0.1
0.32
0.07
Old
41
0.22
0.2
0.22
0.2
Disability
Disabled
22
0.32
0.2
0.45*
0.04
Not disabled
53
0.26
0.07
0.19
0.2
Employment
Employed
31
0.39*
0.03
0.23
0.2
Not employed
44
0.19
0.2
0.32*
0.04
Housing Benefit
Full or partial HB
40
0.20
0.2
0.26
0.1
No HB
35
0.36*
0.04
0.23
0.2
Household Type
Children
23
0.061
0.8
0.17
0.4
No children
52
0.32*
0.03
0.31*
0.03
Previous housing situation
Homeless
19
0.56*
0.02
0.30
0.2
Social housing
13
0.15
0.6
0.36
0.3
PRS
31
0.28
0.1
0.38*
0.04
Other
12
0.40
0.2
0.074
0.8 Table 11 Sub-population analysis related to Hypothesis 4 high-quality service, may be important for all tenants. It
seems plausible to suggest that there may be a causal re-
lationship here, possibly operating through mechanisms
related to the sense of home that tenants can develop in
a secure, stable tenancy with a housing organisation they
trust to provide good service. When tenants feel that
they are being treated well by their housing organisation
and that their overall experience is better than previous
situations, it is plausible that this will help to underpin
their sense of control, autonomy and safety, with positive
impacts on their wellbeing [13, 24]. wellbeing. Whilst recognising that correlation is not
causation and the nature of the independent variables
only enables relatively broad refinements to the hypoth-
eses, this additional specification of the causal pathways
provides a strong basis for further research, particularly
using qualitative data to examine the causal mechanisms
involved. Hypothesis 1 – positive tenancy experience This analysis suggests that tenants’ perceptions of the
quality of service received from their housing provider
may be an important determinant of health and well-
being, supporting the hypothesis from the original ToC
work that a version of CMO-C 1 is in operation. The re-
sults suggest that this is partly about the current service
and partly about comparison with previous rental expe-
riences, although there may be other factors which are
not represented adequately by the available independent
variables. This chimes with the findings from [26] that
housing service satisfaction, as part of what they term
‘empowerment’, is correlated with wellbeing. Whilst the importance of positive tenancy experience
across all groups of tenants is perhaps unsurprising, this
CMO-C also appears to be largely unaffected by the
housing sector/organisation, suggesting that formal se-
curity of tenure may be less important as a contextual
factor than might be expected, at least within the con-
text of these organisations. Importantly, this extends the
existing debate regarding the links between tenure, onto-
logical security and wellbeing which has largely focused
on the distinction between ownership and renting [17,
18, 24, 25, 27]. Just as more recent analyses have sug-
gested that other aspects of security (e.g. financial) may
be more important than ownership, so these findings
suggest that aspects of the tenancy experience may be Importantly, this relationship appears to be near uni-
versal, showing significant correlations across tenants
with different characteristics and backgrounds, suggest-
ing that a positive renting experience, underpinned by a Rolfe et al. BMC Public Health (2020) 20:1138 Page 15 of 19 Table 12 Refinement of CMO-C 1
Version
Contextual factors
Mechanism
Outcome
Original • Security of tenure
• Tenancy support
• Responsiveness of landlord to problems
• Expectations, situation and capacity of tenant
Positive tenancy experience reduces stress and provides
tenants with autonomy and control
Improved health
and wellbeing
Refined
• Previous experience and expectations of
housing service
• Standard of housing service (possibly including
support and responsiveness)
Experience of (comparatively) good housing service reduces
stress and enables tenants to gain benefits from housing as home
Improved health
and wellbeing Association property and those moving into a Letting
Agency property. These possibilities clearly need further
examination. more important than the legal status of the tenancy3 it-
self in some contexts, enabling tenants to feel secure and
at home, with implications for health and wellbeing. 3Note that the research took place before the introduction of the new
Private Residential Tenancy in Scotland, which removes the time limit
on PRS tenancies. 4Local Housing Allowance is the name given to Housing Benefit for
tenants in the PRS. It is limited to the 30th percentile of local rents. Hypothesis 1 – positive tenancy experience Whilst the analysis for this hypothesis included ten-
ants’ rating of maintenance services, there is a reason-
able argument to suggest that this variable could fit
equally well with Hypothesis 1, being at least as much
about service as property quality. Indeed, there are
strong correlations across the four housing variables,
which suggest that these two hypotheses are closely re-
lated in tenants’ real world experiences. Table 12 below summarises these findings, illustrating
how CMO-C 1 has been refined on the basis of this
analysis. Hypothesis 2 – property quality The analysis suggests that the tenant experience of prop-
erty quality may also be an important determinant of
health and wellbeing, supporting the hypothesis that a
version of CMO-C 2 is in operation. The existing evi-
dence base demonstrates that physical housing quality is
a determinant of health where there are negative factors,
such as damp or cold, that directly damage health [4, 6,
7]. It may be the case that some of these issues are rele-
vant within our sample, although there was little evi-
dence of such issues during the face-to-face interviews
at Waves 2 and 3 (to be presented in a later paper). In-
deed, the variations between sub-populations suggest
that there may be different mechanisms involved which
relate to other aspects of property quality beyond the
basic fabric of the building. Table 13 summarises these
findings, illustrating how CMO-C 2 has been refined. Hypothesis 3 – affordability yp
y
Participants’ health and wellbeing is clearly correlated
with their financial situation, as would be expected given
the crucial role of income as a social determinant of
health [2, 3]. However, these findings suggest that, for
tenants in this study, there is a limited impact on health
and wellbeing arising from rent levels. This is likely due
to the particular housing market context in which this
study was located, whereby the majority of rents were
within Local Housing Allowance4 rates and, therefore,
were either covered by Housing Benefit (for those on
very low incomes) or were affordable to those in work. Amongst the three-fifths of participants who did not re-
ceive full Housing Benefit, 80% indicated that they could
cope with paying their rent ‘all of the time’. Hence
CMO-C 3 does not seem to be operating, at least in rela-
tion to rent payments, in this context. The hypothesis is
therefore not presented here, since the lack of variation
in this central element of affordable housing does not fa-
cilitate testing or refinement of the CMO-C, even
though it may operate in different housing market
contexts. There is one particular aspect of the analysis of partici-
pant sub-populations for this pathway that merits fur-
ther investigation. There is a significant correlation
between tenants’ rating of property quality and health
and wellbeing amongst Letting Agency tenants, but not
Housing Association tenants, despite overall ratings of
property quality between the two organisations not be-
ing significantly different (Pearson Chi-Square 2p = 0.3). Given the notion that home is a phenomenon of individ-
ual experience [12] it seems plausible to suggest that
tenants’ previous experiences and personal preferences
are likely to be important in shaping their reaction to
different aspects of property quality. It is possible that
tenants’ previous experiences and expectations vary sys-
tematically between tenants moving into to a Housing Nevertheless,
the
evidence
regarding
(admittedly,
small) changes in tenants’ self-rated financial coping be-
tween Waves 1 and 2 suggests that there may be other
aspects of finance around tenancy transitions which
merit further exploration. Indeed, the strength of the
correlations between overall financial coping and the Page 16 of 19 Rolfe et al. Hypothesis 4 – Neighbourhood and support networks Hypothesis 4
Neighbourhood and support networks
Our analysis suggests that both neighbourhood quality
and social support networks may be important determi-
nants of health and wellbeing, as hypothesised in CMO-
C 4. However, there appear to be notable differences in
terms of which tenants exhibit significant correlations
between health and wellbeing and each of these two as-
pects of the wider environment around housing. Under-
standing the needs and aspirations of different groups of
tenants, including how these might align with demo-
graphic and other characteristics, are important in fur-
ther analysis of the qualitative data from this study. This
is particularly the case given the markedly different op-
portunities that Letting Agents and Community-Based
Housing Associations have to enable choice of area for
tenants, or to make changes to the area in which their
properties are located. Table 14 summarises these find-
ings, illustrating how CMO-C 4 has been refined. Hypothesis 3 – affordability BMC Public Health (2020) 20:1138 Table 13 Refinement of CMO-C 2
Version
Contextual factors
Mechanism
Outcome
Original
• Level of investment in property prior to tenancy
Quality housing provides tenants with a
comfortable space in which to relax and a
sense of status
Improved health
and wellbeing
Refined
• Tenant characteristics (including gender, age, disability, socio-
economic status, household type – these may relate to capacity)
• Previous housing experience
• Property quality
Quality housing provides tenants with a
comfortable space in which to relax and a
sense of status
Improved health
and wellbeing WHO5 wellbeing scores suggests that any impact of
housing on tenants’ financial situation has the potential
to generate significant changes in wellbeing. Again, fur-
ther exploration is required of precisely which aspects of
tenants’
financial
lives,
particularly
around
moving
home, underlie this impact on health and wellbeing. WHO5 wellbeing scores suggests that any impact of
housing on tenants’ financial situation has the potential
to generate significant changes in wellbeing. Again, fur-
ther exploration is required of precisely which aspects of
tenants’
financial
lives,
particularly
around
moving
home, underlie this impact on health and wellbeing. pathway. Indeed, it would be reasonable to suggest that
CMO-C 4 might be more accurately conceptualised as
at least two separate pathways, since there appears to be
something of a divergence in the data patterns related to
neighbourhood quality and social support between dif-
ferent groups of tenants. Hence, whilst the experience of
a good service from a housing provider may be import-
ant for all tenants to gain health and wellbeing benefits,
other aspects of the housing experience and its connec-
tion to a sense of home, such as the connection between
the dwelling and personal relationships [13] may be
more varied between individuals. Strengths and limitations The key strength of this research lies in the longitudinal
approach, offering an insight into change within individ-
ual respondents and the causal dynamics at play. Com-
bining the longitudinal aspect of the study with a realist
approach has enabled us to develop a nuanced picture of
the health and wellbeing impacts of less tangible aspects
of the housing experience. Limitations of the study include the relatively small
sample size and the possibility that the longitudinal ap-
proach may have excluded those tenants with less stable
housing pathways, although comparison of the partici-
pant sample with the wider tenant group suggests min-
imal difference and therefore limited selection bias. A
larger sample size at Wave 3 would also have helped to
explore whether the patterns visible at Wave 2 continue
over a longer timescale. Further study with a larger The complex patterns revealed in this analysis and the
number of factors which potentially underlie the inde-
pendent variables relating to neighbourhood quality and
social support suggest that more evidence is required to
draw firm conclusions regarding this possible causal Table 14 Refinement of CMO-C 4
Version
Contextual factors
Mechanism
Outcome
Original
• Community development activities of landlord
• Opportunities for choice of neighbourhood
• Existing networks of tenants
• Tenancy support
Good neighbourhood environment and supportive
social/community networks around housing location
reduce stress and increase opportunities for socialisation
Improved health
and wellbeing
Refined
• Tenant characteristics (including disability,
socio-economic status and household type)
• Previous experience and expectations of
neighbourhood/community
• Aspects of neighbourhood quality (which
may relate to activities of landlord and/or
choice of neighbourhood)
• Access to social support networks (which
may relate to all of the above)
Good neighbourhood environment and supportive
social/community networks around housing location
reduce stress and increase opportunities for socialisation
Improved health
and wellbeing Page 17 of 19 Rolfe et al. BMC Public Health (2020) 20:1138 Page 17 of 19 Page 17 of 19 group of tenants would be of value in exploring these is-
sues further. correlated with health and wellbeing for all tenants, re-
gardless of demographic characteristics or background. Secondly, aspects of the tenant experience of housing
quality in addition to the basics of weatherproofing seem
to be important for some tenants, in ways that are likely
to be influenced by previous housing experience and
current expectations. Strengths and limitations Thirdly, elements of neighbour-
hood quality and social support in the local area may
have impacts on health and wellbeing, although with
considerable variation between different groups of ten-
ants. It may also be the case that affordability has an ef-
fect on health and wellbeing, but interestingly it appears
to be relatively marginal within the particular housing
market context for this study. Whilst some caution
needs to be exercised in interpreting these refined
CMO-Cs, given the relatively small sample size on which
they are based, the longitudinal nature of the data does
provide a significant insight into the patterns of change
over the first year of participants’ tenancies and the po-
tential causes for the notable improvements in health
and wellbeing. The refined CMO-Cs are summarised in
Fig. 3. The original CMO-C relating to affordability is
included with dotted lines, to indicate its potential ap-
plicability in other housing markets. The use of self-rated measures of health and wellbeing
could also be seen as a limitation, although using more
objective measures of health would likely require a lon-
ger timescale, potentially exacerbating participant reten-
tion issues. The research was also conducted in one
geographical area, so the findings need to be interpreted
in the context of the specific housing policy and market
context of west central Scotland. Moreover, whilst this
study deliberately utilised general outcome measures to
capture impact, further research would be of value using
a range of more specific outcomes measures to examine
differential effects within the broad concept of health
and wellbeing. Funding
Thi
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programme, but had no direct involvement the detailed design of the
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1 1Faculty of Social Sciences, University of Stirling, Stirling FK9 4LA, UK. 2Glasgow Centre for Population Health, Olympia Building, Bridgeton Cross,
Glasgow G40 2QH, UK. 3School of Health and Life Sciences, Glasgow
Caledonian University, Cowcaddens Road, Glasgow G4 0BA, UK. 4Yunus
Centre for Social Business and Health, Glasgow Caledonian University,
Cowcaddens Road, Glasgow G4 0BA, UK. Cowcaddens Road, Glasgow G4 0BA, UK. Supplementary information 6. WHO Europe. Large analysis and review of European housing and health
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Housing Plan Res. 1993;10(2):65–74. SR analysed the data and was the primary author of the manuscript. LG was
a major contributor in writing the manuscript. JG contributed to the analysis
and assisted with writing the findings. IA, PS and CD were all involved in
planning the project and overseeing the research. All authors read and
approved the final manuscript. 13. Després C. The meaning of home: literature review and directions for future
research and theoretical development. J Architectural Plan Res. 1991;8(2):
96–115. 13. Després C. The meaning of home: literature review and directions for future
research and theoretical development. J Architectural Plan Res. 1991;8(2):
96–115. 14. Tomas A, Dittmar H. The experience of homeless women: an exploration of
housing histories and the meaning of home. Hous Stud. 1995;10(4):493–515. Consent for publication Not applicable. 24. Kearns A, Hiscock R, Ellaway A, Macintyre S. ‘Beyond Four Walls’. The
Psycho-social Benefits of Home: Evidence from West Central Scotland. Housing Studies. 2000;15(3):387. 24. Kearns A, Hiscock R, Ellaway A, Macintyre S. ‘Beyond Four Walls’. The
Psycho-social Benefits of Home: Evidence from West Central Scotland. Housing Studies. 2000;15(3):387. Conclusion The basic human need for a home that provides more
than simply shelter from the elements [12] underpins
the need to understand the relationships between hous-
ing, health and wellbeing in ways that go beyond obvious
problems such as damp and cold. Our analysis provides
an important addition to the theoretical understanding
of at least three potential causal pathways through which
housing may affect health and wellbeing. Firstly, a posi-
tive tenancy experience, shaped at least in part by rela-
tionships
with
the
housing
provider
is
strongly Taken together, these CMO-Cs offer an empirically-
informed realist theoretical framework for causal path-
ways connecting housing to health and wellbeing. This Fig. 3 Summary of refined CMO-Cs. Note: Dotted line indicates that this CMOC is not evidenced here, but may be applicable in other
housing markets Page 18 of 19 Page 18 of 19 Page 18 of 19 Rolfe et al. BMC Public Health (2020) 20:1138 Rolfe et al. BMC Public Health (2020) 20:1138 framework provides a lens through which to examine
and potentially improve practice within housing organi-
sations and housing policy, highlighting the ways in
which aspects of housing service can operate as a public
health intervention in the lives of tenants. Moreover, the
framework offers a basis for more research to further re-
fine and test these causal pathways. As Pawson [31, 32,
53] has consistently argued, realist evaluation and re-
search needs to operate in a cyclical fashion, continually
examining and improving theoretical models on the
basis of empirical evidence to enrich our understanding
of causal mechanisms, rather than developing spurious
generalisations [54]. Analysis of the qualitative data from
this study (forthcoming) will help to delineate the mech-
anisms within the framework more accurately and to
examine the contextual factors in more detail, whilst
other studies may also explore the role of these causal
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Evidence of Physiological Decoupling from Grassland Ecosystem Drivers by an Encroaching Woody Shrub
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PloS one
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cc-by
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Abstract Shrub encroachment of grasslands is a transformative ecological process by which native woody species increase in cover
and frequency and replace the herbaceous community. Mechanisms of encroachment are typically assessed using temporal
data or experimental manipulations, with few large spatial assessments of shrub physiology. In a mesic grassland in North
America, we measured inter- and intra-annual variability in leaf d13C in Cornus drummondii across a grassland landscape with
varying fire frequency, presence of large grazers and topographic variability. This assessment of changes in individual shrub
physiology is the largest spatial and temporal assessment recorded to date. Despite a doubling of annual rainfall (in 2008
versus 2011), leaf d13C was statistically similar among and within years from 2008-11 (range of 228 to 227%). A
topography*grazing interaction was present, with higher leaf d13C in locations that typically have more bare soil and higher
sensible heat in the growing season (upland topographic positions and grazed grasslands). Leaf d13C from slopes varied
among grazing contrasts, with upland and slope leaf d13C more similar in ungrazed locations, while slopes and lowlands
were more similar in grazed locations. In 2011, canopy greenness (normalized difference vegetation index – NDVI) was
assessed at the centroid of individual shrubs using high-resolution hyperspectral imagery. Canopy greenness was highest
mid-summer, likely reflecting temporal periods when C assimilation rates were highest. Similar to patterns seen in leaf d13C,
NDVI was highest in locations that typically experience lowest sensible heat (lowlands and ungrazed). The ability of Cornus
drummondii to decouple leaf physiological responses from climate variability and fire frequency is a likely contributor to the
increase in cover and frequency of this shrub species in mesic grassland and may be generalizable to other grasslands
undergoing woody encroachment. Citation: Nippert JB, Ocheltree TW, Orozco GL, Ratajczak Z, Ling B, et al. (2013) Evidence of Physiological Decoupling from Grassland Ecosystem Drivers by an
Encroaching Woody Shrub. PLoS ONE 8(12): e81630. doi:10.1371/journal.pone.0081630 Editor: Carl J. Bernacchi, University of Illinois, United States of America Received August 16, 2013; Accepted October 23, 2013; Published December 5, 2013 Copyright: 2013 Nippert 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 NSF-LTER award to the Konza Prairie (DEB-0823341). Jesse B. Nippert1*, Troy W. Ocheltree1,2, Graciela L. Orozco1, Zak Ratajczak1, Bohua Ling3,
Adam M. Skibbe1 Jesse B. Nippert1*, Troy W. Ocheltree1,2, Graciela L. Orozco1, Zak Ratajczak1, Bohua Ling3,
Adam M. Skibbe1 1 Division of Biology, Kansas State University, Manhattan, Kansas, United States of America, 2 Department of Forest and Rangeland Stewardship, Colorado State University,
Fort Collins, Colorado, United States of America, 3 Department of Geography, Kansas State University, Manhattan, Kansas, United States of America Abstract Publication of this article was funded in part by the Kansas State
University Open Access Publishing Fund. GLO was supported by the KSU McNair scholars program. 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: nippert@ksu.edu December 2013 | Volume 8 | Issue 12 | e81630 Introduction KPBS is divided into 57
experimental landscape units (over 3487 ha) representing combi-
nations of varying fire year return frequencies (1, 2, 4, 10, and 20)
and grazing (bison, cattle, or no large mammalian grazers)
treatments. Most prescribed fires occur in the spring (late March –
early May) of each year, and have been imposed for the past 30+
years. The bison herd exists within the central 1012 ha of KPBS,
maintained at a density of ,300 adults since 1997, which results in
a ,25% removal of graminoid biomass [41]. Cattle occupy
844 ha on the eastern side of the site in cow-calf pairs from mid-
May to September. No endangered or federally protected species
were impacted by the research described here. The site topography at KPBS is variable over small scales
(,0.1 km). Soil depth and structure varies with topography, as
uplands have thin, rocky soil (Florence series) and lowlands have
deeper silty-clay loams (Tully series). Changes in available soil
moisture as a consequence of topographic position have been
shown to result in lower aboveground net primary productivity in
upland locations [24]. We used the stable carbon isotopic ratio (d13C) of leaf tissue,
which most broadly represents the integrated water-use efficiency
(iWUE) and associated stomatal regulation during leaf develop-
ment [38–39]. At each sample date, we revisited the same
populations of clonal dogwood individuals (e.g., a ‘shrub island’). Using leaf d13C we assessed leaf physiological responses over the
broadest landscape gradients possible at this grassland site, with a
monthly frequency during the summer from 2008–2011 (Table 1). We hypothesized that greater reliance on deeper soil-water by this
woody shrub [13,40] would minimize physiological variability to
intra- and inter-annual climate variability because deep-water is
less variable through time and typically more available than
shallow soil moisture [40]. Access to water deep in the soil profile is
not related to the treatment contrasts at this site (i.e., grazer
presence, fire frequency) but does vary according to topography
with uplands having shallower soils than lowlands. Thus, we also
hypothesized that individual shrubs from locations with more bare
soil and/or higher sensible heat (i.e., uplands, grazed locations,
and recently burned locations) would have higher iWUE than
shrubs from locations embedded in the herbaceous grass matrix
with little bare soil and/or greater access to deep-soil water. The regional climate for KPBS is characterized as mid-
continental, with warm-wet summers, and cool-dry winters. Introduction Daily
mean air temperature (1982-2011) varies between the coolest
(21.460.5uC - January) and warmest (26.160.3uC - July) months. From 1982–2011, mean annual rainfall (61 SE) for KPBS was
805635 mm, with ,75% of the total amount occurring during
the growing season (April–September). Sampling frequency/procedure This research was conducted across 25 watersheds at KPBS,
capturing the full suite of burn frequency by grazing by
topography combinations at this site (Table 1). 76 C. drummondii
islands—isolated clonal shrubs of many stems in a grassland
matrix—were selected at random during the first sampling period
in 2008, and then subsequently sampled for the duration of the
project. Clonal shrub size was variable across the landscape, from
,6–100 m2. The topography of the location for each sampled
shrub was classified based on its relative position within each
watershed (upland/ slope/lowland). Leaf samples were collected at
approximate 4-week intervals during the growing season (May–
September) from 2008–2010. In 2011, samples were collected in
July and August only. Sample collection always occurred across
the entirety of KPBS within a single day. Upon visiting each
dogwood island, six emergent leaves were collected from the outer Introduction Permission to conduct
this research and the associated research permit was approved by
the KPBS director, Dr. John M. Briggs. KPBS is divided into 57
experimental landscape units (over 3487 ha) representing combi-
nations of varying fire year return frequencies (1, 2, 4, 10, and 20)
and grazing (bison, cattle, or no large mammalian grazers)
treatments. Most prescribed fires occur in the spring (late March –
early May) of each year, and have been imposed for the past 30+
years. The bison herd exists within the central 1012 ha of KPBS,
maintained at a density of ,300 adults since 1997, which results in
a ,25% removal of graminoid biomass [41]. Cattle occupy
844 ha on the eastern side of the site in cow-calf pairs from mid-
May to September. No endangered or federally protected species
were impacted by the research described here. reported to occur in the transition zones and ecotones between C4-
dominanted grasslands and riparian deciduous forests [32]. Previously, it has been shown that C. drummondii uses deeper soil-
water sources compared to the coexisting C4 grass community
[13,33–34]. Using the stable isotopic signature of water from C. drummondii, a C4 grass (Andropogon gerardii), and soils at varying
depths, Ratajczak et al. [13] showed that reliance on deep-water
released C. drummondii from competition for water with the
dominant grasses on site, which rely exclusively on surface soil
moisture [35]. Use of water from deeper soil depths occurred in
large shrub stems, as well as developing small stems, which lack a
developed root profile [34], highlighting the rhizomatous-transfer
of deep water among stems within the shrub island [13]. In
locations with shallow soils (,50 cm depth), C. drummondii has
patchy occurrence, does not increase in cover over time, and
exhibits ,50% mortality from a combination of water limitation
and/or competition with grasses (Ratajczak unpublished). Thus,
reliance on deeper soil moisture has the potential to minimize the
negative consequences of short-term drought and high air
temperature on the physiology and growth dynamics of C. drummondii (sensu: [36,37]). a LTER (Long-Term Ecological Research) site dedicated to the
study of tallgrass prairie ecology, jointly owned by the Nature
Conservancy and Kansas State University. Permission to conduct
this research and the associated research permit was approved by
the KPBS director, Dr. John M. Briggs. Introduction [21–22]. These disturbances regulate ecosystem dynamics, main-
taining grassland that is floristically diverse [23], productive [24],
and capable of supporting large mammalian grazers [25]. Previous
work has shown that the removal of fire from the system facilitates
the rapid transition to a closed-canopy evergreen forest (Juniperus
virginiana) within decades [5,26], resulting in altered site biogeo-
chemistry and reduced floristic diversity [26–28]. If fire frequency
is changed (but not removed altogether), deep-rooted clonal shrubs
(e.g., Cornus drummondii, Rhus glabra) increase in density and cover,
with the most pronounced responses occurring in locations with
soils greater than 0.5 m deep [4,13]. Similar to the consequences
of grassland conversion to an evergreen forest, conversion from
mesic grassland to clonal shrubland alters ecosystem dynamics [5],
occurs rapidly [13,29], and results in semi-permanent ecosystem
change (requiring mechanical removal and herbicide application
for shrub removal [30–31]). The worldwide encroachment of grassland systems by woody
plants
has been
documented
extensively [1],
with specific
examples from semi-arid [2–3], mesic [4–5], sub-arctic [6] and
alpine grasslands [7], as well as savannas [8–10]. The often abrupt
and non-linear transitions from grassland to woodland can
typically be explained by positive feedbacks [11–15]. The diverse
characteristics of ecosystems, attributes of encroaching species,
rates of change, and local feedback dynamics have made the
identification of general patterns difficult to isolate, resulting in
many paradigms for shrub encroachment worldwide (e.g., elevated
CO2, nutrient enrichment/depletion, ecohydrological change,
altered
disturbance
regimes
[11,16–20]). Despite
a
similar
theoretical trajectory of shrub encroachment into grasslands
(e.g., positive feedbacks), we still lack commonalities in the local
mechanism(s) facilitating woody encroachment across grasslands. The clonal shrub rough-leaf dogwood (Cornus drummondii) is a
grassland-encroaching species showing the greatest expansion in
cover (from ,0% to .30% cover) along the western edge of mesic
prairie in central North America (Nebraska, Kansas, Oklahoma,
USA). C. drummondii is a native species to this region, historically Within central North America, mesic grassland structure and
function is commonly regulated by the interplay of multiple
intermittent disturbances - fire, grazing, and climatic variability -
which prevents the transition from grassland to woodland or forest December 2013 | Volume 8 | Issue 12 | e81630 1 PLOS ONE | www.plosone.org Dogwood Leaf d13C in a Tallgrass Prairie Dogwood Leaf d13C in a Tallgrass Prairie a LTER (Long-Term Ecological Research) site dedicated to the
study of tallgrass prairie ecology, jointly owned by the Nature
Conservancy and Kansas State University. Site Description Research was conducted in mesic grassland at the Konza
Prairie Biological Station (KPBS), located in the Flint Hills
ecoregion of eastern Kansas, USA (39u059 N, 96u359 W). KPBS is Table 1. Shrub island sample sizes among landscape contrasts. Statistical Analysis where R is the ratio of the heavy to light isotope for the sample and
standard, respectively. The within-run variability estimated as the
SD of working standards was always ,0.05%, and the between-
run variability, estimated as the difference between the measured
value of a working standard and its calibrated value, was always
,0.05%. All statistical analyses were performed using the open-source R
statistical software package [42]. Climate data from the KPBS
headquarters were harvested from the ClimDB database (http://
www.fsl.orst.edu/climhy/) and were summarized for growing
season statistics only, which was defined as DOY 105–258 (April
15- September 15). Stable isotope and N content data were
verified for normality, and any outliers (defined as 2 standard
deviations beyond the overall mean) were removed from the data
set. Linear mixed-effects models were used to analyze the data
using the ‘nlme’ package in R [43]. Linear mixed-effects models
accommodate unbalanced and grouped data by associating the
random effects of individual experimental units to the observations
of interest [44]. For the leaf d13C data, fixed-effects included all
spatial contrasts- grazing (grazed/ungrazed), topographic position
(upland, slope, lowland), fire frequency (1, 2, 4, 10, 20 years), as
well as temporal contrasts - year and day of year sampled. Because
day of year is nested within year, this interaction term was not
included as a fixed effect. In the random effects structure of the
model, watershed (location of shrub sampled) within DOY
(sampling day) was used. For the NDVI data, fixed-effects
included grazing, fire, topography and day of year and their
interactions, and watershed was a random effect. To assess the
relationship between leaf d13C and %N(mass), a simple linear
regression was fit to all data from 2008–2011. Integrated Water-Use Efficiency (iWUE) is related to d13C of
plant tissue through the ratio of internal to atmospheric CO2
concentration (ci/ca). iWUE is a measure of the amount of carbon
fixed during photosynthesis (A) relative to stomatal conductance (g)
and can be estimated from ci/ca: WUE~ A
g ~
ca 1{ ci
ca
1:6
ð2Þ ð2Þ The 13C of plant tissue is determined by the 13C of the CO2
source and ci/ca, and is expressed as: d13Cp~d13Ca{az b{a
ci
ca
ð3Þ ð3Þ Where d13C p and d13Ca are the carbon isotope ratios of the
plant tissue and atmospheric air, respectively. Results Cumulative growing season precipitation (from DOY 105–258)
averaged 520.3 mm from 1982–2011, but differed between the 4
years studied (Fig. 1a). The amount of growing season precipita-
tion was above the long-term average (520.31 mm from 1982–
2011) during 2008–2010. Precipitation in 2008 was the greatest for
the 4 years of this study (848.6 mm), followed by 2009 (630.0 mm)
and 2010 (647.6 mm), although the temporal pattern of precip-
itation and corresponding intra-annual variability differed among
these two years (Fig. 1a). The amount of precipitation received in
2011 was the lowest of the 4 years (406.1 mm). The long-term
average for growing season temperature from 1982–2011 was
22.05uC. 2008 & 2009 were cooler than average, and 2011 was
1.1uC higher than average during the growing season. 2011 was
2.83uC warmer than the next warmest year (2010) from DOY
191–211 (Fig. 1b). During this same period of time, VPD during
2011 was on average 0.72 kPa greater than 2010 (Fig. 1c). Table 1. Shrub island sample sizes among landscape contrasts. Table 1. Shrub island sample sizes among landscape contrasts. Fire Frequency (yr)
Topo. position
1
2
4
10
20
Upland
Slope
Lowland
Grazing
Yes
9
7
8
–
4
7
8
13
No
17
5
15
4
7
10
10
28
Topo. position
Upland
4
1
7
2
3
Slope
5
4
6
–
3
Lowland
17
7
10
2
5
76 total shrub islands were visited each sampling period. doi:10.1371/journal.pone.0081630.t001
PLOS ONE | www.plosone.org
2
December 2013 | Volume 8 | Issue 12 | e81630 Table 1. Shrub island sample sizes among landscape contrasts. Fire Frequency (yr)
Topo. position
1
2
4
10
20
Upland
Slope
Lowland
Grazing
Yes
9
7
8
–
4
7
8
13
No
17
5
15
4
7
10
10
28
Topo. position
Upland
4
1
7
2
3
Slope
5
4
6
–
3
Lowland
17
7
10
2
5
76 total shrub islands were visited each sampling period. doi:10.1371/journal.pone.0081630.t001 76 total shrub islands were visited each sampling period. doi:10.1371/journal.pone.0081630.t001 December 2013 | Volume 8 | Issue 12 | e81630 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 2 Dogwood Leaf d13C in a Tallgrass Prairie perimeter of each shrub island. Once collected, leaves were stored
in coin envelopes and dried at 60uC for one week. NDVI~ NIR{red
NIRzred
ð4Þ ð4Þ Stable isotopic analysis We
measured
the
stable
carbon
isotope
ratios
and
N
concentrations of dried and ground leaf material from C. drummondii in the Stable Isotope Mass Spectrometry Laboratory
(SIMSL) at Kansas State University. Samples were combusted
with a CE1110 elemental analyzer (Carlo Erba Instruments,
Milan, Italy) and coupled to a Delta Plus mass spectrometer
(Thermo Electron Corporation, Bremen, Germany) for isotope
analysis using a ConFlo II Universal Interface (Thermo Electron
Corporation, Bremen, Germany). The isotopic ratio of samples
was calculated using delta notation as: Due to variations in spectral resolution over the season in 2011,
the values used for red (620–750 nm) and NIR (750–1400 nm)
varied by date (Red = R684.02 nm, NIR = R871.17 nm, for May–
August, and Red = R685.2 nm, NIR = R875.96 nm, for Septem-
ber). These values were chosen to represent each red and NIR
spectrum edge respectively. NDVI was calculated at each shrub
island centroid (4 m2) for each of the same 76 individuals
previously measured for leaf d13C analysis. Because all shrubs
were larger than the resolution of the imagery collected, no
herbaceous vegetation from the circumference of the shrub was
included in the NDVI data, and little to no herbaceous vegetation
existed under the shrub canopy [13]. d~
Rsample
Rs tan dard
{1
1000
ð1Þ ð1Þ Statistical Analysis ‘a’ and ‘b’ are
discrimination factors due to diffusion through the stomata and
carboxylation, which are relatively constant values of 4.4% and
29% [38]. ci/ca responds to changes in photosynthetic rate and
stomatal conductance, just as iWUE, and can be solved using eq. 3. When using d13C as a proxy for iWUE, smaller values indicate
lower iWUE. December 2013 | Volume 8 | Issue 12 | e81630 NDVI calculation Using ArcGIS 10.0 Spatial Analyst Tools (ESRI 2012) we
extracted values from a calculated NDVI (normalized difference
vegetation index) product derived from four hyperspectral flights
of KPBS in 2011. These hyperspectral data were collected by an
aircraft mounted AISA camera and atmospherically corrected
with FLAASH resulting in a 2 m62 m resolution data product. NDVI was calculated as: Leaf d13C in C. drummondii did not vary among years (p = 0.287,
F = 2.07) or days sampled within years (p = 0.512, F = 0.47) PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e81630 3 Dogwood Leaf d13C in a Tallgrass Prairie Table 2. Mixed-effects model results for leaf d13C (%) among
landscape contrasts including: grazing (grazed/ungrazed),
topographic position (upland, slope, lowland), and burn
frequency (1, 2, 4, 10, 20) and temporal contrasts (year and
day of year). numDF
denDF
F-value
p-value
Grazing
1
682
3.85
0.05
Position (topo)
2
682
12.81
,.0001
Burn_freq
1
682
3.37
0.0669
Year
1
2
2.07
0.2872
DOY
1
9
0.47
0.5118
G:P
2
682
7.7
0.0005
G:Bf
1
682
1.58
0.2087
P:Bf
2
682
2.26
0.105
G:Year
1
682
0.58
0.4471
G:DOY
1
682
1.18
0.2777
P:Year
2
682
0.39
0.6768
P:DOY
2
682
0.97
0.3783
Bf:Year
1
682
0.82
0.3648
Bf:DOY
1
682
2.05
0.1531
G:P:Bf
2
682
0.7
0.4962
G:P:Year
2
682
0.03
0.9666
G:P:DOY
2
682
0.06
0.9449
G:Bf:Year
1
682
1.7
0.1931
G:Bf:DOY
1
682
0.63
0.4279
P:Bf:Year
2
682
0.36
0.6986
P:Bf:DOY
2
682
1.08
0.3395
G:P:Bf:Year
2
682
1.07
0.3443
G:P:Bf:DOY
2
682
0.57
0.5677
Random effects include the watershed within day sampled. doi:10.1371/journal.pone.0081630.t002 Figure 1. Konza climate from DOY 105–259 for the four years
of this study: a – cumulative precipitation (mm); b – mean daily
air temperature (6C); c – mean daily vapor pressure deficit
(kPa). doi:10.1371/journal.pone.0081630.g001 Table 2. Mixed-effects model results for leaf d13C (%) among
landscape contrasts including: grazing (grazed/ungrazed),
topographic position (upland, slope, lowland), and burn
frequency (1, 2, 4, 10, 20) and temporal contrasts (year and
day of year). Table 2. Mixed-effects model results for leaf d13C (%) among
landscape contrasts including: grazing (grazed/ungrazed),
topographic position (upland, slope, lowland), and burn
frequency (1, 2, 4, 10, 20) and temporal contrasts (year and
day of year). Random effects include the watershed within day sampled. doi:10.1371/journal.pone.0081630.t002 Random effects include the watershed within day sampled. doi:10.1371/journal.pone.0081630.t002 (Table 2, Fig. Discussion Measuring the leaf d13C across 4 growing seasons and a broad
spatial template allowed us to assess the integrated physiological
dynamics for C. drummondii, the predominate woody encroacher for
this mesic grassland. Compared across years, patterns in leaf d13C
were similar (Fig. 2), despite a doubling in inter-annual precipi-
tation and variable air temperature and vapor pressure deficits
(Fig. 1). The similarity in leaf d13C across years suggests that
integrated leaf physiological function was insensitive to ambient
annual climate dynamics. This result directly contrasts previous
studies on the herbaceous species at KPBS, which have shown this
grassland community to be very responsive to intra- and inter-
annual variability in climate [24,45–46]. remainder of the season (Fig. 4). The period with the highest
NDVI here corresponds with previous work showing the highest
photosynthetic rates for C. drummondii mid-summer [51]. Previously, a shrub d13C isoscape was conducted along a
topographic gradient in subtropical Texas savanna [53]. Bai et al. [53] reported a ,1.5% range in d13C across landscape and
temporal contrasts, with the greatest differences between species
and among contrasts of topographic positions. In our results here
from 2008–11, the site-wide seasonal range of was also narrow
(,1.5%) despite individuals being measured across the full suite of
site treatments (topography, fire, grazing) (Table 1). By compar-
ison, a site-wide sampling of 310 separate C3 species was
conducted on Konza Prairie in 2009–10 across similar contrasts
in landscape gradients [54]. In the supplemental data of Craine et
al. [54], leaf d13C varied from 235 to 225% (mean = 229.7%,
SD = 1.5%) illustrating the role of species differences, seasonal and
landscape variability as a driver of leaf d13C at this site. It was
previously shown that leaf %N accounted for some of the
topographic variability in leaf d13C for subtropical woody shrubs
[53,55]. In this study, a significant statistical relationship between
%N and leaf d13C was present (p = 0.001), but explained very little
of the overall variability (r2 = 0.01). y
[
,
]
Leaf d13C did not vary intra-annually (Table 2), but leaf d13C
values were highest early the growing season, lowest mid-summer
(early July), and then increased to higher values later in the
growing season (Fig. 2). Using small plots, McCarron and Knapp
[33] measured instantaneous water-use efficiency for C. NDVI calculation 2), despite the varying climate histories from 2008–
2011 (Fig. 1). A significant grazing by topographic position
interaction was present (p,0.001) for leaf d13C (Table 2, Fig. 3),
with leaves from slope positions in grazed watersheds having
smaller d13C values than leaves from ungrazed slope positions. When assessed by topographic position on the landscape, leaves
from C. drummondii in upland locations had larger d13C values (i.e. more enriched in 13C) compared to individuals from topographic
slopes and lowland positions, respectively (Fig. 3). A similar
difference in leaf d13C existed in leaves collected from grazed
locations, with leaves from grazed sites tending to be more
enriched in 13C than ungrazed sites (Fig. 3). Leaf d13C did not vary
(p.0.05) according to burn frequency at KPBS for the years
studied (Table 2). A significant, but weak, positive correlation was
present (p = 0.001; r2 = 0.01; y = 0.26x227.99) between leaf d13C
and %N using data from all years combined (data not shown). NDVI from each shrub island varied significantly (p,0.001)
among the 4 time periods measured in 2011 (Table 3) with a
parabolic shape, with peak values near DOY 180 (Fig. 4). No
differences were present based on burn frequency across the
landscape, but NDVI was significantly lower in grazed areas
(Fig. 4a) and upland topographic positions (Fig. 4b). Figure 1. Konza climate from DOY 105–259 for the four years
of this study: a – cumulative precipitation (mm); b – mean daily
air temperature (6C); c – mean daily vapor pressure deficit
(kPa). doi:10.1371/journal.pone.0081630.g001 doi:10.1371/journal.pone.0081630.g001 December 2013 | Volume 8 | Issue 12 | e81630 PLOS ONE | www.plosone.org 4 Dogwood Leaf d13C in a Tallgrass Prairie Discussion
Figure 3. Changes in leaf d13C (%) by topographic positions
(upland, slope, lowland) and according to grazing contrasts
(grazed, ungrazed). Data are mean values (61SE) from 2008–2011. Figure 2. Changes in mean leaf d13C (%) (±1SE) by day of year
across the growing season from 2008–2011. doi:10.1371/journal.pone.0081630.g002 Figure 2. Changes in mean leaf d13C (%) (±1SE) by day of year
across the growing season from 2008–2011. doi:10.1371/journal.pone.0081630.g002 Figure 3. Changes in leaf d13C (%) by topographic positions
(upland, slope, lowland) and according to grazing contrasts
(grazed, ungrazed). Data are mean values (61SE) from 2008–2011. doi:10.1371/journal.pone.0081630.g003 Figure 2. Changes in mean leaf d13C (%) (±1SE) by day of year
across the growing season from 2008–2011. doi:10.1371/journal.pone.0081630.g002 Figure 3. NDVI calculation Changes in leaf d13C (%) by topographic positions
(upland, slope, lowland) and according to grazing contrasts
(grazed, ungrazed). Data are mean values (61SE) from 2008–2011. doi:10.1371/journal.pone.0081630.g003 December 2013 | Volume 8 | Issue 12 | e81630 Discussion If future climate changes alter seasonal rainfall distribution
resulting in more rainfall events and amounts of rainfall in the
non-growing season (winter), greater infiltration to deeper soil
depths is likely to occur. In this scenario with greater soil moisture
at deeper soil depths, the shrub communities may be buffered from
future summer-time droughts. landscape [57]. At Konza Prairie, a drought of sufficient
magnitude to lower water availability at soil depths below 1 m
are unknown, as are the potential physiological and stress-
responses by woody shrubs reliant on these deeper water sources. If future climate changes alter seasonal rainfall distribution
resulting in more rainfall events and amounts of rainfall in the
non-growing season (winter), greater infiltration to deeper soil
depths is likely to occur. In this scenario with greater soil moisture
at deeper soil depths, the shrub communities may be buffered from
future summer-time droughts. Reliance on a deeper water source provides a physiological
advantage for woody plants by minimizing the impacts of within-
season drought and allowing for longer periods of growth than the
herbaceous community [47,50]. In addition, the clonal redistri-
bution of water via rhizomes supports the production of new stems
and
the
expansion
of
shrub
islands
across
the
landscape
independent of short-term variability in climate [13,34]. Indeed,
with access to deep-soil water a hysteresis loop may exist in this
system because the severity of drought that governs seedling
establishment is expected to be lower than that required to cause
adult shrub mortality. Woody shrubs without access to deep-water
sources (existing in locations with shallow soils) have greater
susceptibility to drought and stress-induced physiological adjust-
ment [56–57]. g
In combination with physiological studies from other sites [47–
48,50,53], the results of this study suggest that deep-rooted woody
species like C. drummondii may have growth and cover dynamics
that are decoupled from several of the local environmental drivers
that govern the dynamics of the herbaceous grassland communi-
ties (here, climate variability and fire). Bai et al. [53] concluded
that the greater reliance on deeper soil-water sources may allow
shrub species to be decoupled from microclimate and temporal
variability in near-surface soil moisture. This decoupling from key
ecosystem drivers results in a fundamentally different response for
the woody encroaching species compared to the herbaceous
grassland community, allowing the woody species to avoid
competition for water in both time and space. Discussion drummondii,
and reported non-significant seasonal changes (but a trend with
lowest WUE mid-season and highest WUE late in the growing
season). Similar subtle responses for instantaneous WUE across the
growing season despite changes in surface soil moisture have been
shown for other grassland-encroaching woody species, including
Prosopis glandulosa [47], and Juniperus virginiana [48] and for shrubs
coexisting in grasslands (Amorpha canescens – [49]; Artemisia cana and
Rhus trilobata – [50]). The seasonal pattern in leaf d13C here (Fig. 2)
may reflect contributions of stored carbohydrates in the produc-
tion of new leaf biomass early in the growing season. In this
scenario, the first leaves produced utilize a greater proportion of
stored carbohydrates, with an isotopic signature reflecting carbon
fixed at the end of the previous growing season. This leaf d13C
would vary from carbon fixed mid-summer when leaves are at
peak photosynthetic activity [51] and leaf d13C would likely reflect
recently fixed carbon. Remobilization of stored carbohydrates into
developing leaves has been shown previously using d13C in shrub
species [52]. NDVI calculated for individual shrub islands in 2011
had a significant seasonal trend (Table 3), with a maximum
‘canopy greenness’ in late June and subsequent declines over the We hypothesized that the use of deep soil-water by C. drummondii
would minimize variability in leaf d13C across years with varying
rainfall patterns (and microclimates) and among fire, grazing, and
topographic contrasts, because a reliable water source may
minimize the negative physiological effects of short-term drought,
high temperature, and high evaporative demand (i.e. high VPD). As noted previously, leaf d13C did not vary according to sample
date or year, but a topography*grazing interaction exists (Fig. 3). C. drummondii from lowlands regions (with deeper soils) had smaller
leaf d13C (and lower iWUE) than individuals from uplands, which
typically have shallower, coarse soils and reduced soil moisture
[24]. Leaf d13C was typically larger in grazed versus ungrazed
locations, suggesting higher iWUE for shrubs in grazed locations. December 2013 | Volume 8 | Issue 12 | e81630 December 2013 | Volume 8 | Issue 12 | e81630 PLOS ONE | www.plosone.org 5 Dogwood Leaf d13C in a Tallgrass Prairie Figure 4. Changes in mean NDVI (±1SE) by day of year in 2011. Each estimate of NDVI represents the image captured at ,262 m
resolution for each individual dogwood island on the landscape. Discussion Panel
‘a’ shows differences according to topographic positions, while panel ‘b’
shows differences by grazing contrasts. doi:10.1371/journal.pone.0081630.g004 Table 3. Mixed-effects model results for NDVI by day of year
and by landscape contrasts: grazing (grazed/ungrazed),
topographic position (upland, slope, lowland), and burn
frequency (1, 2, 4, 10, 20). numDF
denDF
F-value
p-value
Grazing
1
238
6.968
0.0088
Position (topo)
2
238
3.2293
0.0413
Burn_freq
1
238
0.3121
0.5769
DOY
1
238
22.1902
,.0001
G:P
2
238
3.7687
0.0245
G:B
1
238
0.0334
0.8551
P:B
2
238
0.1706
0.8432
G:DOY
1
238
0.0711
0.7899
P:DOY
2
238
1.3037
0.2735
B:DOY
1
238
0.01
0.9203
G:B:P
2
238
1.2241
0.2959
G:P:DOY
2
238
0.4158
0.6603
G:B:DOY
1
238
0.6971
0.4046
DOY:B
2
238
0.2106
0.8103
4-way
2
238
0.0732
0.9295
Random effects include the watershed sampled in 2011. doi:10.1371/journal.pone.0081630.t003 Random effects include the watershed sampled in 2011. doi:10.1371/journal.pone.0081630.t003 On Konza Prairie, ‘slope’ positions typically represent mid-points
between thin-soil uplands and deep-soil lowlands. Thus, soil-depth
on slopes is variable site-wide. Here, leaf d13C on slopes was higher
in ungrazed versus grazed locations (Fig. 3), likely reflecting
differences in soil depth among slopes rather than specific impacts
of grazing at these locations. Uplands and grazed locations
typically have less aboveground herbaceous biomass and greater
occurrence of bare soil, resulting in higher sensible heat fluxes
compared to locations with greater herbaceous biomass surround-
ing each individual shrub island. It is likely that higher iWUE
reflects physiological responses to higher sensible heat at upland
and grazed locations. Similar to leaf d13C, NDVI for C. drummondii
varied according to topographic position and by grazing contrasts
(Fig. 4) with the highest canopy greenness in ungrazed and
lowland topographic positions. Figure 4. Changes in mean NDVI (±1SE) by day of year in 2011. Each estimate of NDVI represents the image captured at ,262 m
resolution for each individual dogwood island on the landscape. Panel
‘a’ shows differences according to topographic positions, while panel ‘b’
shows differences by grazing contrasts. doi:10.1371/journal.pone.0081630.g004 landscape [57]. At Konza Prairie, a drought of sufficient
magnitude to lower water availability at soil depths below 1 m
are unknown, as are the potential physiological and stress-
responses by woody shrubs reliant on these deeper water sources. December 2013 | Volume 8 | Issue 12 | e81630 References Sheffer M (2009) Critical transitions in nature and society. Princeton, NJ,
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Midl Nat 153: 217–231. doi: 10.1674/0003-0031. 7. Acknowledgments This work was inspired by the ISOSCAPES 2008 workshop in Santa
Barbara, CA. We thank Teall Culbertson and Whitley Jackson for field and
laboratory assistance. Nate Brunsell, and John Briggs provided thoughtful
discussion and comments that improved this manuscript. We thank Tony
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rates of woody encroachment in some regions may occur because
post-establishment encroaching species become decoupled from
the typical system dynamics that regulate the dominant species of
the ecosystem. The spatial and temporal partitioning of water resources
between coexisting woody plants and grasses has been previously
shown in a variety of North American grasslands [35,58–59]. In
karst landscapes of the Edwards Plateau, TX, an extended drought
(and reduced availability of deep soil water) lowered carbon uptake
and resulted in physiological stress by woody plants in a savanna December 2013 | Volume 8 | Issue 12 | e81630 PLOS ONE | www.plosone.org 6 Dogwood Leaf d13C in a Tallgrass Prairie maintenance of the long-term experimental design. GLO was supported by
the KSU McNair Scholars Program. maintenance of the long-term experimental design. GLO was supported by
the KSU McNair Scholars Program. Author Contributions Conceived and designed the experiments: JBN TWO. Performed the
experiments: JBN TWO GLO ZR. Analyzed the data: JBN TWO GLO
ZR AMS. Contributed reagents/materials/analysis tools: JBN TWO GLO
ZR BL AMS. Wrote the paper: JBN TWO GLO ZR BL AMS. References Knapp AK, Briggs JM, Hartnett DC, Collins SL (1998) Grassland Dynamics:
Long-Term Ecological Research in Tallgrass Prairie. Oxford University Press,
NY. 47. Throop HL, Reichmann LG, Sala OE, Archer SR (2012) Response of dominant
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plant species richness in tallgrass prairie. Oikos 99: 571–579. doi: 10.1034/
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Seasonal changes in depth of water uptake for encroaching trees Juniperus
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Linking plant growth responses across topographic gradients in tallgrass prairie. Oecologia 166: 1131–1142. doi: 10.1007/s00442-011-1948-6. December 2013 | Volume 8 | Issue 12 | e81630 7 PLOS ONE | www.plosone.org Dogwood Leaf d13C in a Tallgrass Prairie Dogwood Leaf d13C in a Tallgrass Prairie Dogwood Leaf d13C in a Tallgrass Prairie 49. Nippert JB, Fay PA, Knapp AK (2007) Photosynthetic traits in C3 and C4
grassland species in mesocosm and field environments. Environ Exp Bot 60:
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ecosystem function of savannas: a case study from the Edwards Plateau Karst,
Texas. In: Hill MJ, Hanan NP (eds) Ecosystem Function in Savannas:
measurement and modeling at landscape to global scales. Boca Raton, FL,
USA, CRC Press, pp 117–135. j
52. Aranjuelo I, Ebbets AL, Evans RD, Tissue DT, Nogue´s van Gestel N, et al. (2011) Maintenance of C sinks sustains enhanced C assimilation during long-
term exposure to elevated [CO2] in Mojave Desert shrubs. Oecologia 167: 339–
354. doi:10.1007/s00442-011-1996-y. 58. Weltzin JF, McPherson GR (1997) Spatial and temporal soil moisture resource
partitioning by trees and grasses in a temperate savanna, Arizona, USA. Oecologia 112: 156–164. doi: 10.1007/s004420050295. 53. Bai E, Bouton TW, Liu F, Wu XB, Archer SR (2008) Variation in woody plant
d13C along a topoedaphic gradient in a subtropical savanna parkland. Oecologia
156: 479–489. doi:10.1007/s00442-008-1003-4. Oecologia 112: 156–164. doi: 10.1007/s004420050295. 59. Dodd MB, Lauenroth WK, Welker JM (1998) Differential water resource use by
herbaceous and woody plant life-forms in a shortgrass steppe community. Oecologia 117: 504–512. doi: 10.1007/s004420050686. 54. Craine JM, Towne EG, Ocheltree TW, Nippert JB (2012) Community
traitscape of foliar nitrogen isotopes reveals N availability patterns in a tallgrass
prairie. Plant Soil 356: 395–403. doi:10.1007/s11104-012-1141-7. December 2013 | Volume 8 | Issue 12 | e81630 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8
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Beyond the diffraction limit of optical/IR interferometers
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Astronomy & astrophysics
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To cite this version: M. Hadjara, A. Domiciano de Souza, F. Vakili, S. Jankov, F. Millour, et al.. Beyond the diffraction
limit of optical/IR interferometers. II. Stellar parameters of rotating stars from differential phases. Astronomy and Astrophysics - A&A, 2014, 569, 10.1051/0004-6361/201424185. insu-03618542 Beyond the diffraction limit of optical/IR
interferometers. II. Stellar parameters of rotating stars
from differential phases
M. Hadjara, A. Domiciano de Souza, F. Vakili, S. Jankov, F. Millour, A. Meilland, Z. Khorrami, A. Chelli, C. Baffa, K. -H. Hofmann, et al. Distributed under a Creative Commons Attribution 4.0 International License ABSTRACT They were
found to be compatible with previously published values from differential phase and visibility measurements, while we were able to
determine, for the first time, the inclination angle i of Fomalhaut (i = 90◦± 9◦) and δ Aquilae (i = 81◦± 13◦), and the rotation-axis
position angle PArot of δ Aquilae. Results. The derived parameters for each star were Req = 11.2±0.5 R⊙, Veq sin i = 290±17 km s−1, PArot = 35.4◦±1.4◦, for Achernar;
Req = 2.0 ± 0.2 R⊙, Veq sin i = 226 ± 34 km s−1, PArot = −65.5◦± 5.5◦, for Altair; Req = 2.2 ± 0.3 R⊙, Veq sin i = 74 ± 35 km s−1,
PArot = −101.2◦± 14◦, for δ Aquilae; and Req = 1.8 ± 0.2 R⊙, Veq sin i = 93 ± 16 km s−1, PArot = 65.6◦± 5◦, for Fomalhaut. They were
found to be compatible with previously published values from differential phase and visibility measurements, while we were able to
determine, for the first time, the inclination angle i of Fomalhaut (i = 90◦± 9◦) and δ Aquilae (i = 81◦± 13◦), and the rotation-axis
position angle PArot of δ Aquilae. p
g
rot
q
Conclusions. Beyond the theoretical diffraction limit of an interferometer (ratio of the wavelength to the baseline), spatial super
resolution is well suited to systematically estimating the angular diameters of rotating stars and their fundamental parameters with a
few sets of baselines and the Earth-rotation synthesis provided a high enough spectral resolution. Key words. stars: rotation – stars: general – methods: observational – methods: numerical – techniques: interferometric –
techniques: high angular resolution ⋆Based on observations performed at the European Southern
Observatory, Chile, under ESO AMBER-consortium GTO program IDs
084.D-0456 081.D-0293 and 082.C-0376. II. Stellar parameters of rotating stars from differential phase M. Hadjara1,2, A. Domiciano de Souza1, F. Vakili1, S. Jankov3, F. Millour1, A. Meilland1, Z. Khorrami1, A. Chelli
C. Baffa5, K.-H. Hofmann6, S. Lagarde1, and S. Robbe-Dubois1 M. Hadjara1,2, A. Domiciano de Souza1, F. Vakili1, S. Jankov3, F. Millour1, A. Meilland1, Z. Khorrami1, A. Chelli4,
C. Baffa5, K.-H. Hofmann6, S. Lagarde1, and S. Robbe-Dubois1 1 Laboratoire J.L. Lagrange −UMR 7293 – Observatoire de la Côte d’Azur (OCA), Université de Nice-Sophia Antipolis (UNS),
Centre National de la Recherche Scientifique (CNRS), Campus Valrose, 06108 Nice Cedex 2, France
e-mail: Massinissa Hadjara@oca eu 1 Laboratoire J.L. Lagrange −UMR 7293 – Observatoire de la Côte d’Azur (OCA), Université de Nice-Sophia
Centre National de la Recherche Scientifique (CNRS), Campus Valrose, 06108 Nice Cedex 2, France e-mail: Massinissa.Hadjara@oca.eu
2 e mail: Massinissa.Hadjara@oca.eu
2 Centre de Recherche en Astronomie, Astrophysique et Géophysique (CRAAG), Route de l’Observatoire, BP 63, Bouzareah,
16340 Alger, Algérie j
de Recherche en Astronomie, Astrophysique et Géophysique (CRAAG), Route de l’Observatoire, BP 63, Bouzareah,
Alger, Algérie 2 Centre de Recherche en Astronomie, Astrophysique et Géophysique (CRAAG), Route de l’Observatoire, BP 63, Bouzareah,
16340 Alger, Algérie 3 Astronomical Observatory, Volgina 7, PO Box 74 11060 Belgrade, Serbia
4 UJF-Grenoble 1/CNRS-INSU, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG) UMR 5274, 38041 Grenoble,
F Astronomical Observatory, Volgina 7, PO Box 74 11060 Belgrade, Serbia
4 UJF-Grenoble 1/CNRS-INSU, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG) UMR 5274, 38041 Grenoble,
France y
g
g
4 UJF-Grenoble 1/CNRS-INSU, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG) UMR 5274, 3804
France 4 UJF-Grenoble 1/CNRS-INSU, Institut de Planétologie et d’Astrophysique de Gre
France 4 UJF-Grenoble 1/CNRS-INSU, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG) UMR 5274, 38041 Grenoble,
France 5 INAF−Osservatorio Astrofisico di Arcetri, Largo E. Fermi, 5, 50125 Firenze, Italy
6 Max-Planck-Institut für Radioastronomie, Auf dem Hügel 69, 53121 Bonn, Germany 5 INAF−Osservatorio Astrofisico di Arcetri, Largo E. Fermi, 5, 50125 Firenze, Italy
6 Max-Planck-Institut für Radioastronomie, Auf dem Hügel 69, 53121 Bonn, Germany Received 12 May 2014 / Accepted 4 July 2014 ⋆⋆Figure 5 is available in electronic form at
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Astrophysics A&A 569, A45 (2014)
DOI: 10.1051/0004-6361/201424185
c⃝ESO 2014 A&A 569, A45 (2014)
DOI: 10.1051/0004-6361/201424185
c⃝ESO 2014 ABSTRACT Context. As previously demonstrated on Achernar, one can derive the angular radius, rotational velocity, axis tilt, and orientation
of a fast-rotating star from the differential phases obtained by spectrally resolved long baseline interferometry using earth-rotation
synthesis. y
Aims. We applied this method on a small sample of stars for different spectral types and classes, in order to generalize the technique
to other rotating stars across the H-R diagram and determine their fundamental parameters. Aims. We applied this method on a small sample of stars for different spectral types and classes, in order to generalize the technique
to other rotating stars across the H-R diagram and determine their fundamental parameters. Methods We used differential phase data from the AMBER/VLTI instrument obtained prior to refurbishing its spectrometer in 2010 Methods. We used differential phase data from the AMBER/VLTI instrument obtained prior to refurbishing its spectrometer in 2010. With the exception of Fomalhaut, which has been observed in the medium-resolution mode of AMBER (λ/δλ ≈1500), our three
other targets, Achernar, Altair, and δ Aquilae offered high-resolution (λ/δλ ≈12 000) spectro-interferometric data around the Brγ
absorption line in K band. These data were used to constrain the input parameters of an analytical, still realistic model to interpret the
observations with a systematic approach for the error budget analysis in order to robustly conclude on the physics of our 4 targets. We
applied the super resolution provided by differential phases φdiffto measure the size (equatorial radius Req and angular diameter /⃝eq),
the equatorial rotation velocity (Veq), the inclination angle (i), and the rotation axis position angle (PArot) of 4 fast-rotating stars:
Achernar, Altair, δ Aquilae, and Fomalhaut. The stellar parameters of the targets were constrained using a semi-analytical algorithm
dedicated to fast rotators SCIROCCO. Results. The derived parameters for each star were Req = 11.2±0.5 R⊙, Veq sin i = 290±17 km s−1, PArot = 35.4◦±1.4◦, for Achernar;
Req = 2.0 ± 0.2 R⊙, Veq sin i = 226 ± 34 km s−1, PArot = −65.5◦± 5.5◦, for Altair; Req = 2.2 ± 0.3 R⊙, Veq sin i = 74 ± 35 km s−1,
PArot = −101.2◦± 14◦, for δ Aquilae; and Req = 1.8 ± 0.2 R⊙, Veq sin i = 93 ± 16 km s−1, PArot = 65.6◦± 5◦, for Fomalhaut. ⋆Based on observations performed at the European Southern
Observatory, Chile, under ESO AMBER-consortium GTO program IDs
084.D-0456 081.D-0293 and 082.C-0376.
⋆⋆Figure 5 is available in electronic form at
http://www.aanda.org 2.1. The sample stars The data of the four studied stars in this paper are avail-
able from ESO archive. Achernar (α Eri, HR 472, HD 10144)
is a rotationnally flattened Be star with an oblateness ratio
reported between 1.41 and 1.56 typically with 3% accuracy
(Domiciano de Souza et al. 2003). Subsequent studies (Kervella
& Domiciano de Souza 2006) considered a polar mass-loss com-
ponent added to the photospheric disk, for a star with rota-
tional parameter Veq sin i = 225 km s−1 (from previous spec-
troscopic estimates by Slettebak 1982). Then a small residual
disk (Carciofiet al. 2008) and/or the presence of a companion
by Kervella et al. (2008) were invoked to explain the apparent
strong flattening for Achernar from interferometric assymetric
data. However, Kanaan et al. (2008) considered the circumstel-
lar environment (CSE) contribution as important for explaining
the strong flatness. Overall, Domiciano de Souza et al. (2012,
Paper I), gave the recent parameters of Achernar as for its equa-
torial radius to be 11.6 R⊙, its equatorial velocity 298 km s−1, the
inclination 101◦and the position angle 34.7◦. Optical-long baseline interferometry (OLBI) is a key ob-
serving technique for directly constraining the stellar diam-
eter and the so-called von Zeipel effect (von Zeipel 1924;
Domiciano de Souza et al. 2003; McAlister et al. 2005;
Aufdenberg et al. 2006; Peterson et al. 2006; Monnier et al. 2007; Zhao et al. 2009; Che et al. 2011; van Belle 2012; Delaa
et al. 2013). The study of rotation across the H-R diagram may
also give information on the internal structure of stars of differ-
ent spectral types and luminosity classes and the physics of their
atmosphere (Espinosa Lara & Rieutord 2011). OLBI is classically based on measuring visibility amplitudes
at different baselines to determine the angular size of the tar-
get being observed. However, when a rotating star is studied
with spectrally resolved interferometry, the Doppler shift across
a line, which implicitly contains spatial information, together
with the change in the photocenter relative to this shift (Lagarde
1994), can measure the angular size of the visible hemisphere of
the star. Altair (α Aql, HR 7557, HD 187642) is a bright (V = 0.77)
A7IV-V star (Johnson & Morgan 1953) that is known to be a
rapid rotator (240 km s−1 from Slettebak 1955). 2.1. The sample stars These charac-
teristics, together with its location (close to celestial equator,
therefore observable in both the northern and southern hemi-
spheres), make Altair a prime target among the fast rotators. For
example, Buzasi et al. (2005) show that it is a variable of the
δ Scuti type by detecting several oscillating frequencies in this
star. Several interferometric and spectroscopic observations in-
dicate a Veq sin i value between 190 km s−1 and 250 km s−1 (Abt
& Morrell 1995; van Belle et al. 2001; Royer et al. 2002; Reiners
& Royer 2004b, among others). Domiciano de Souza et al. (2005) used the interferometer VINCI/VLTI and a model for
fast rotators, including Roche approximation, limb-darkening,
and von Zeipel gravity-darkening, to find the same value of
Veq sin i = 227 km s−1 as in Reiners & Royer (2004a), who
used spectroscopy for determine the constraints on Altairs incli-
nation angle and differential rotation from the global rotational
broadening profile derived from about 650 spectral absorption
lines. Most recently, Monnier et al. (2007) have reconstructed
an image of the surface of Altair from CHARA observations,
inferring several fundamental parameters: inclination, position
angle, effective temperature, and radii of the pole and of the
equator. They derived the values of Veq sin i = 240 km s−1
and 241 km s−1. In addition, spectrally resolved long-baseline interferome-
try – also called differential interferometry (DI; Petrov 1989;
Lachaume 2003) once combined with Earth-rotation synthesis
and/or the use of simultaneous baselines from three or more tele-
scopes, offers new possibilities for observing the details of ro-
tationally flattened stars and directly checking whether the von
Zeipel effect applies to different spectral types and luminosity
classes (van Belle 2012). The application of DI in practice de-
mands, however, special data analysis to access the so-called
differential phase (Vakili et al. 1998), which in turn needs ap-
propriate calibration procedures that go beyond the usual tra-
ditional angular diameter estimates versus visibility curves ex-
trema and/or zeros. This paper scrutinizes four stars of different spectral types,
namely: – Achernar (α Eridani, B6Vep spectral type), – Altair (α Aquilae, A7V spectral type), q
p
yp
– δ Aquilae (HD182640, F0IV spectral type) – Fomalhaut (α Piscis Austrini, A4V spectral type). These stars were observed using DI with the AMBER focal in-
strument of the VLTI (Petrov et al. 2007). 1. Introduction be ignored in the case of the stars studied in this paper present-
ing low luminosity. Fast-rotating stars exhibit a number of pecu-
liar characteristics, such as geometrical flattening, coupled with
gravitational darkening (von Zeipel 1924), making the poles ap-
parently hotter than the equator. Gravity darkening has a pro-
found impact on the physics of the star, with important obser-
vational consequences. For example, the models from Collins
& Sonneborn (1977) indicate a two-component spectral energy
distribution (SED) for these stars, with an infrared (IR) excess
due to gravity darkening. It is therefore not easy to place these
stars in one single spectral classification, since the observed The rotation of stars can reach more than 80% of their critical or
breakup velocity vc = √(GM/Rc) in some specific cases. Here,
G is the gravitational constant, M the mass of the star, and Rc
the equatorial radius at this velocity. The Eddington factor can Article published by EDP Sciences A45, page 1 of 14 A&A 569, A45 (2014) 2. Observations and data reduction SED depends on their rotational velocity and inclination an-
gle (Maeder & Peytremann 1972). In addition, rotation-induced
mechanisms, such as meridional circulation or turbulence, may
affect the internal structure of the star and its evolution (Meynet
2009). Thus, fast rotators can be considered as a physics labo-
ratory for studying the impact of rotation on the stellar structure
and evolution. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. −200
−150
−100
−50
0
50
100
150
200
−200
−150
−100
−50
0
50
100
150
200
E <−−−−− U (m)
V (m) −−−−−> N
∆ 2008−05−04> K0−G1 | G1−A0 | A0−K0 −> Altair
∇ 2008−05−05> K0−G1 | G1−A0 | A0−K0 −> δ Aquilae
o 2008−12−05> G1−D0 | D0−H0 | H0−G1 −> Fomalhaut
o 2008−12−05> G1−D0 | D0−H0 | H0−G1 −> Fomalhaut
o 2008−12−05> G1−D0 | D0−H0 | H0−G1 −> Fomalhaut
x 2008−12−08> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut
* 2008−12−09> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut
* 2008−12−09> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut
* 2008−12−09> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut
−200
−150
−100
−50
0
50
100
150
200
−200
−150
−100
−50
0
50
100
150
200
E <−−−−− U (m)
V (m) −−−−−> N
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
Fig. 1. Left: baselines and the corresponding (u, v) coverage of VLTI/AMBER observations of Altair, δ Aquilae, and Fomalhaut. Earth-rotation
synthesis spanned over ∼1.5 h/night for δ Aquilae and Altair and ∼5 h/night for Fomalhaut. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. Right: same data for Achernar, where Earth-rotation
synthesis spanned over ∼5 h/night. Notice the rather dense sampling of the Fourier space for the target stars. −200
−150
−100
−50
0
50
100
150
200
−200
−150
−100
−50
0
50
100
150
200
E <−−−−− U (m)
V (m) −−−−−> N
∆ 2008−05−04> K0−G1 | G1−A0 | A0−K0 −> Altair
∇ 2008−05−05> K0−G1 | G1−A0 | A0−K0 −> δ Aquilae
o 2008−12−05> G1−D0 | D0−H0 | H0−G1 −> Fomalhaut
o 2008−12−05> G1−D0 | D0−H0 | H0−G1 −> Fomalhaut
o 2008−12−05> G1−D0 | D0−H0 | H0−G1 −> Fomalhaut
x 2008−12−08> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut
* 2008−12−09> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut
* 2008−12−09> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut
* 2008−12−09> K0−G1 | G1−A0 | A0−K0 −> Fomalhaut −200
−150
−100
−50
0
50
100
150
200
−200
−150
−100
−50
0
50
100
150
200
E <−−−−− U (m)
V (m) −−−−−> N
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
o 2009−10−25> D0−HO | H0−K0 | K0−D0
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
x 2009−10−26> G1−D0 | D0−H0 | H0−G1
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
* 2009−10−30> K0−G1 | G1−A0 | A0−K0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0
+ 2009−11−01> H0−G0 | G0−E0 | E0−H0 Fig. 1. Left: baselines and the corresponding (u, v) coverage of VLTI/AMBER observations of Altair, δ Aquilae, and Fomalhaut. Earth-rotation
synthesis spanned over ∼1.5 h/night for δ Aquilae and Altair and ∼5 h/night for Fomalhaut. 2.2. The observations All the above stars were observed with the AMBER/VLTI in-
strument located at Cerro Paranal, Chile, with the Auxiliary
Telescopes (ATs). They were observed using the high spectral
resolution mode of AMBER (λ/∆λ ≈12 000) with the excep-
tion of Fomalhaut, observed using the medium spectral resolu-
tion mode (λ/∆λ ≈1500). Table 1 provides the observation log
of Altair, δ Aquilae, and Fomalhaut. The UV coverage for all
studied stars is provided in Fig. 1. The Achernar’s observations
(that are not included in Table 1) are the same as in Paper I. Notes. The calibration stars are hd184406, 88Aqr, and γScl. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. Right: same data for Achernar, where Earth-rotation
synthesis spanned over ∼5 h/night. Notice the rather dense sampling of the Fourier space for the target stars. Table 1. VLTI/AMBER observations of Altair, δ Aquilae, and
Fomalhaut with details on the dates, times, and baseline triplets. Table 1. VLTI/AMBER observations of Altair, δ Aquilae, and
Fomalhaut with details on the dates, times, and baseline triplets. Object
Date and time
Baseline length
Baseline PA
Bproj(m)
PA(◦)
Altair −→Observation with AT Triplet K0-G1-A0:
Altair
2008-05-04T09:50
80, 87, 127
−148, −68, −106
hd184406
2008-05-04T10:28
85, 79, 127
−145, −67, −108
δ Aquilae −→Observation with AT Triplet K0-G1-A0:
hd184406
2008-05-05T09:08
79, 89, 125
−150, −67, −106
δ Aquilae
2008-05-05T09:49
85, 84, 128
−148, −66, −107
Fomalhaut −→Observation with AT Triplet G1-D0-H0:
Fomalhaut
2008-12-05T00:20
70, 59, 71
−32, 83, 18
88Aqr
2008-12-05T01:13
66, 55, 71
−26, 82, 20
Fomalhaut
2008-12-05T01:30
68, 50, 69
−20, 91, 23
γScl
2008-12-05T01:48
69, 51, 68
−20, 93, 23
Fomalhaut
2008-12-05T02:06
68, 44, 68
−13, 96, 25
88Aqr
2008-12-05T02:24
63, 43, 70
−13, 87, 25
Fomalhaut −→Observation with AT Triplet K0-G1-A0:
Fomalhaut
2008-12-08T02:15
76, 76, 81
−138, −24, −81
88Aqr
2008-12-08T02:31
80, 68, 79
−141, −25, −91
Fomalhaut
2008-12-09T00:10
87, 86, 117
−144, −49, −97
88Aqr
2008-12-09T00:27
89, 83, 117
−145, −51, −100
Fomalhaut
2008-12-09T00:40
85, 84, 110
−142, −43, −93
88Aqr
2008-12-09T00:58
87, 78, 107
−143, −44, −97
Fomalhaut
2008-12-09T01:31
80, 79, 94
−139, −33, −87
γScl
2008-12-09T01:51
79, 81, 95
−138, −32, −84
Notes. The calibration stars are hd184406, 88Aqr, and γScl. idisk = 65.◦9±0.4◦and the position angle of PAdisk = 156◦±0.3◦. Kalas et al. (2008) confirm the presence of a planetary com-
panion. Royer et al. (2007) estimated the equatorial velocity
Veq = 93 km s−1 and Di Folco et al. (2004) find the equatorial ra-
dius Req = 1.1 mas (1.8 R⊙). Recently, Le Bouquin et al. (2009)
used the spectrally resolved near-IR long baseline interferome-
try to obtain spectro-astrometric measurements across the Brγ
absorption line, to find a position angle PAstar = 65◦± 0.3◦for
the stellar rotation axis, perpendicular to the literature measure-
ment for the disk position angle (PAdisk = 156◦.0 ± 0.3◦), and
they suggest backward-scattering properties for the circumstel-
lar dust grains. However, they did not infer the star’s radius from
their measurements, which we do in this article with the same
dataset. 2.1. The sample stars This comparative
study focuses on the rotational characteristics of these stars. To interpret these data, we developed a dedicated analytical
model called SCIROCCO (Simulation Code of Interferometric-
observations for ROtators and CirCumstellar Objects, Hadjara
et al. 2012, 2013) in order to derive the fundamental and geo-
metrical parameters of our sample of stars. The source δ Aquilae A (δ Aql, HR 7377, HD 182640)
is a subgiant F0IV star (Cowley & Fraquelli 1974). Using a
model atmosphere analysis of spectroscopic binaries and mul-
tiple star systems, Fuhrmann (2008) determined that the mass
of this star is 1.65 M⊙, and its radius is 2.04 R⊙. Looking for
signatures of differential rotation in line profiles, Reiners (2006)
found an effective temperature of 7016 K. The star δ Aquilae A
presents non-radial pulsations and is a Delta Scuti variable that
in addition is spinning rapidly with a Veq sin i estimated around
89 km s−1 (Mantegazza & Poretti 2005). The present paper is organized as follows: we first intro-
duce the stars, their observations, and data reduction (Sect. 2). We recall the principles of differential phase measurements and
present the SCIROCCO model (Sect. 3). In Sect. 4, we vali-
date the SCIROCCO model, comparing it with previously re-
ported measures of Achernar. We then determine the parameters
of Altair, δ Aquilae, and Fomalhaut in Sect. 5. These results, to-
gether with future applications of SCIROCCO, are discussed for
a broader study of fast-rotating stars with AMBER/VLTI inter-
ferometry across the HR diagram (Sect. 6). Fomalhaut (αPsA, HR 8728, HD 216956) is a young A4V
star. As the nineteenth brightest star in the sky, it is one of
the closest main sequence star surrounded by a spatially re-
solved debris disk with an inclined ring of about 140 AU
in radius (Holland et al. 2003; Stapelfeldt et al. 2004). With
HST/ACS imaging, under the assumption that the disk is intrin-
sically circular, Kalas et al. (2005) estimated the disk inclination A45, page 2 of 14 M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. 2.3. Instrumental biais trouble-shooting and data calibration The differential phase φdiffobtained from AMBER data reduc-
tion algorithm is related to the object’s Fourier phase φobj by
(e.g., Millour et al. 2011, 2006): v = Bproj cos (PA)/λ). The parameters a and b correspond to an
offset and a slope, given in appropriate units. Data have been reduced using version 3.1 of the amdlib
software (Chelli et al. 2009; Tatulli et al. 2007). We adopted a
standard frame selection based on fringe signal-to-noise ratio
(Millour et al. 2007) and kept the 50% best frames calibrated
later using appropriate software by Millour et al. (2008). The
visibility amplitudes could not be reliably calibrated due to too
chaotic a transfer function. Indeed, FINITO did not provide the φdiff(u, v) = φobj(u, v) −a(u, v) −b(u, v)/λ,
(1) (1) where the spatial frequency coordinates u and v depend on the
wavelength λ, the projected baseline length Bproj and the baseline
position angle PA (from north to east; u = Bproj sin (PA)/λ and A45, page 3 of 14 A45, page 3 of 14 A&A 569, A45 (2014) Fig. 2. Top: AMBER instrumental fringing effect on the differential phases as a function of wavelength. Left: Achernar’s differential phases showing
high-frequency beating (black line), and the same filtered out (red line). Right: same as for Altair. Bottom: left: Achernar’s dynamical differential
phases before removing the fringing effect, for the observing night of 2009 Nov. 01, for the E0-H0 baseline. Center: after bias removal, the rotation
effect around the Brackett γ line becomes more visible as a dark vertical strip next to a bright vertical strip. Right: by projecting the differential
phases as a function of wavelength, one gets the typical s-shaped effect as a function of wavelength. Fig. 2. Top: AMBER instrumental fringing effect on the differential phases as a function of wavelength. Left: Achernar’s differential phases showing
high-frequency beating (black line), and the same filtered out (red line). Right: same as for Altair. Bottom: left: Achernar’s dynamical differential
phases before removing the fringing effect, for the observing night of 2009 Nov. 01, for the E0-H0 baseline. Center: after bias removal, the rotation
effect around the Brackett γ line becomes more visible as a dark vertical strip next to a bright vertical strip. Right: by projecting the differential
phases as a function of wavelength, one gets the typical s-shaped effect as a function of wavelength. 2.3. Instrumental biais trouble-shooting and data calibration The high spectral resolution mode of AMBER (λ/∆λ = 12000)
leads to a velocity resolution of ≃25 km s−1, while the medium-
resolution mode (λ/∆λ = 1500) leads to the velocity resolu-
tion of ≃200 km s−1. Projected equatorial rotational velocities
Veq sin i above ∼150 km s−1 would ensure that inside the Brγ
line, the attained resolution is ∼12 individual spectral canals in
high resolution and two spectral canals in the middle resolution
observational mode. In such cases, rotation effects need to be
accounted for when modeling phase signatures in order to be
consistent with the physics of the studied star. additional data necessary at that time to properly calibrate the ab-
solute visibilities. Our final dataset include the source spectrum,
differential visibilities, differential phases, and closure phases. Owing to the defective IRIS-feeding dichroic plate in the
VLTI optical train in front of AMBER, a high-frequency beat-
ing affected all our measurements (spectrum, visibilities, phases)
and was also present in the calibrated data (Fig. 2). Owing
to the polarizing prisms of AMBER, a low-frequency beating
(AMBER “socks”) was also present in our data. The high-
frequency beating appears at a specific frequency in the wave-
number domain. We removed it by Fourier filtering at that spe-
cific frequency. The second beating was corrected by subtracting
a sine wave fit to the continuum signal. We performed a wavelength calibration by using the posi-
tions of telluric lines. We normalized the spectrum by adjust-
ing and dividing a fourth order polynomial to the continuum. Figure 2 (top) shows an comparative example of the AMBER
differential phases before and after processing, while Fig. 2 (bot-
tom) shows dynamical differential phases before and after re-
moving of the instrumental biases. 1 MATrix LABoratory. 3. SCIROCCO code for modeling rotating stars To interpret these spectro-interferometric observations, we de-
veloped a chromatic semi-analytical model for fast rotators:
Simulation Code of Interferometric-observations for ROtators
and CirCumstellar Objects (SCIROCCO). The code, imple-
mented in Matlab1, allows computing monochromatic intensity
maps of uniformly rotating, flattened, and gravity-darkened stars
from a semi-analytical approach. SCIROCCO is described be-
low, and in the next section it is used to investigate the depen-
dence of observed interferometric differential phases of the four
stars on the relevant input physical parameters. Our final data set consists of: – Altair: 6 (= 2 × 3 baselines) φdiff(λ) curves centered on Br γ, – Altair: 6 (= 2 × 3 baselines) φdiff(λ) curves centered on Br γ, – δ Aquilae: 6 (=2 × 3 baselines) φdiff(λ) curves centered on
Br γ, – Fomalhaut: 18 (= 6 × 3 baselines) φdiff(λ) curves centered on
Brγ, γ
– Achernar: the same 84 (= 28 × 3 baselines) φdiff(λ) curves as
in Paper I, used here for reference to our analytical model. A45, page 4 of 14 M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. Fig. 3. Left: 3D Brackett γ local line profile representation for a star with [T eff, log g]pole = [10 500 K, 30 cm/s2] at the poles and [T eff, log g]eq =
[19 000 K, 35 cm/s2] at the equator from Kurucz/Synspec model. The polar line profiles (in the board) have less amplitude than the equatorial one
in the middle. Right: 3 panels of 1D cuts of the same 3D figure at different latitudes; at the poles (red line), at the equator (blue) and the average
(in green). Fig. 3. Left: 3D Brackett γ local line profile representation for a star with [T eff, log g]pole = [10 500 K, 30 cm/s2] at the poles and [T eff, log g]eq =
[19 000 K, 35 cm/s2] at the equator from Kurucz/Synspec model. The polar line profiles (in the board) have less amplitude than the equatorial one
in the middle. Right: 3 panels of 1D cuts of the same 3D figure at different latitudes; at the poles (red line), at the equator (blue) and the average
(in green). 3.3. Continuum intensity map Once the local effective temperature Teff(θ) is defined, the spe-
cific intensity maps in the continuum Ic(θ, φ) (continuum specific
intensity at each visible pixel of the stellar surface) are given by The stellar surface is analytically described by an oblate ellip-
soid of revolution with the semi-minor axis Rp in the direction
of the rotation axis and semi-major axis given by the equatorial
radius Req. The polar-to-equatorial radii ratio is taken from the
Roche model (Domiciano de Souza et al. 2002): Ic(λ, θ, φ)
I0(λ, Teff) = 1 −
4
X
k=1
ak(λ)
1 −µ(θ, φ)
K
2
(5) (5) Rp
Req
= 1 −
V2
eqRp
2GM =
1 +
V2
eqReq
2GM
−1
(2) (2) where (θ, φ) are the (co-latitude, longitude) coordinates on the
stellar surface, I0 is approximated by the analytical (black body)
Planck function, and µ(θ, φ) is the cosine of the angle between
the normal to the surface at a given point and the observer’s
direction. Limb darkening is analytically included following
the four-parameter prescription from Claret (2000a), where the
limb-darkening coefficients ak are chosen from tabulated values
for a given photometric band, local effective gravity, and temper-
ature. In this paper we consider solar metallicity and turbulent
velocity of 2 km s−1 for the limb-darkening parameters. where G is the gravitational constant, Veq the equatorial linear
rotation velocity, and M the mass of the star. The minor-to-major axis ratio of the oblate spheroid pro-
jected onto the sky plane (apparent ellipse) depends on the polar
inclination angle i and the orientation of the ellipse, which is
defined by the position angle PArot of the minor (rotation) axis
projected onto the sky (position angle of the minor axis). 3.2. Gravity darkening The specific intensity maps I(λ, θ, φ) across photospheric ab-
sorption lines are computed as the product of the continuum
specific intensity Ic(λ, θ, φ) defined above and a local normal-
ized intensity profile H, which can vary over the stellar surface
both because of rotational Doppler shift, and gravity darkening
I is thus given by Following the recent spectro-interferometric works on fast rota-
tors, we consider a von Zeipel-like (von Zeipel 1924) form for
the gravity darkening effect with a β coefficient: Teff(θ) =
C
σ
0.25
gβ
eff(θ)
(3) (3) I(λ, θ, φ) = Ic(λ, θ, φ)H
λ + λ0
Vproj(θ, φ)
c
, θ, φ
! (6) (6) where Teff(θ) and geff(θ) are the effective surface temperature and
gravity, which depend on the co-latitude θ; β is the gravity dark-
ening coefficient; and σ is the Stefan-Boltzmann constant. The
constant C is given by where Vproj is the surface rotational velocity projected onto the
observer’s direction, c is the vacuum light velocity, and λ0 the
central wavelength of the line profile. C =
σT
4
effS ⋆
R
g4β
eff(θ) dS
(4) C =
σT
4
effS ⋆
R
g4β
eff(θ) dS The local profile H over the stellar surface is interpolated
from synthetic spectra calculated with the SYNSPEC code based
on stellar atmosphere models from the Kurucz ATLAS9 grid
(Kurucz 1970; Hubeny & Lanz 1995), computed with the tab-
ulated closest values [Teff, log g] according to the co-latitude de-
ducted by SCIROCCO. Figure 3 depicts examples of local pro-
files interpolated from the adopted spectral synthesis code. (4) where T effis the average effective temperature over the stel-
lar surface (ellipsoidal surface in our model) and S ⋆the stel-
lar surface. We adopt the Roche model expression for the ef-
fective gravity geff(θ), but considering the equation of the oblate
ellipsoid of revolution for the dependence of the radius with co-
latitude θ. Given this definition of geff(θ), the gravity darkening is
totally defined by the above equations with input parameters T eff
and β. Once the specific intensity maps on the photospheric lines
are computed, the spectro-interferometric observables are di-
rectly obtained as spectra and photocenters, and by Fourier trans-
formations, which provide visibility amplitudes, phases, and clo-
sure phases (examples are given in the bottom row of Fig. 4). 3.2. Gravity darkening A45, page 5 of 14 A45, page 5 of 14 A&A 569, A45 (2014)
Top-left: velocity map of the same model (inclination 60◦, orientation 0◦). Then for the simulated rotation a rigid body
ty around 90% of the critical velocity of the star. Top-right: simulated intensity map in the continuum assuming von Ze
rkening effects where the poles are brighter than at the equator. Center-left: corresponding 2D map of the visibility modulus
ometric baselines cited above are represented by circles: ([75 m, 45◦] in red circle, [150 m, 45◦] in green circle, [75 m, 90
0 m, 90◦] in magenta circle). Center-right: same as for the 2D map of the visibility phase. ([75 m, 45◦] in red circle, [150
75 m, 90◦] in blue circle and [150 m, 90◦] in magenta circle). Bottom-left: monochromatic intensity map for a given Doppler
t spectral line. Bottom-right: top: photo-center along the Z axis. (Z axis represents the declination and Y axis the right asce 45 (2014) A&A 569, A45 (2014) A&A 569, A45 (2014) Fig. 4. Top-left: velocity map of the same model (inclination 60◦, orientation 0◦). Then for the simulated rotation a rigid body is assumed, and
a velocity around 90% of the critical velocity of the star. Top-right: simulated intensity map in the continuum assuming von Zeipel gravity and
limb-darkening effects where the poles are brighter than at the equator. Center-left: corresponding 2D map of the visibility modulus, where the four
interferometric baselines cited above are represented by circles: ([75 m, 45◦] in red circle, [150 m, 45◦] in green circle, [75 m, 90◦] in blue circle
and [150 m, 90◦] in magenta circle). Center-right: same as for the 2D map of the visibility phase. ([75 m, 45◦] in red circle, [150 m, 45◦] in green
circle, [75 m, 90◦] in blue circle and [150 m, 90◦] in magenta circle). Bottom-left: monochromatic intensity map for a given Doppler shift across the
Brackett spectral line. Bottom-right: top: photo-center along the Z axis. (Z axis represents the declination and Y axis the right ascension.) Middle:
photo-center along Y (the photo-centers are in radians). Bottom: normalized spectrum with the Brackett γ rotationally broadened line. Table 2. Limb-darkening parameters adopted for Achernar. 2 http://www.mathworks.com/matlabcentral/fileexchange/
9592-generalized-nonlinear-non-analytic-chi-square-
fitting 3.2. Gravity darkening – the stellar distance d (to convert from linear to angular sizes). Altair
δ Aquilae
Fomalhaut
Parameters
Values
Turbulent velocity VT
2 km s−1
2 km s−1
2 km s−1
log g
4 cm/s2
3.5 cm/s2
4.5 cm/s2
7250
8750
Claret Teff
to
7250 K
to
9000 K
9000 K
Metallicity
−0.2
0
0
Spectral filter
K
K
K For Fig. 4 we define a reference using the stellar model similar
to Achernar (exactly as in our Paper I): Req = 11 R⊙, d = 50 pc,
Veq sin i = 250 km s−1, i = 60◦, M = 6.1 M⊙, T eff= 15 000 K,
PArot = 0◦(north direction), β = 0.25 (theoretical value for ra-
diative stellar envelopes; von Zeipel 1924), no differential ro-
tation, with gravity darkening and limb darkening effects and
Kurucz/Synspec line profile. These parameters correspond to Veq
equal to 90% of the critical velocity Vcrit, Req/Rp = 1.4 and
an equatorial angular diameter /⃝eq = 2Req/d = 2 mas. To be
compatible with the AMBER observations presented in Paper I,
the simulations were performed around the hydrogen Brγ line,
Bproj = 75 m and 150 m, PA = 45◦and 90◦, and λ/∆λ = 12 000. adapted for our needs using the generalized nonlinear non-
analytic chi-square fitting code, developed by N. Brahms2. This
code performs a fit to the measurements with known errors, and
can use several Matlab library toolbox, such as the Levenberg-
Marquardt (LM) algorithm for faster convergence. We used the
Monte Carlo method to confirm that the parameter uncertainties
are normally distributed (Bevington & Robinson 2002) in order
to fit the simulated differential phases to the observations and to
constraint the free parameters and their uncertainties. The free
parameters are Req, Veq, i, and PArot. For our four stars cited in
the introduction (Sect. 1), the VLTI/AMBER φdiffobservations
were analyzed with the numerical model SCIROCCO presented
in Sect. 3. p
j
Examples of maps of projected velocity and specific inten-
sity in the continuum and in the line are shown in the top row
of Fig. 4. The second row represents the 2D maps of the visibil-
ity modulus and visibility phase, photocenters (or centroid: the
first-order term of the phase according to Mac Lauren develop-
ment, Jankov et al. 2001), and normalized spectra deduced from
the monochromatic intensity maps. 3.2. Gravity darkening Parameters
Value
Turbulent velocity VT
2 km s−1
log g
3 to 3.5 cm/s2
Claret Teff
11 500 to 19 000 K
Metallicity
0
Spectral filter
K
Thus we can give feedback on the input model parameters
by comparing the synthesized observable quantities to spectro-
interferometric measures within the error bars. The relevant in-
put parameters of our model for fast-rotating stars are
– the equatorial radius Req,
– the stellar mass M,
– the inclination angle i and the equatorial rotation velocity Veq
(or alternatively Veq sin i),
A45, page 6 of 14 Table 2. Limb-darkening parameters adopted for Achernar. Parameters
Value
Turbulent velocity VT
2 km s−1
log g
3 to 3.5 cm/s2
Claret Teff
11 500 to 19 000 K
Metallicity
0
Spectral filter
K
Thus we can give feedback on the input model parameters
by comparing the synthesized observable quantities to spectro-
interferometric measures within the error bars. The relevant in-
put parameters of our model for fast-rotating stars are
– the equatorial radius Req,
– the stellar mass M,
– the inclination angle i and the equatorial rotation velocity Veq
(or alternatively Veq sin i),
A45, page 6 of 14 Table 2. Limb-darkening parameters adopted for Achernar. Thus we can give feedback on the input model parameters
by comparing the synthesized observable quantities to spectro-
interferometric measures within the error bars. The relevant in-
put parameters of our model for fast-rotating stars are – the equatorial radius Req, – the stellar mass M, – the inclination angle i and the equatorial rotation velocity Veq
(or alternatively Veq sin i), A45, page 6 of 14 A45, page 6 of 14 M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. ainties estimated from a Levenberg-Marquardt fit of our model to the VLTI/AMBER φdiffobserved on studied stars,
und in the literature. Table 3. Parameters and uncertainties estimated from a Levenberg-Marquardt fit of our model to the VLTI/AMBER φdiffobserved on studied stars,
and compared with what we found in the literature. Table 3. Parameters and uncertainties estimated from a Levenberg-Marquardt fit of our model to the VLTI/AMBER φdiffobserved on studied stars,
and compared with what we found in the literature. Table 3. Parameters and uncertainties estimated from a Levenberg-Marquardt fit of our model to the VLTI/AMBER φdiffobserved on studied stars,
and compared with what we found in the literature. 3.2. Gravity darkening Previously studied stars
Parameters
Achernar
Comparison of best fit values those from literature
Best-fit parameter
In this paper
In the literature
Equatorial radius Req
11.2 ± 0.5 R⊙
11.6 ± 0.6 R⊙a
Equatorial rotation velocity Veq
295 ± 10 km s−1
298 ± 9 km s−1a
Rotation-axis inclination angle i
100.0 ± 2.5◦
101.5 ± 5.2◦a
Rotation-axis position angle PArot
35.4 ± 1.4◦
34.9 ± 1.6◦a
χ2
2.3003
Fixed parameter
Value
Distance d
44.1 pca
Mass M
6.1 M⊙a
Surface mean effective temperature T eff
15 000 Ka
Gravity-darkening coefficient β
0.20a
Derived parameter
Value
Equatorial angular diameter /⃝eq
2.38 ± 0.10 mas
Equatorial-to-polar radii Req/Rp
1.42 ± 0.06
Veq sin i
290 ± 10 km s−1
Veq/Vcrit
0.94 ± 0.03
Polar effective temperature Tpol
18 728 ± 327 K
Equatorial effective temperature Teq
11 243 ± 1308 K
Luminosity log L/L⊙
3.632 ± 0.044
References. (a) Domiciano de Souza et al. (2012), and references there in. and compared with what we found in the literature. Previously studied stars
Parameters
Achernar
Comparison of best fit values those from literature
Best-fit parameter
In this paper
In the literature
Equatorial radius Req
11.2 ± 0.5 R⊙
11.6 ± 0.6 R⊙a
Equatorial rotation velocity Veq
295 ± 10 km s−1
298 ± 9 km s−1a
Rotation-axis inclination angle i
100.0 ± 2.5◦
101.5 ± 5.2◦a
Rotation-axis position angle PArot
35.4 ± 1.4◦
34.9 ± 1.6◦a
χ2
2.3003
Fixed parameter
Value
Distance d
44.1 pca
Mass M
6.1 M⊙a
Surface mean effective temperature T eff
15 000 Ka
Gravity-darkening coefficient β
0.20a
Derived parameter
Value
Equatorial angular diameter /⃝eq
2.38 ± 0.10 mas
Equatorial-to-polar radii Req/Rp
1.42 ± 0.06
Veq sin i
290 ± 10 km s−1
Veq/Vcrit
0.94 ± 0.03
Polar effective temperature Tpol
18 728 ± 327 K
Equatorial effective temperature Teq
11 243 ± 1308 K
Luminosity log L/L⊙
3.632 ± 0.044
References. (a) Domiciano de Souza et al. (2012), and references there in. References. (a) Domiciano de Souza et al. (2012), and references there in. Table 4. Limb-darkening parameters adopted, respectively, left to right,
for Altair, δ Aquilae, and Fomalhaut. Table 4. Limb-darkening parameters adopted, respectively, left to right,
for Altair, δ Aquilae, and Fomalhaut. – the mean effective temperature on the stellar surface T eff, – the gravity-darkening coefficient β, – the stellar distance d (to convert from linear to angular sizes). 4. Validation of SCIROCCO on VLTI/AMBER
differential phases of Achernar To test and validate our semi-analytical code, we performed a
χ2 minimization using the VLTI/AMBER differential phase data
recorded on Achernar (cf. Sect. 2.1) and previously analyzed by
Domiciano de Souza et al. (2012, Paper I). The limb-darkening parameters are fixed assuming the
Claret function (Claret 2000a), as cited in the Sect. 3 and The χ2 minimization was performed using a free Matlab
implementation (from Matlab central file exchange) that we A45, page 7 of 14 A&A 569, A45 (2014) Table 5. Parameters and uncertainties estimated from a Levenberg-Marquardt fit of our model to the VLTI/AMBER φdiffobserved on studied stars, and compared with values found in the literatur
New studied stars
Parameters
Altair
δ Aquilae
Fomalhaut
Comparison of best fit values those from literature
Best-fit parameter
In this paper
In the literature
In this paper
In the literature
In this paper
In the literature
Equatorial radius Req
2.0 ± 0.2 R⊙
2.029 ± 0.007 R⊙a
2.2 ± 0.3 R⊙
Rmean = 2.04 R⊙b
1.8 ± 0.2 R⊙
Rmean = 1.8 R⊙f
Equatorial rotation velocity Veq
269 ± 14 km s−1
285.8 ± 12 km s−1a
75 ± 23 km s−1
Veq sin i = 68.4 km s−1e to 89 km s−1c
93 ± 7 km s−1
93 km s−1d
Rotation-axis inclination angle i
57.3 ± 5.6◦
57.2 ± 1.9◦a
81 ± 13◦
?◦
90 ± 9◦
?◦
Rotation-axis position angle PArot
−65.5 ± 5.5◦
−61.8 ± 0.8◦a
−101.2 ± 13.6◦
?◦
65.6 ± 4.7◦
65 ± 3◦d
χ2
1.8775
1.3297
0.44132
Fixed parameter
Value
Distance d
5.14 pca
15.5 pcb
7.66 pc f
Mass M
1.791 M⊙a
1.65 M⊙b
2 M⊙f
Surface mean effective temperature T eff
7700 Ka
7119 Ke
8760 K f
Gravity-darkening coefficient β
0.19a
0.20
0.20
Derived parameter
Value
Equatorial angular diameter /⃝eq
3.69 ± 0.36 mas
1.27 ± 0.17 mas
2.19 ± 0.24 mas
Equatorial-to-polar radii Req/Rp
1.22 ± 0.12
1.02 ± 0.14
1.02 ± 0.11
Veq sin i
226 ± 12 km s−1
74 ± 23 km s−1
93 ± 7 km s−1
Veq/Vcrit
0.74 ± 0.04
0.26 ± 0.08
0.25 ± 0.02
Polar effective temperature Tpol
8912 ± 234 K
7233 ± 510 K
8909 ± 342 K
Equatorial effective temperature Teq
7372 ± 937 K
7123 ± 1107 K
8764 ± 1024 K
Luminosity log L/L⊙
1.025 ± 0.028
1.002 ± 0.037
1.192 ± 0.038
References. (a) Monnier et al. 6. Conclusions and discussion summarized in the Table 2. The gravity darkening coefficient β
has been fixed using the predicted theoretical relation between β
and temperature from Claret (2000b, 2012), as well as recent
observational results. Based on the similar β value found for
studied rapid rotators (including β Cas, which has spectral type
F2III-IV, close to δ Aql), Che et al. (2011) recommend adopting
a new standard β = 0.19 for future modeling of rapidly rotat-
ing stars with radiative envelopes. However, here (as well as in
Paper I) we use the value β = 0.2, since this number of signifi-
cant digits is consistent with our results, showing that φdiffis not
very sensitive to β and to the relative uncertainties ∼10% in the
derived parameters. The model parameters that were held fixed,
following Paper I, are shown in Table 3. Following Espinosa
Lara & Rieutord (2011), which established the relation between
the gravity darkening coefficient and the flatness of the star (see
Fig. 4 of this paper), we deduced β = 0.15 for Achernar, with
which we have obtained similar results with β = 0.2. summarized in the Table 2. The gravity darkening coefficient β
has been fixed using the predicted theoretical relation between β
and temperature from Claret (2000b, 2012), as well as recent
observational results. Based on the similar β value found for
studied rapid rotators (including β Cas, which has spectral type
F2III-IV, close to δ Aql), Che et al. (2011) recommend adopting
a new standard β = 0.19 for future modeling of rapidly rotat-
ing stars with radiative envelopes. However, here (as well as in
Paper I) we use the value β = 0.2, since this number of signifi-
cant digits is consistent with our results, showing that φdiffis not
very sensitive to β and to the relative uncertainties ∼10% in the
derived parameters. The model parameters that were held fixed,
following Paper I, are shown in Table 3. Following Espinosa
Lara & Rieutord (2011), which established the relation between
the gravity darkening coefficient and the flatness of the star (see
Fig. 4 of this paper), we deduced β = 0.15 for Achernar, with
which we have obtained similar results with β = 0.2. Acknowledgements. This research made use of the SIMBAD database, operated
at the CDS, Strasbourg, France, and of the NASA Astrophysics Data System
Abstract Service. The author, M. Hadjara, acknowledges the support from the
CRAAG Institue (Algeria), the Lagrange and OCA financial grants to carry
on the present work, as well as the grants from the Fizeau European inter-
ferometry initiative (I2E). This research made use of the Jean-Marie Mariotti 6. Conclusions and discussion In this work we applied the super-resolution capabilities pro-
vided by differential interferometry to measuring size (equato-
rial radius and angular diameter), projected velocity, and posi-
tion angle of rotation axis of four fast-rotating stars: Achernar,
Altair, δ Aquilae, and Fomalhaut. Our analysis was based
on spectro-interferometric differential phases recorded with
the VLTI/AMBER beam combiner, operating in its high and
medium spectral resolution modes and centered on the Br γ line. All targets are only partially resolved by the interferometer in
terms of fringe contrast (angular sizes ∼2−5 lower than the the-
oretical angular resolution λ/Bmax). The stellar parameters of the targets were constrained
using a semi-analytical model to interpret the observations:
SCIROCCO. This simplified algorithm dedicated to fast rotators
was validated by showing that it leads to results in agreement
with previous values obtained from an independent analysis of
the same differential phase observations of Achernar (Paper I). Figure 5 shows the best-fit φdiff, together with the corre-
sponding observations of Achernar. The best-fit values of the
free parameters found with the LM algorithm are given in
Table 3. As shown in these table and figures, best-fit model
parameters and differential phases agree with the results from
Paper I, within the measured uncertainties. The observations of Altair, δ Aquilae, and Fomalhaut were
fitted with SCIROCCO to determine their Req (and
/⃝eq),
Veq sin i, and PArot from the VLTI/AMBER differential phases
alone. These parameters are in good agreement (within the un-
certainties) with values found in the literature and derived from
different observables, such as spectra, squared visibilities, and
differential phases. In this work, we were able to deduce the rotation-axis in-
clination angle i for δ Aquilae, and Fomalhaut (i = 81 ± 13◦
and 90 ± 9◦, respectively) and the rotation-axis position angle
PArot = −101.2 ± 14◦for δ Aquilae, information that we have
not found in the literature. 4. Validation of SCIROCCO on VLTI/AMBER
differential phases of Achernar (2007); (b) Fuhrmann (2008); (c) Mantegazza & Poretti (2005); (d) Le Bouquin et al. (2009); (e) do Nascimento et al. (2003); (f) Di Folco et al. (2004). A45, page 8 of 14 M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. Fig. 6. The 6 VLTI/AMBER φdiffmeasured on Altair and δ Aquilae around Brγ at 2 different observing times (format YYYY-MM-DDTHH MM
SS) and, for each time, three different projected baselines and baseline position angles, as indicated in the plots. The smooth curves superposed on
the observations are the best-fit φdiffobtained without a differential rotation. M. Hadjara et al.: Beyond the diffraction limit of optical/IR interferometers. II. Fig. 6. The 6 VLTI/AMBER φdiffmeasured on Altair and δ Aquilae around Brγ at 2 different observing times (format YYYY-MM-DDTHH MM
SS) and, for each time, three different projected baselines and baseline position angles, as indicated in the plots. The smooth curves superposed on
the observations are the best-fit φdiffobtained without a differential rotation. 5. SCIROCCO fitting of Altair, δ Aquilae,
and Fomalhaut The free parameters adopted for Altair, δ Aquilae, and
Fomalhaut model-fitting are Req, Veq, i, and PArot. They were
calculated from previous works cited in the Sect. 3. The limb-
darkening parameters are fixed assuming the Claret function
(Claret 2000a), as described in Sect. 3, and are summarized in
Table 4. The remaining model parameters were held fixed, as
shown in Table 5. Still following Espinosa Lara & Rieutord
(2011), we deduced β = 0.19 for Altair (exactly what we and
Monnier et al. 2007, used). For δ Aquilae, and Fomalhaut we
computed β around 0.24, and for these values we also obtained
similar results to β = 0.2 for these two last stars. This work demonstrates that we can use the methodology
shown in our Paper I for large specimens of fast rotators of
different spectral types, and for several spectral interferometric
observations. The results from this work thus open new perspectives for
using spectro-interferometry to study partially resolved fast-
rotating stars with present interferometers in order to simulta-
neously measure their sizes, projected velocities, and position
angles. The best-fit values of the free parameters found with the LM
algorithm are given in Table 5 as well. Figures 6 and 7 show
the best-fit φdiff, together with the corresponding observations of
δ Aquilae, and Fomalhaut. The uncertainties of the parameters
estimated by the LM algorithm are ≈1%. A45, page 9 of 14 A45, page 9 of 14 A&A 569, A45 (2014)
Fig. 7. The 18 VLTI/AMBER φdiffmeasured on Fomalhaut around Brγ at 7 different observing times (format YYYY-MM-DDTHH MM SS)
and, for each time, three different projected baselines and baseline position angles, as indicated in the plots. The smooth curves superposed to
the observations are the best-fit φdiffobtained without a differential rotation (Domiciano de Souza et al. 2003), gravity-darkened Roche model, as
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each time, three different projected baselines and baseline position angles, as indicated in the plots. The smooth curves superposed to
ervations are the best-fit φdiffobtained without a differential rotation (Domiciano de Souza et al. 2003), gravity-darkened Roche mode
cribed in Sect. 3. All the observed φdiffpoints are shown here, knowing that the fit has been performed using all the wavelength points. A&A 569, A45 (2014) A&A 569, A45 (2014) Fig. 5. The 84 VLTI/AMBER φdiffmeasured on Achernar around Brγ at 28 different observing times (format YYYY-MM-DDTHH MM SS) and,
for each time, three different projected baselines and baseline position angles, as indicated in the plots. The smooth curves superposed to the
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y
p
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Fig. 5. continued. A&A 569, A45 (2014) Fig. 5. continued. A45, page 14 of 14
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Psychometric properties of the Sindhi version of the Mood and Feelings Questionnaire (MFQ) in a sample of early adolescents living in rural Pakistan
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eCommons@AKU
eCommons@AKU
Centre of Excellence in Women and Child
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11-17-2022
Psychometric properties of the sindhi version of the mood and
Psychometric properties of the sindhi version of the mood and
feelings questionnaire (MFQ) in a sample of early adolescents
feelings questionnaire (MFQ) in a sample of early adolescents
living in rural Pakistan
living in rural Pakistan
Janavi Shetty
University of Toronto, Canada
Florence Perquier
Centre for Addiction and Mental Health, Toronto, Canada
Susan C. Campisi
Hospital for Sick Children, Canada
Yaqub Wasan
Aga Khan University, yaqub.wasan@aku.edu
Madison Aitken
University of Toronto, Canada
See next page for additional authors
Follow this and additional works at: https://ecommons.aku.edu/coe-wch
Part of the Maternal and Child Health Commons, Patient Safety Commons, Pediatrics Commons,
Psychiatry Commons, Psychology Commons, and the Women's Health Commons
Recommended Citation
Recommended Citation
Shetty, J., Perquier, F., Campisi, S. C., Wasan, Y., Aitken, M., Korczak, D. J., Monga, S., Soofi, S. B., Szatmari,
P., Bhutta, Z. A. (2022). Psychometric properties of the sindhi version of the mood and feelings
questionnaire (MFQ) in a sample of early adolescents living in rural Pakistan. PLOS Global Public Health.
Available at:
Available at: https://ecommons.aku.edu/coe-wch/47 eCommons@AKU
eCommons@AKU eCommons@AKU
eCommons@AKU Centre of Excellence in Women and Child
Health Centres of Excellence Psychometric properties of the sindhi version of the mood and
Psychometric properties of the sindhi version of the mood and
feelings questionnaire (MFQ) in a sample of early adolescents
feelings questionnaire (MFQ) in a sample of early adolescents
living in rural Pakistan
living in rural Pakistan Psychometric properties of the sindhi version of the mood and
Psychometric properties of the sindhi version of the mood and
feelings questionnaire (MFQ) in a sample of early adolescents
feelings questionnaire (MFQ) in a sample of early adolescents
living in rural Pakistan
living in rural Pakistan Recommended Citation
Recommended Citation Shetty, J., Perquier, F., Campisi, S. C., Wasan, Y., Aitken, M., Korczak, D. J., Monga, S., Soofi, S. B., Szatmari,
P., Bhutta, Z. A. (2022). Psychometric properties of the sindhi version of the mood and feelings
questionnaire (MFQ) in a sample of early adolescents living in rural Pakistan. PLOS Global Public Health. Available at:
Available at: https://ecommons.aku.edu/coe-wch/47 Shetty, J., Perquier, F., Campisi, S. C., Wasan, Y., Aitken, M., Korczak, D. J., Monga, S., Soofi, S. B., Szatmari,
P., Bhutta, Z. A. (2022). Psychometric properties of the sindhi version of the mood and feelings
questionnaire (MFQ) in a sample of early adolescents living in rural Pakistan. PLOS Global Public Health. Available at:
Available at: https://ecommons.aku.edu/coe-wch/47 Janavi Shetty, Florence Perquier, Susan C. Campisi, Yaqub Wasan, Madison Aitken, Daphne J. Korczak,
Suneeta Monga, Sajid Bashir Soofi, Peter Szatmari, and Zulfiqar Ahmed Bhutta OPEN ACCESS OPEN ACCESS
Citation: Shetty J, Perquier F, Campisi SC, Wasan
Y, Aitken M, Korczak DJ, et al. (2022)
Psychometric properties of the Sindhi version of
the Mood and Feelings Questionnaire (MFQ) in a
sample of early adolescents living in rural Pakistan. PLOS Glob Public Health 2(11): e0000968. https://
doi.org/10.1371/journal.pgph.0000968 Abstract There is a need for reliable and valid screening tools that assess depressive symptoms in
adolescents in Pakistan. To address this need, the present study examined the psychomet-
ric properties and factor structure of a Sindhi-translated and adapted version of the child-
report Mood and Feelings Questionnaire (MFQ-C) and the Short Mood and Feelings Ques-
tionnaire (SMFQ-C) in a community sample of adolescents living in Matiari, Pakistan. Ques-
tionnaires were translated into Sindhi and administered by study psychologists to 1350
participants (52.3% female) 9.0 to 15.9 years old. Measurement structure was examined
using confirmatory factor analysis. Internal consistency was estimated, and convergent and
divergent validity were explored using subscales from the Strengths and Difficulties Ques-
tionnaire and the Screen for Child Anxiety Related Emotional Disorders. The unidimensional
structure of the MFQ-C was found to be adequate, but a four-factor structure comprising
core mood, vegetative, cognitive and agitated distress symptoms best fit the data (CFI =
0.97, TLI = 0.97, RMSEA = 0.05). The original unidimensional structure of the SMFQ-C was
supported (CFI = 0.97, TLI = 0.96, RMSEA = 0.07). The MFQ-C and the SMFQ-C respec-
tively showed excellent (α = 0.92) and good internal consistency (α = 0.87) as well as satis-
factory construct validity with some differences observed across the MFQ-C subscales. The
SMFQ-C and the adapted MFQ-C appear to be reliable and valid measures of depressive
symptoms among early adolescents living in rural Pakistan. Both total and subscale scores
can be derived from the MFQ-C to assess general and specific dimensions of depressive
symptoms in this population. Authors
Authors This article is available at eCommons@AKU: https://ecommons.aku.edu/coe-wch/47 PLOS GLOBAL PUBLIC HEALTH RESEARCH ARTICLE
Psychometric properties of the Sindhi version
of the Mood and Feelings Questionnaire
(MFQ) in a sample of early adolescents living
in rural Pakistan
Janavi ShettyID1,2*, Florence PerquierID1, Susan C. CampisiID3,4, Yaqub Wasan5,
Madison AitkenID1,6, Daphne J. KorczakID3,6, Suneeta MongaID3,6, Sajid Bashir SoofiID5,
Peter Szatmari1,3,6, Zulfiqar A. Bhutta4,5*
1 Cundill Centre for Child and Youth Depression, Centre for Addiction and Mental Health, Toronto, Ontario,
Canada, 2 Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada,
3 Department of Psychiatry, Hospital for Sick Children, Toronto, Ontario, Canada, 4 Centre for Global Child
Health; Hospital for Sick Children, Toronto, Ontario, Canada, 5 Centre of Excellence in Women and Child
Health, Aga Khan University, Karachi, Sindh, Pakistan, 6 Department of Psychiatry, Temerty Faculty of
Medicine, University of Toronto, Toronto, Ontario, Canada
* shettyjanavi@hotmail.com (JS); zulfiqar.bhutta@sickkids.ca, zulfiqar.bhutta@aku.edu (ZAB) Psychometric properties of the Sindhi version
of the Mood and Feelings Questionnaire
(MFQ) in a sample of early adolescents living
in rural Pakistan Janavi ShettyID1,2*, Florence PerquierID1, Susan C. CampisiID3,4, Yaqub Wasan5,
Madison AitkenID1,6, Daphne J. KorczakID3,6, Suneeta MongaID3,6, Sajid Bashir SoofiID5,
Peter Szatmari1,3,6, Zulfiqar A. Bhutta4,5* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Cundill Centre for Child and Youth Depression, Centre for Addiction and Mental Health, Toronto, Ontario,
Canada, 2 Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada,
3 Department of Psychiatry, Hospital for Sick Children, Toronto, Ontario, Canada, 4 Centre for Global Child
Health; Hospital for Sick Children, Toronto, Ontario, Canada, 5 Centre of Excellence in Women and Child
Health, Aga Khan University, Karachi, Sindh, Pakistan, 6 Department of Psychiatry, Temerty Faculty of
Medicine, University of Toronto, Toronto, Ontario, Canada * shettyjanavi@hotmail.com (JS); zulfiqar.bhutta@sickkids.ca, zulfiqar.bhutta@aku.edu (ZAB) Introduction requests can be directed to Amjad Hussain
(Database Manager, Aga Khan University, Karachi,
PAKISTAN, email: amjad.hussain@aku.edu). Depression in youth is common worldwide and has been identified as one of the leading
causes of disability among those 10- to 24- years old [1]. However, the burden of depression
and depressive symptoms in youth living in low- and middle-income countries (LMICs) is still
difficult to estimate due to a lack of available data [2]. Depression in adolescence is associated
with deleterious behaviours and outcomes. In LMICs, relative to non-depressed adolescents,
adolescents with depressive symptoms are more likely to engage in risky sexual behaviour, sub-
stance use, delinquent activity, self-harm, and suicidal behaviour [3]. Youth who experience
depression or depressive symptoms are also at a greater risk for future affective disorders [4]
and ill health during adulthood [5]. Funding: This work was funded by the Cundill
Centre for Child and Youth Depression, Centre for
Addiction and Mental Health, Toronto, Ontario,
Canada. JS, FP, SCC, and MA received support
from the Cundill Centre for Child and Youth
Depression. 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. Pakistan is classified as an LMIC and is the sixth-largest country in the world, with youth
between the ages of 10 to 19 years old accounting for approximately 23% of the total popula-
tion [6, 7]. In a school-based study of urban youth ages 11 to 18 years old living in Rawalpindi,
the estimated prevalence of depression measured by the Hospital Anxiety and Depression
scale was 17.2% [8]. Similar results were obtained among urban youth 12 to 14 years old living
in Hyderabad: the prevalence of depression as measured by the Children’s Depression Inven-
tory was estimated to be 14.9% in girls and 19.9% in boys [9]. To our knowledge, the only
study that has examined the prevalence of depression in youth living in rural Pakistan was con-
ducted in a sample of unmarried 16- to 18-year-old girls living in rural Punjab [10]. The esti-
mated prevalence of clinical depression as measured by the Structured Clinical Interview for
DSM-IV Disorders was 4.4% in this sample. Editor: Chunxiao Li, South China Normal
University, CHINA Received: May 3, 2022
Accepted: September 23, 2022
Published: November 17, 2022 Copyright: © 2022 Shetty 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: There are ethical
restrictions on sharing a de-identified dataset
because the data contain sensitive patient
information related to the mental health of children. The demographic data, even if de-identified, may
potentially be identifying when viewed as a line list
rather than in aggregate because the participants
are from a singular district in Pakistan. These
restrictions have been imposed by The Aga Khan
University, The Hospital for Sick Children, and The
Centre for Addiction and Mental Health. Data 1 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 Introduction Among PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 2 / 18 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan scales developed specifically for youth, the Strengths and Difficulties Questionnaire (SDQ)
which measures internalizing more broadly [22] and the Centre for Epidemiological Studies
Depression Scale for Children have been translated to Urdu [23]. To our knowledge, there are
no scales available in Sindhi, the most common language spoken in the province of Sindh [24]. The Mood and Feelings Questionnaire (MFQ) is one of the most useful tests for screening
childhood depression in children and adolescents [25]. It has been used and validated in many
epidemiological, clinical and non-clinical studies [26–28], and is recommended by the National
Institute for Health and Clinical Excellence [29]. The instrument was developed to reflect symp-
toms of depression specified in the DSM-III-R, in addition to several items of clinical relevance
related to loneliness and feeling unloved [30] but has also been validated against DSM-IV(-TR)
and DSM 5 depression diagnoses [31, 32]. The validated parent- (MFQ-P; 34-items) and self-
report (MFQ-C; 33-items) forms are free of use [31]. Regarding the MFQ factor structure, both
single- and five-factor structures (cognitive, vegetative, core mood, suicidality, and agitated dis-
tress symptoms) have been supported in HICs [19, 28, 33]. Also available is the short 13-item
version of the MFQ (SMFQ), which has also shown good psychometric properties in commu-
nity and clinical samples [34–36]. Although there are benefits of parents and youth as infor-
mants when assessing depressive symptoms, it has been frequently noted that parents tend to
underreport their child’s depressive symptoms on screening tools [37–39]. As such, information
self-reported by the adolescents themselves is often preferred [40]. To our knowledge, only two studies have examined the psychometric properties of the
MFQ-C (long and short forms) in community samples of adolescents living in LMICs. In
Thailand, the full-form MFQ-C was recently validated in a school-based study of youth 12 to
18 years old. The Thai MFQ-C showed excellent internal consistency (a = .92), convergent
validity with related measures of depression (the Childhood Depression Inventory and the
SDQ-emotional symptoms subscale), and good test-retest reliability [32]. However, the factor
structure of the scale was not examined. In Bangladesh, the psychometric properties of the
short form (SMFQ-C) were studied in a convenience sample of 9 to 17-year-old adolescents
living in rural and urban areas [41]. Introduction However, one-third of the sample had high levels
of psychological distress as measured by the Self Reporting Questionnaire, with higher scores
being associated with poverty, physical abuse, family stress, and adverse recent life events [10]. Despite the need for mental health care, in Pakistan, the ratio of psychiatrists per 100,000
people is 0.18, which is much lower than the world median ratio of 1.27 [11]. Further, most
mental health services for youth are provided in adult psychiatric facilities, located mainly in
urban areas [12]. In primary care settings, many health workers have limited time and/or
insufficient training to administer diagnostic interviews to all at-risk individuals [13]. Access
to free, validated, and easy to administer tools for screening depressive symptoms in youth
may thus contribute to the improvement of detection, surveillance, and early intervention of
youth depression in LMICs. Most existing screening tools for youth depression originate from high-income countries
(HICs), and few psychometric studies have been conducted in LMICs [14]. However, the clini-
cal presentation of depression and depressive symptoms can vary based on culture and con-
text. Culture may define and create specific sources of distress, which in turn impact the
manifestation and self-report of psychiatric symptoms [15]. For example, compared to West-
ern cultures, Eastern cultures have been found to stigmatize mental health problems more and
show more concern over the impact that disclosure of a mental illness may have on family
social and economic status [16]. In some LMICs, such as Pakistan, suicidal behaviours are con-
sidered criminal offences punishable by law, which may further impact disclosure [17, 18]. Low levels of endorsement of items can then affect the validity of the measurement tool they
are part of. These considerations are especially salient when attempting to administer screen-
ing tools developed in HICs in other contexts and it is unclear whether variation in prevalence
estimates across countries may represent actual differences in prevalence or differences in
measurement [19]. As such, there is a need for the adaptation of existing scales to cultural spec-
ificities and the investigation of the psychometric properties of screening tools prior to their
use in different cultures and contexts [20]. Several indigenous and culturally adapted scales
that measure adult depression are available in Urdu, Pakistan’s national language [21]. Method Analyses were conducted using data from the Nash-wo-Numa study, a cross-sectional study of
adolescents living in Matiari, Pakistan, between January 2019 and February 2020. Information
relevant to the current study is presented. Further details regarding sampling strategies, proce-
dures, study measures, and objectives are described elsewhere [42]. Introduction The unidimensional structure of the SMFQ-C translated
in Bangla was supported. The brief scale showed good internal consistency (α = .84) and ade-
quate convergent validity with the Spence Children’s Anxiety Scale-Parent version [41]. Both the MFQ-C and SMFQ-C show limited but promising evidence of good psychometric
properties in LMICs. This study aims to investigate the psychometric properties of a culturally
adapted version of the MFQ-C, translated into Sindhi, in adolescents living in the rural district
of Matiari, Pakistan. Given the benefits that a shorter screening tool may provide in general
populations or low-resource settings, the psychometric properties of the original SMFQ-C will
also be examined. Procedure Written consent from legal guardians and assent from participants were obtained following
standard procedures [42]. Mental health measures were administered verbally by study psy-
chologists through structured interviews conducted at the study site. Several steps were taken
to address cultural sensitivities. Firstly, participant questionnaires were administered in the
presence of a parent or chaperone. Secondly, four suicidality-related questions were adminis-
tered only to participants who endorsed specific items and were thus identified to be at risk of
self-harm. These questions were administered in a private module without a chaperone pres-
ent and safety protocols were initiated by research staff if there was an indication of immediate
risk of self-harm. Participants and mothers were informed that they could refuse to answer
any questions. All questionnaires were translated from English to Sindhi (the primary lan-
guage spoken in the province). Translations were reviewed and discussed with study psycholo-
gists and staff familiar with both languages, and pilot tested in the community [42]. Participants The participants were youth and their mothers living in Matiari. The estimated population of
Matiari is 770,040 of which approximately 48,000 are school-aged children between 9.0 and
15.9 years old [24, 42]. Matiari is primarily rural, with about 85% of the population living in
rural areas [24]. 3 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan The primary objective of the Nash-wo-Numa study was to determine the prevalence of and
risk factors associated with impaired linear growth in adolescents. The age ranges of partici-
pants varied between females (9.0 to 14.9 years old) and males (10.0 to 15.9 years old) to
account for variation in the onset of puberty. Several steps were taken to ensure that the sample
was representative of the population of Matiari. Briefly, data regarding age, sex (assigned at
birth), and the number of occupants in each household obtained from a companion study
were used in the selection process. Using these data, computer-assisted random sampling was
employed on 53,000 households within the Lady Health Workers catchment population of 26
health facilities in the Matiari District [42]. If households had more than one adolescent, one
was randomly selected to participate. Inclusion criteria for the principal study specified that participants must be within the age
ranges of the study, and their birthmothers must be available and cognitively able to partici-
pate. Female participants could not currently be or previously have been pregnant. Lastly, par-
ticipants with chronic or genetic illnesses known to impact growth (e.g., heart disease,
diabetes, Down syndrome) were excluded. For the present study, participants were also
excluded if they had no responses to any MFQ items. Ethics approval for the principal study
was obtained from Aga Khan University, Pakistan, and The Hospital for Sick Children, Can-
ada. Ethics approval for the present study was also granted by the Centre for Addiction and
Mental Health Research Ethics Committee, Canada. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 Measures Mood and Feelings Questionnaires-Child Report (MFQ-C and SMFQ-C). The original
MFQ-C is a 33-item questionnaire designed to assess depressive symptoms in children and
youth ages 8 to 18 [30]. Respondents are asked to rate how they have been feeling or acting
recently (e.g., “I felt miserable or unhappy”) on a 3-point Likert scale: 0 “not true,” 1 “sometimes
true,” 2 “true.” Total scores for the MFQ typically range from 0 to 66. Due to cultural sensitivi-
ties, four suicidality items from the original MFQ-C were administered only to participants
who were deemed at-risk for self-harm. Therefore, the psychometric properties of our modi-
fied version of the MFQ-C was examined considering the remaining 29-item scale, with scores
ranging from 0 to 58. Moreover, one school-related item (“I didn’t have any fun in school”),
was not administered to adolescents not attending school and was conservatively coded as “0”
for those participants. The Short Mood and Feelings Questionnaire (SMFQ-C) consists of
13-items from the MFQ-C that assess affective and cognitive symptoms of depression [36], 4 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan with total scores ranging from 0 to 26. Higher scores indicate more severe depressive symp-
toms on both long and short MFQ measures. The Strengths and Difficulties Questionnaire (SDQ) is a 25-item questionnaire which has
been originally validated among children 4 to 16 years old and contains five subscales: emo-
tional symptoms, conduct problems, hyperactivity, peer problems, and prosocial behaviour
[22, 43]. Higher scores indicate greater difficulties on all scales except for the prosocial behav-
iour subscale. The parent SDQ translated to Urdu was previously validated in an urban, clini-
cal sample of youth ages 4 to 16 years old in Pakistan and displayed adequate discriminant
validity across all subscales [22]. The Screen for Child Anxiety Related Emotional Disorders-child version (SCARED-C)
was used to assess symptoms of anxiety and its expected correlation with depressive symptoms. It has been validated for use in children 8 to 18 years old and consists of 41 items relating to
five distinct subscales: separation anxiety, panic/somatic symptoms, social anxiety, generalized
anxiety, and school avoidance [44, 45]. Items describe first-person statements (e.g., “I am ner-
vous”). Measures Adolescents were asked to rate items on a 3-point Likert scale: 0 “not true or hardly ever
true”, 1 “somewhat or sometimes true”, 2 “very true or often true”. The SCARED-C has not pre-
viously been validated in Pakistan, but has shown adequate face and content validity, and good
internal consistency in a community sample of rural youth living in India [46]. Statistical analyses All analyses were conducted in R studio (RStudio Team, 2015). The significance threshold was
set at α = 0.05. The data were assessed for missing observations. Descriptive analyses for demo-
graphic variables and scale items were conducted, including examining correlations (using
Spearman correlations for ordinal variables). The Kaiser-Meyer-Olkin (KMO) measure of
sample adequacy was used to assess whether the sample was adequate for factor analysis
(KMO 0.60 is acceptable) [47]. The factor structure and psychometric properties of the modified MFQ-C and the SMFQ-C
were assessed using confirmatory factor analyses (CFA). Due to the ordinal nature of the MFQ
scale items, the mean-and-variance adjusted weighted least squares (WLSMV) estimator was
used [48]. For the modified MFQ-C, we considered a unidimensional structure (29-items) as
well as a four-factor solution first described by Jeffreys et al., (2016) comprising of core mood
symptoms (4 items), vegetative symptoms (6 items), agitated distress (4 items), and cognitive
symptoms (12 items; 26-items total). In accordance with Jeffreys et al. (2016)’s study, three
items were not included in the four-factor solution (“I ate more than usual”, “I did not want to
see friends”, and “I worried about aches and pains”). Moreover, one item originally included in
Jeffreys et al. (2016)’s solution (“she or he wasn’t as happy as usual”) was specific to the MFQ
parent version. As this item is not included in the MFQ-C, it was not considered in the present
analysis. Regarding the SMFQ-C, we considered the unidimensional solution supported by
previous studies [36]. Item factor loadings below 0.32 were not interpreted and a threshold of 0.70 was used to
characterize items with high factor loadings [47]. Model fit was assessed using the Compara-
tive Fit Index (CFI 0.90 indicates adequate and 0.95 indicates good fit); Tucker- Lewis
index (TLI 0.90 for adequate and 0.95 for good fit), root mean square error of approxima-
tion (RMSEA 0.08 for adequate and RMSEA 0.06 for good fit) [49]. Chi-Square/Degrees
of Freedom (χ2 /df) was reported for each model but the χ2 test was not used to assess model
fit due to its sensitivity to large sample sizes [50]. Subgroup analyses were conducted separately
for males and females and measurement invariance analyses were conducted to determine
whether or not the results from the CFAs differed based on sex. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 Description of the MFQ scales The mean total score on our modified 29-item MFQ-C scale was 5.6 (SD = 6.6) for the total
sample and ranged from 0 to 50. Mean total scores were significantly higher in females
(M = 5.9, SD = 7.0) than males (M = 5.2, SD = 6.2, W = 173801, p< 0.001). The mean total
score on the SMFQ-C was 2.5 (SD = 3.4) for the full sample and ranged from 0 to 24 with
females also scoring significantly higher (M = 2.7, SD = 3.5) than males (M = 2.3, SD = 3.2,
W = 207147, p< 0.001). Total score distributions for the full sample were right-skewed
(MFQ-C: skew = 2.28, kurtosis = 6.65; SMFQ-C: skew = 2.18, kurtosis = 5.84). Spearman’s cor-
relations between items are available in Fig B in S1 File. Statistical analyses Configural (no constraints), 5 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan metric (constrained loadings), and scalar (constrained loadings and thresholds) models were
examined. Changes in model fit indices and Δ χ2 were assessed for evidence of measurement
invariance [49, 51]. To evaluate internal consistency, which provides an indication of the extent to which the
items of a scale measure the same construct [52]. Cronbach’s alpha was calculated for the full
instrument and each identified subscale. Corrected item-total correlations and Cronbach’s
alpha when removing each item (α-i) were also assessed. Corrected item-total correlations
were considered adequate when equal or higher than 0.30 [53]. Convergent and divergent validity were examined using Spearman’s rank correlations
between the MFQ-C/SMFQ-C and other scales. Depression symptoms often overlap with anx-
iety and emotional symptoms. All have been shown to cluster together on the larger construct
of symptoms of internalising disorders, separately from symptoms of externalizing disorders
[54, 55]. In accordance with this conceptualization, convergent validity was assessed using the
SCARED-C and the SDQ emotional symptoms subscale scores and divergent validity was
examined using scores on the SDQ conduct problems, hyperactivity and prosocial behaviour
subscales. Additional analyses were also conducted to compare the results of the main analysis
to the results obtained in the subgroup of participants who attended school (n = 850) and
assess whether school attendance may have affected our findings. Sample characteristics Only one participant was excluded from our analyses due to missing responses to all MFQ
items (see Fig A in S1 File for participant flow-chart). All other participants had complete data
on the MFQ. The final sample consisted of 1350 participants (52.3% female). Participants’ demographics are presented for the whole sample and by sex in Table 1. The
mean age of participants was 12.4 (SD = 1.7) years, 12.8 (SD = 1.6) years for males, and 12.0
(SD = 1.7) years for females. The majority of participants attended school and lived in rural
areas. Compared to males, females were younger, were less likely to attend school but more
likely to live in an urban area. Their mothers were also slightly younger and they were more
likely to live in a Muslim family (all p0.01, see Table 1). Factor structure of the MFQ-C and the SMFQ-C The overall KMO measure of sampling adequacy was 0.93 for the full scale MFQ and 0.88 for
the SMFQ, indicating that the sample was suitable for factor analysis. Fit indices for all models
are presented in Table 2. The unidimensional structure from the original MFQ-C (29 items)
showed adequate fit (CFI = 0.94, TLI = 0.94, RMSEA = 0.06) with item factor loadings ranging
from 0.34 (“I didn’t have any fun in school”) to 0.97 (“Talking more slowly”) (Fig 1). However, 6 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan Table 1. Demographic and family characteristics of participants. https://doi.org/10.1371/journal.pgph.0000968.t001 Factor structure of the MFQ-C and the SMFQ-C Fit indices for confirmatory factor analyses models of the Mood and Feelings Questionnaire (MFQ-C) and Short Mood and Feelings Questionnaire
(SMFQ-C). MFQ-C
SMFQ
Fit Index
Four-Factor (26 items)
Unidimensional (29 items)
Unidimensional
CFI
0.971
0.941
0.969
TLI
0.968
0.937
0.963
RMSEA
0.047
0.059
0.069
[95% CI]
[0.044, 0.050]
[0.057,0.062]
[0.063, 0.075]
χ2 /df
3.98
5.71
7.45
Note: Robust estimates are reported. CFI = Comparative Fit Index; TLI = Tucker-Lewis Index; RMSEA = Root Mean Square Error of Approximation; CI = Confidence
Interval; χ2 = Chi-Square Statistic; df = degrees of freedom. ctor analyses models of the Mood and Feelings Questionnaire (MFQ-C) and Short Mood and Feelings Questionnaire Table 2. Fit indices for confirmatory factor analyses models of the Mood and Feelings Questionnaire (MFQ-
(SMFQ-C). Table 2. Fit indices for confirmatory factor analyses models of the Mood and Feelings Questionnaire (MFQ-C) and Shor
(SMFQ C) ood and Feelings Questionnaire (MFQ-C) and Short Mood and Feelings Questionnaire omparative Fit Index; TLI = Tucker-Lewis Index; RMSEA = Root Mean Square Error of Approximation; CI = Confidence
es of freedom. Note: Robust estimates are reported. CFI = Comparative Fit Index; TLI = Tucker-Lewis Index; RMSEA = Root Mean Square Error of Approximation; CI = Confidence
Interval; χ2 = Chi-Square Statistic; df = degrees of freedom. the 26-item four-factor model showed an excellent and overall better fit than what was
observed for the unidimensional factor model (CFI = 0.97, TLI = 0.97, RMSEA = 0.05). All fac-
tor loadings were greater than the specified cut-off of 0.32 [47] (Fig 2). Again, the lowest factor
loading was observed for the item “I didn’t have any fun in school” on the ‘cognitive’ subscale
(0.35). In a sensitivity analysis conducted in the subgroup of participants who attended school,
this item loaded higher but still had the lowest factor loading (0.46), and there was minimal
change in model fit indices. Finally, the unidimensional model for the SMFQ-C also showed
excellent fit (CFI = 0.97, TLI = 0.96, RMSEA = 0.07), with all factor loadings being greater
than 0.49 (Fig 1). Measurement invariance across sexes was assessed for the MFQ-C four factor and for the
SMFQ-C unidimensional models. There was evidence of measurement invariance. The Measurement invariance across sexes was assessed for the MFQ-C four factor and for the
SMFQ-C unidimensional models. There was evidence of measurement invariance. The Fig 1. Factor structure of the MFQ-C and the SMFQ-C Participant’s Sex
Male
(N = 644)
Female
(N = 706)
Overall
(N = 1350)
Variable
N(%) or M[SD]
N(%) or M[SD]
N(%) or M[SD]
W or χ2
Participant age, years
12.8 [1.6]
12.0 [1.7]
12.4 [1.7]
W = 289414, p<0.001
School Attendance
No
160 (24.8)
340 (48.2)
500 (37.0)
χ2
(1) = 77.50, p<0.001
Yes
484 (75.2)
366 (51.8)
850 (63.0)
Urbanicity
Urban
31 (4.8)
84 (11.9)
115 (8.5)
χ2
(2) = 21.70, p <0.001
Rural
521 (80.9)
530 (75.1)
1051 (77.9)
Peri Urban
92 (14.3)
92 (13.0)
184 (13.6)
Mother’s Age, years
40.2 [5.8]
39.2 [5.8]
39.7 [5.8]
W = 251143, p<0.001
Mother’s Native Language
Urdu
7 (1.1)
10 (1.4)
17 (1.3)
χ2
(2) = 0.68, p = 0.75
Sindhi
618 (96.0)
671 (95.0)
1289 (95.5)
Other
19 (3.0)
25 (3.5)
44 (3.3)
Mother’s marital status
Married
607 (94.3)
656 (92.9)
1263 (93.6)
χ2
(1) = 1.03, p = 0.60
Widowed/
divorced/separated
37 (5.7)
50 (7.1)
87 (6.4)
Mother’s Parity
6.56 [2.6]
6.35 [2.5]
6.45 [2.5]
W = 236012, p = 0.22
Presence of Father at Home
Living together
604 (93.8)
657 (93.1)
1261 (93.4)
χ2
(2) = 0.32, p = 0.85
Living elsewhere
10 (1.6)
13 (1.8)
23 (1.7)
Died
30 (4.7)
36 (5.1)
66 (4.9)
Mother’s Employment
Unskilled labour
15 (2.3)
15 (2.1)
30 (2.2)
χ2
(6) = 5.14, p = 0.53
Skilled labour
219 (34.0)
206 (29.2)
425 (31.5)
Agriculture
6 (0.9)
8 (1.1)
14 (1.0)
Domestic service
8 (1.2)
12 (1.7)
20 (1.5)
Professional
8 (1.2)
7 (1.0)
15 (1.1)
Stay-at-home parent
381 (59.2)
446 (63.2)
827 (61.3)
Sales/Other
7 (1.1)
12 (1.7)
19 (1.4)
Father’s Employment
Unskilled labour
21 (3.3)
18 (2.5)
39 (2.9)
χ2
(6) = 0.84, p = 0.99
Skilled labour
467 (72.5)
509 (72.1)
976 (72.3)
Agriculture
38 (5.9)
44 (6.2)
82 (6.1)
Sales
26 (4.0)
30 (4.2)
56 (4.1)
Professional
58 (9.0)
67 (9.5)
125 (9.3)
Unemployed
7 (1.1)
7 (1.0)
14 (1.0)
Other
27 (4.2)
31 (4.4)
58 (4.3)
Family’s Religion
Muslim
520 (80.7)
608 (86.1)
1128 (83.6)
χ2
(1) = 6.69, p = 0.01
Hindu
124 (19.3)
98 (13.9)
222 (16.4)
Note: χ2 = Chi-Square statistic; W = Wilcoxon W statistic. Table 1. Demographic and family characteristics of participants. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 7 / 18 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan Table 2. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 Factor structure of the MFQ-C and the SMFQ-C Factor loadings from the unidimensional confirmatory factor analysis of the modified Mood and Feelings
Questionnaire (child-version, 29 items) and the Short Mood and Feelings Questionnaire (child-version, 13 items
https://doi.org/10.1371/journal.pgph.0000968.g001 Fig 1. Factor loadings from the unidimensional confirmatory factor analysis of the modified Mood and Feelings
Questionnaire (child-version, 29 items) and the Short Mood and Feelings Questionnaire (child-version, 13 items). https://doi.org/10.1371/journal.pgph.0000968.g001 8 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan Fig 2. Factor loadings from the four-factor solution in confirmatory factor analysis applied to data obtained with
the modified Mood and Feelings Questionnaire (child-version, 26-items). https://doi.org/10.1371/journal.pgph.0000968.g002 Fig 2. Factor loadings from the four-factor solution in confirmatory factor analysis applied to data obtained with
the modified Mood and Feelings Questionnaire (child-version, 26-items). https://doi.org/10.1371/journal.pgph.0000968.g002 Fig 2. Factor loadings from the four-factor solution in confirmatory factor analysis applied to data obtained with
the modified Mood and Feelings Questionnaire (child-version, 26-items). https://doi.org/10.1371/journal.pgph.0000968.g002 changes in χ2 /DF across the configural, metric, and scalar models were non-significant and
models had acceptable fit for males and females (see Table A in S1 File). changes in χ2 /DF across the configural, metric, and scalar models were non-significant and
models had acceptable fit for males and females (see Table A in S1 File). PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 Reliability Cronbach’s alphas and corrected-item total correlations are presented in Table 3. Internal con-
sistency for the total MFQ-C score was excellent (α = 0.92) and the corrected item-total corre-
lations were adequate ( 0.30) for all items except for: “I didn’t have any fun in school (r =
0.23) and “I slept a lot more than usual” (r = 0.24). Regarding MFQ-C subscales, internal consistency was good for the ‘cognitive’ and the ‘veg-
etative’ subscales (respectively α = 0.87 and α = 0.81) and acceptable for the ‘core mood’ and
‘agitated distress’ subscales (respectively α = 0.62 and α = 0.66). Again, low item-total correla-
tions were found for the items “I didn’t have any fun in school” (r = 0.17) on the ‘cognitive’
scale and “I slept a lot more than usual” on the ‘vegetative’ subscale (r = 0.24). Removal of these
items only slightly improved the internal consistency of their corresponding subscales. In sen-
sitivity analyses conducted among adolescents attending school, the item-total correlation for
the item “I didn’t have any fun in school” remained modest on the unidimensional scale
(r = 0.31) and the “vegetative” subscale (r = 0.28). 9 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan Table 3. Reliability indices for items of the Mood and Feelings Questionnaire and the Short Mood and Feelings
Questionnaire. MFQ-C
MFQ-C
SMFQ
(four-factor
structure)
(unidimensional)α
= 0.92
(unidimensional)α
= 0.87
Item
r c
it
α–i
r c
it
α–i
r c
it
α–i
Core mood
α = 0.62
1. Miserable or unhappy
0.48
0.48
0.56
0.91
0.56
0.86
2. Didn’t enjoy
0.37
0.57
0.56
0.91
0.51
0.86
11. Grumpy with parents
0.36
0.58
0.41
0.91
14. Cried a lot
0.38
0.57
0.35
0.91
0.36
0.87
Vegetative
α = 0.81
3. Less hungry
0.41
0.81
0.44
0.91
5. Sat and did nothing
0.58
0.77
0.64
0.91
0.56
0.86
6. Moving/walking slowly
0.73
0.74
0.68
0.91
12. Talking less
0.72
0.74
0.67
0.91
13. Talking more slowly
0.74
0.73
0.70
0.91
29. Slept more than usual
0.24
0.85
0.24
0.91
Cognitive
α = 0.87
8. Felt no good
0.60
0.85
0.61
0.91
0.63
0.86
9. Blamed self
0.30
0.87
0.35
0.91
15. Nothing good in future
0.55
0.86
0.58
0.91
18. Bad things happen
0.60
0.85
0.57
0.91
19. Reliability Hated myself
0.76
0.84
0.67
0.91
0.67
0.85
20. Felt like bad person
0.75
0.84
0.67
0.91
0.64
0.85
21. Looked ugly
0.66
0.85
0.61
0.91
23. Felt lonely
0.52
0.86
0.59
0.91
0.55
0.86
24. Nobody loved me
0.50
0.86
0.49
0.91
0.50
0.86
25. No fun in school
0.17
0.88
0.23
0.91
26. Not as good other kids
0.55
0.86
0.49
0.91
0.52
0.86
27. Did everything wrong
0.64
0.85
0.65
0.91
0.62
0.86
Agitated distress
α = 0.66
7. Restless
0.37
0.64
0.47
0.91
0.45
0.86
10. Hard to makeup mind
0.54
0.52
0.58
0.91
17. Hard to concentrate
0.49
0.55
0.56
0.91
0.51
0.86
28. Didn’t sleep as well
0.35
0.66
0.51
0.91
Items not included in the four factor solution
4. Ate more
0.30
0.91
16. Did not want to see friends
0.47
0.91
22. Worried about aches and pain
0.32
0.91
Note: r c
it = Corrected item-total correlation; α = Cronbach’s alpha; α–i = Cronbach’s alpha if item is removed Table 3. Reliability indices for items of the Mood and Feelings Questionnaire and the Short Mood and Feelings
Questionnaire. The internal consistency of the SMFQ-C was good (α = 0.87). Item-total correlations ran-
ged from 0.36 (“I cried a lot”) to 0.67 (“I hated myself”) and Cronbach’s alpha when items were
removed remain comparable (α-i between 0.85 and 0.87). the SCARED-C subscales (Table 4). Similar results were observed for the SMFQ-C total
scores. the SCARED-C subscales (Table 4). Similar results were observed for the SMFQ-C total
scores. In terms of divergent validity, the MFQ-C scale, its core mood, vegetative, and agitated dis-
tress subscales and the SMFQ-C scale demonstrated a small but significant negative correlation
with the SDQ-prosocial behaviour subscale (-.20 ρ -.10). However, no significant correla-
tion was found between the SDQ-prosocial behaviour and the MFQ-C cognitive symptoms
subscales. Surprisingly, the MFQ-C total and subscale scores, as well as the SMFQ-C scores,
demonstrated low-to-moderate correlations with scores on the SDQ-hyperactivity and SDQ-
conduct problems subscales (.08 ρ .39). The magnitude of the correlations varied greatly
across the MFQ-C subscales, with a moderate correlation observed for core mood symptoms
and low correlations observed for the vegetative, cognitive and agitated distress subscales. Convergent and divergent validity Convergent validity was supported by moderate-to-strong positive correlations between
MFQ-C (total and subscale) scores and scores on the SDQ-emotional symptoms and on PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 10 / 18 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan Table 4. Spearman’s correlations between the Mood and Feelings Questionnaire scales and subscales and similar psychometric scales. Scale
N
MFQ-C
SMFQ-C
Core Mood
Vegetative
Cognitive
Agitated Distress
total
total
SDQ
Emotional symptoms
1350
0.53
0.50
0.46
0.36
0.38
0.47
Conduct problems
1350
0.34
0.33
0.39
0.08
0.20
0.23
Hyperactivity
1350
0.39
0.36
0.30
0.36
0.34
0.34
Prosocial behaviour
1350
-0.17
-0.16
-0.20
-0.11
-0.04
-0.10
SCARED
Panic/somatic
1336
0.57
0.54
0.46
0.41
0.45
0.51
Social anxiety
1323
0.40
0.38
0.31
0.31
0.31
0.35
Separation anxiety
1289
0.49
0.46
0.38
0.37
0.38
0.45
Generalized anxiety
1326
0.64
0.63
0.53
0.38
0.55
0.55
School avoidance
850
0.49
0.44
0.41
0.37
0.40
0.45
Note
<0.05
<0.001; MFQ-C = Mood and Feelings Questionnaire, child version (29 items); SMFQ-C = Short Mood and Feelings Questionnaire, child version (13 items);
SDQ = Strengths and Difficulties Questionnaire; SCARED = Screen for Child Anxiety Related Emotional disorders. All MFQ and SCARED scales were child-report. SDQ subscales were parent-report. tions between the Mood and Feelings Questionnaire scales and subscales and similar psychometric scales. <
<0.001; MFQ-C = Mood and Feelings Questionnaire, child version (29 items); SMFQ-C = Short Mood and Feelings Questionnaire, child version (13 items);
SDQ = Strengths and Difficulties Questionnaire; SCARED = Screen for Child Anxiety Related Emotional disorders. All MFQ and SCARED scales were child-report. SDQ subscales were parent-report. <0.001; MFQ-C = Mood and Feelings Questionnaire, child version (29 items); SMFQ-C = Short Mood and Feelings Questionnaire, child version (13 items);
SDQ = Strengths and Difficulties Questionnaire; SCARED = Screen for Child Anxiety Related Emotional disorders. All MFQ and SCARED scales were child-report. SDQ subscales were parent-report. https://doi.org/10.1371/journal.pgph.0000968.t004 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 <0.05
<0.001; MFQ-C = Mood and Feelings Questionnaire, child version (29 items); SMFQ-C = Short Mood and Feelings Questionnaire, child version (13 items);
SDQ = Strengths and Difficulties Questionnaire; SCARED = Screen for Child Anxiety Related Emotional disorders.
All MFQ and SCARED scales were child-report. SDQ subscales were parent-report. Discussion Our study is the first to examine the psychometric properties and factor structure of instru-
ments assessing depressive symptoms in early adolescents from a community sample in rural
Pakistan. Though Urdu is considered the national language, Pakistan is a multilingual country
with each province linked to a specific ethnic group with its own language [56]. Sindhi is the
first language of over 30 million people in Pakistan [24] yet there are no psychometric scales
that are available in the language which may be problematic, particularly in rural communities
where bilingualism is less common. Our results showed that the modified MFQ-C and the
standard SMFQ-C translated to Sindhi have good psychometric properties in a primarily rural
population of adolescents. To our knowledge, our study is also the first to examine the multiple-factor structure of the
child version of the MFQ. We found the MFQ-C unidimensional structure to be adequate but
the four-factor structure comprising core mood, vegetative, cognitive and agitated distress
symptoms best fit the data. Past research has examined the factor structure of the MFQ-C in
HICs, with all supporting the original unidimensional solution, however, alternative structures 11 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan have not been considered [19, 57]. The hypothesis of a five-factor solution was developed with
data collected using the MFQ-P. It was first suggested by Jeffreys et al. (2016) and has since
been supported in a study based on the Spanish version of the MFQ-P, in addition to a modi-
fied 33-item single-factor structure [28, 33]. Similar to our findings, both the unidimensional
and the five-factor solution fit the data well, however, a slightly better fit was observed for the
five-factor solution [28]. Four items of the original MFQ-C, assessing suicidal behaviours, were not included in the
modified version of the tool used in the Nash-wo-Numa Study. The exclusion of these items
reflects the cultural norm of youth in younger age groups being accompanied by a parent or
chaperone when sensitive questionnaires are administered in Pakistan or in other countries
where suicidal behaviour is criminalized. Administering these questions in community sam-
ples may result in unnecessary distress and may impact accurate disclosure [17]. Discussion However, the
need for this adaptation is not limited to cultural considerations and can also be necessary
when adequate follow-up cannot be provided to participants who endorse suicidal items. In a
study conducted in a sample of youth from the United States, three suicidality related-items of
the MFQ-C were eliminated for this reason [19]. Similar to our results, the unidimensional
solution was still supported for the 30-item scale [19]. Though the exclusion of suicidality
items can be justified in various contexts and cultures, their exclusion prevents cross-cultural
comparisons from being made when total scores are computed as normative cut-off values
cannot be applied. In this case, a four-factor solution is particularly useful when examining the
MFQ factor structure as it allows for the exclusion of the four suicidality items contained on a
single subscale, but does not prevent reasonable comparisons to be made across the remaining
subscales [33]. As such, we recommend that future users of the scale examine the subscale
scores as opposed to total scores from the 29-item MFQ. Factor loadings and item-total correlations for the item “I didn’t have fun in school” in both
the unidimensional and four-factor structures of our modified MFQ-C were lower than those
observed in HICs [28, 33]. Similar findings were noted in the subgroup of participants attend-
ing school. For comparability, we retained the item in our analyses but future research regard-
ing the usefulness of inquiring about school-related symptoms when assessing depression in
LMICs is warranted. The reliability of the full-scale MFQ-C in this sample was similar to previously reported
estimates from other LMICs (α = 0.90–0.92) [27, 32]. Our results were also comparable to that
of studies examining the MFQ-P, with the exception of the ‘core mood’ subscale in which the
internal consistency (α = 0.62) was lower than previously observed (α = 0.78) [33]. This dis-
crepancy may be attributed to the number of items included in the ‘core mood’ subscale,
which differs between the MFQ-P (five items) and the MFQ-C (four items). Our results provide strong support for the unidimensional structure of the SMFQ-C and its
reliability and validity in a sample of youth in Pakistan. The internal consistency and unidi-
mensional factor structure of the SMFQ-C was in accordance with results obtained in past
studies conducted in LMICs [41, 58]. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 Discussion These findings provide strong support for the use of this
brief measure in community-based and/or low-resource settings in which long-form question-
naires are not feasible to administer. However, the mean SMFQ-C scores were lower in our
sample compared to what was observed in a similar sample of adolescents in Bangladesh (M =
8.67, SD = 5.61) [41]. This discrepancy may reflect a true difference in the severity of depres-
sive symptoms in adolescents living in rural Pakistan compared to those living in Bangladesh. Alternatively, the variation could be attributed to the younger age range and the use of random
sampling in the present study, or because participants were not interviewed alone. Both
depressive symptomatology and risky behaviours have been negatively associated with 12 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan disclosure when an adult is present, thus adolescents may have underreported depressive
symptoms in our study [59]. The convergent validity of our modified MFQ-C (full scale and subscales) and the SMFQ-C
was supported. The observed positive correlations with scores on the SDQ-emotional symp-
toms subscale are consistent with past research examining the MFQ-C and MFQ-P [27, 28]. Similarly, correlations between the SCARED-C subscales and the SMFQ-C and MFQ-C total
score and subscale scores were consistent with past research examining the MFQ-P [33] and
are in agreement with the body of literature emphasizing the high comorbidity between
depression and anxiety [60]. The SDQ-emotional symptoms and SCARED-C do not measure
the exact same constructs as the MFQ-C and SMFQ-C, which explains the moderate-to-strong
correlations observed. However, these correlations reflect the expected associations between
depressive symptoms emotional problems and anxiety symptoms, suggesting that the MFQ-C
total and subscale scores and SMFQ-C scores effectively capture the characteristics shared
between these constructs. Regarding divergent validity, surprisingly significant correlations were found between
scores on the MFQ-C (full scale and subscales), the SMFQ-C, and scores on the SDQ-conduct
problems and SDQ-hyperactivity subscales. However, the associations observed between
depressive symptoms and conduct problems are in agreement with previous results obtained
in community samples from HICs and could be explained by the overlap of symptoms of both
conditions, by their mutual influence on one another, or by shared underlying etiological fac-
tors such as socioeconomic factors [61]. Discussion In LMICs, this association may be even stronger as
lower socioeconomic status has been associated with both higher levels of depression [62] and
conduct problems [63]. The significant correlations observed between hyperactivity and
depressive symptoms has also been noted in past research [27]. This association could be
attributed to the similarity in items between the two scales. The SDQ-hyperactivity subscale
enquires about problems such as restlessness, fidgeting, being easily distracted, and finishing
tasks [43] much of which is reflected in the MFQ-C. However, divergent validity was sup-
ported through low negative correlations between all MFQ-C scales and SDQ- prosocial
behaviour, analogously to results of past research examining the MFQ-P [28]. The main strength of our work lies in the use of a representative sample and of standardized
methods that limit selection and information biases and allow us to generalize our results to
the population of adolescents living in Matiari. Nevertheless, our findings should be consid-
ered in light of several limitations. Firstly, because our modified version of the MFQ-C
excluded four suicidal items, the total scores were computed by summing the score of 29
items, rather than 33. As such the mean total MFQ-C score obtained in our sample cannot be
compared to scores obtained in other studies. Secondly, in accordance with Jeffreys et al. (2016)’s analysis, our four-factor solution did not include the following three items: “I ate
more than usual”, “I worried about aches and pains” and “I did not want to see friends”. Modest
item-total correlations were found for “I ate more than usual” and “I worried about aches and
pains” when assessing the unidimensional structure of our MFQ-C (0.30 and 0.32, respec-
tively), with similar findings also noted in a Danish school-based study [33, 38]. Further, in a
study conducted in Thailand, the item-total correlation for the item “I ate more than usual”
was also low (r = 0.20) [32]. This item may not be as relevant in low socioeconomic status pop-
ulations as it is in HICs when assessing depressive symptoms, particularly when the prevalence
of food insecurity is high. In the rural districts of the province of Sindh, food insecurity is esti-
mated to affect 52.4% of adolescents [64]. However, we cannot exclude the possibility of these
three items being clinically relevant in this population. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 Conclusion Overall, both the Sindhi MFQ-C and SMFQ-C appear to be reliable measures of depressive
symptoms in early adolescents living in rural Pakistan. While the unidimensional structures of
the MFQ-C and SMFQ-C provide a global measure of depressive symptom severity, the symp-
tom-specific subscale scores of the MFQ-C allow for the assessment of specific dimensions of
depressive symptoms. The brevity of the SMFQ-C further emphasizes its usefulness in low-
resource settings, where limited time and/or trained staff prevent long-form questionnaires
from being administered. Our findings also support the use of these instruments in future pop-
ulation-based studies, which may contribute to the surveillance and the identification of covar-
iates of adolescent depressive symptoms in rural Pakistan. Research regarding reasonable cut-
off scores that indicate probable depression in this population would provide further insight
into its usefulness as a diagnostic or screening tool. More generally, the MFQ questionnaires
may also be used in other LMIC populations that share similar characteristics and cultures. Acknowledgments We appreciate the ongoing assistance and support provided Fariha Shaheen, Imran Ahmed,
Arjumand Rizvi and the entire team at the Aga Khan University. We express gratitude for the
participating youth of Matiari and their families to advance adolescent research in rural
Pakistan. Discussion Thirdly, due to the cross-sectional
design of the study, we were unable to assess the test-retest reliability of the MFQ-C and
SMFQ-C. Fourthly, since a mostly rural community sample was examined, it is unclear if these PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 13 / 18 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan findings are generalizable to clinical or urban populations in Pakistan. Lastly, as no formal
diagnostic interviews were conducted, the discriminant validity of the modified Sindhi
MFQ-C and SMFQ-C could not be analyzed and meaningful cut-off scores could not be deter-
mined in our sample. Consequently, we recommend future users of these scales to consider
continuous scores, in addition to dichotomized measures of depression. Supporting information S1 File. Fig A. Participant Flow-chart. Fig B. Spearman’s Correlations between items from the
Mood and Feelings Questionnaire—child version. Table A. Measurement Invariance Analysis
by Sex of the Child Report Version of the 26-item Four Factor Mood and Feelings Question-
naire (MFQ-C) and the Short Mood and Feelings Questionnaire (SMFQ-C)—child versions. Table B. Factor loadings for the four-factor and unidimensional structure of the adapted
Mood and Feelings Questionnaire- child version and the unidimensional structure of the
Short Mood and Feelings Questionnaire-child version. (DOCX) S2 File. Mood and Feelings Questionnaire (Sindhi).
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ken, Daphne J. Korczak, Suneeta Monga, Sajid Bashir Soofi, Peter Szatmari, Zulfiqar A. Bhutta. PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
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17405629.2012.686740 52. Anselmi P, Colledani D, Robusto E. A Comparison of Classical and Modern Measures of Internal Con-
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scales Instead of the Hypothesised Five Subscales on the Strengths and Difficulties Questionnaire 17 / 18 PLOS Global Public Health | https://doi.org/10.1371/journal.pgph.0000968
November 17, 2022 PLOS GLOBAL PUBLIC HEALTH Psychometric properties of the MFQ in Pakistan (SDQ): Data from British Parents, Teachers and Children. J Abnorm Child Psychol [Internet]. 2010; 38
(8):1179–91. Available from: https://doi.org/10.1007/s10802-010-9434-x PMID: 20623175 56. Ashraf MA, Turner DA, Laar RA. Multilingual Language Practices in Education in Pakistan: The Conflict
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November 17, 2022 References Validity and Reliability of the Thai Version of the
Short Mood and Feelings Questionnaire. East Asian Arch Psychiatry. 2020; 30(2):48–51. https://doi. org/10.12809/eaap1875 PMID: 32611827 59. Herrera A v., Benjet C, Me´ndez E, Casanova L, Medina-Mora ME. How Mental Health Interviews Con-
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cence. Clin Child Fam Psychol Rev. 2006; 9(3–4):201–20. https://doi.org/10.1007/s10567-006-0011-3
PMID: 17053962 62. Lorant V, Deliège D, Eaton W, Robert A, Philippot P, Ansseau M. Socioeconomic Inequalities in
Depression: A Meta-Analysis. Am J Epidemiol [Internet]. 2003 Jan 15; 157(2):98–112. Available from:
https://doi.org/10.1093/aje/kwf182 PMID: 12522017 63. Piotrowska PJ, Stride CB, Croft SE, Rowe R. Socioeconomic status and antisocial behaviour among
children and adolescents: A systematic review and meta-analysis. Clin Psychol Rev [Internet]. 2015;
35:47–55. Available from: https://doi.org/10.1016/j.cpr.2014.11.003 PMID: 25483561 64. Sheikh S, Iqbal R, Qureshi R, Azam I, Barolia R. Adolescent food insecurity in rural Sindh, Pakistan: A
cross-sectional survey. BMC Nutr. 2020; 6(1):1–9. https://doi.org/10.1186/s40795-020-00343-w PMID:
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Membrane separation techniques– removal of inorganic and organic admixtures and impurities from water environment – review
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Archives of Environmental Protection
Vol. 45 no. 4 pp. 4–19 © Copyright by Polish Academy of Sciences Membrane separation techniques
– removal of inorganic and organic admixtures
and impurities from water environment – review Michał Bodzek Institute of Environmental Engineering, Polish Academy of Sciences, Zabrze, Poland Institute of Environmental Engineering, Polish Academy of Sciences, Zabrze, Poland * Corresponding author’s e-mail: michal.bodzek@ipis.zabrze.pl Keywords: membrane techniques, water desalination and softening, natural organic matter, removal of inorganic
and organic micropollutants. Abstract: Introduction and development of membrane techniques in the production of drinking water and
purifi cation of wastewaters, in the last 40 years, was important stage in the fi eld of water treatment effectiveness. Desalination of sea and brackish water by RO is an established way for drinking water production. Signifi cant
improvements in design of RO, the application of alternative energy sources, modern pretreatment and new
materials have caused the success of the process. NF is the method of water softening, because NF membranes can
retain di- and multivalent ions, but to a limited extend monovalent. Drinking water containing viruses, bacteria and
protozoa, as well as other microorganisms can be disinfected by means of UF. Viruses are retained by UF membranes,
whereas bacteria and protozoa using both UF and MF membranes. For the removal of NOM it is possible to use
direct NF or integrated systems combining UF or MF with coagulation, adsorption and oxidation. The use of
NF, RO and ED, in the treatment of water containing micropollutants for drinking and industrial purposes, can
provide more or less selective removal of the pollutants. The very important are disinfection byproducts, residue
of pharmaceuticals and endocrine disrupting compounds. For endocrine disrupting compounds, special attention
is paid onto polycyclic aromatic hydrocarbons and surface-active substances, chlorinated pesticides, phthalates,
alkylphenols, polychlorinated biphenyls, hormones, synthetic pharmaceuticals and other substances disposed to
the environment. The application of MF and UF in the removal of inorganic and organic micropollutants is possible
in integrated systems with: coagulation, adsorption, complexion with polymers or surfactants and biological
reactions. PL ISSN 2083-4772
DOI 10.24425/aep.2019.130237 Archives vol 45 no 4 a4 srodki_kor 1.indd 4
Archives vol 45 no 4 a4 srodki_kor 1.indd 4 Introduction to the problem of water contamination is the development and
implementation of new technologies (Sozański et al. 2009),
and membrane separation techniques are highly recommended
(Bodzek and Konieczny 2010). Above 70% of Earth is covered with water, but 97.5% of this
is water with salt content above 1 g/L. Among the remaining
2.5% of water sources, 69% is glacier and permafrost ice,
while less <1% of total fresh water sources, i.e. around 0.01%
of total water sources, is available for humans (Bodzek and
Konieczny 2017). The disproportional distribution of water
sources is one of the most serious problems infl uencing the
growth and development of the societies. Water is a very
important raw material, its resources are renewable, but, on the
other hand, limited. The limited access to fresh water is mainly
caused by the pollution of the natural waters by industry and
agriculture as well as municipal wastewaters, increase of
human population accompanied with the growing life standard
and climate changes (Bodzek and Konieczny 2011a). In the treatment of drinking water and wastewater,
pressure-driven membrane techniques are frequently applied. The choice of an appropriate membrane process depends
on the types of effl uents and admixtures present in water
(Bodzek and Konieczny 2017, 2006). Reverse osmosis (RO)
retains monovalent ions and the majority of low-molecular
compounds and principally is used for water desalination/
demineralization and the removal of inorganic and organic
micropollutants. Nanofi ltration (NF) retains bivalent ions
and low-molecular organic/inorganic compounds and is
applied to water softening and removal of micropollutants. Ultrafi ltration (UF) and microfi ltration (MF) are able to
direct the removal of colloids, suspended substances and
microorganisms and can be used for water clarifi cation
and disinfection, but in integrated systems for the removal
of organic and inorganic pollutants/micropollutants. In
addition, other membrane techniques also are applied to The choice of the technology for water and wastewater
treatment is depended on its type. Ground water may be simply
purifi ed before its distribution to the water system, while
surface water and wastewater treatment are more complex due
to the presence of dangerous contaminants. The partial solution 2019-11-14 10:50:45
2019-11-14 10:50:45 Archives vol 45 no 4 a4 srodki_kor 1.indd 4
Archives vol 45 no 4 a4 srodki_kor 1.indd 4 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... Water desalination/demineralization Desalination of seawater and salty groundwater is a common
method of potable and industrial water production in regions
with dry climate. At the end of 2015, there were approximately
18,000 desalination plants worldwide, with a total production
capacity of 86.55 million m3/day (Voutchkov 2016). The
number of desalination installation constantly increases not
only in the Middle East or Northern Africa (44%), but also in
regions in which one would never suppose the possibility of
application the method, e.g. Spain and Australia (i.e. dry and
semi dry areas) (Ghaffour et al. 2013, Voutchkov 2016). Despite the fact that the desalination installations are
increasing fast, their price is still quite high for most of people,
but desalination is still a cheaper solution than e.g. long
distance water transport (Ghaffour et al. 2013, Subramani et al. 2011). Nowadays, investment costs of large water desalination
plants are comparable with costs of conventional drinking
water treatment plant (Bodzek and Konieczny 2011a). The scheme of a typical installation for water desalination
by RO is shown in Figure 2 (Al-Karaghouli and Kazmerski
2013, Bodzek and Konieczny 2017, 2011a, El-Ghonemy
2012, Wilf 2007). It is divided into: water intake, raw water
pretreatment, high-pressure pump with membrane modules
and energy recovery from retentate, and treatment of desalted
water before distribution sections. Seawater is an unlimited source of the feed for desalination
processes, and brackish water, most often collected from
ground water, is also important in water desalination. The salt
concentration in seawater varies from 24,000 to 42,000 mg/L,
depending on the localization (Table 1) (World Bank 2004). The concentration of salts in brackish water is much lower and
varies from 2,000 to 5,000 mg/L. The present RO installations are equipped with polyamide
composite membranes (TFC) which comprise of three layers:
polyester support layer (thickness 120–150 μm), microporous
transient layer i.e. polysulphone UF membrane (thickness about
40 μm) and a very thin active layer (skin thickness – 0.2 μm) Fig. 1. Performance of desalination installations according
to the type of feed Table 1. Salinity of seawater and brackish water
Water
Total salinity, g/m3
Oceans
35,000
The Red Sea
41,000
Persian Gulf
45,000
The Mediterranean Sea
38,000
Aral Sea
29,000
The Baltic Sea
7,000
Low saline brackish water
1,000–5,000
Highly saline brackish water
5,000–15,000 Table 1. Salinity of seawater and brackish water Fig. 1. Performance of desalination installations according
to the type of feed Table 2. Introduction 5 5 Desalination can be performed using different methods,
fi rst of all RO and ED as well as various types of distillation
(Bodzek and Konieczny 2017, El-Ghonemy 2012, Subramani
et al. 2011). The latest research shows that forward osmosis
can be used for desalination (Chung et al. 2012, Gryta 2012,
Linares et al. 2015, Subramani et al. 2011). Globally, 65% of
water produced via desalination is obtained using membrane
processes and 28% by means of thermal methods (Kaselain et al. 2019). In the last 10 years, seawater and brackish water reverse
osmosis desalination have come to dominate desalination
markets, at costs comparable to other fresh water production
methods (Malaeb and Ayoub 2011). Nowadays, more than
90% of RO installations are involved in the production of
drinking and potable water as well as to treatment of water for
energy, semiconductors, etc. Figure 1 shows the percentage of
RO installations capacities used for different types of water
and wastewater treatment (Bodzek and Konieczny 2017). The main desalination feed is seawater (55%), next brackish
ground water (25%), and the rest are surface and wastewater
(Ghaffour et al. 2013). However, this data is very unstable due
to the dynamics of desalination market. In Table 2 the most
important operational parameters of thermal and membrane
methods are compared (Fritzman et al. 2007). pressure-driven processes. For example, forward osmosis
(FO) is recently proposed for desalination and the removal
of some micropollutants and electrodialysis (ED) and liquid
membranes for inorganic compound removal. The above techniques can be used for the removal of
effl uents as independent process or combined with unit
processes, e.g. bioreactors, forming hybrid processes. Water desalination/demineralization Comparison of the operating parameters of thermal and membrane desalination methods
Parameter
MSF
RO
ED
Thermal energy demand, kWh/m3
12
–
–
Electrical energy demand, kWh/m3
35
0.4–0.7
1
Salinity of raw water, g/L
30–100
1–45
0.1–3
Desalted water, mg/L TDS
<10
<500
<500 ble 2. Comparison of the operating parameters of thermal and membrane desalination methods 2019-11-14 10:50:46
2019-11-14 10:50:46 Archives vol 45 no 4 a4 srodki_kor 1.indd 5
Archives vol 45 no 4 a4 srodki_kor 1.indd 5 M. Bodzek 6 Final
treatment
Membrane modules
High-pressure pump
Low-pressure
pump
Pretreatment
Raw
water
Desalted
water
Energy
recovery
turbine
Fig. 2. Performance of desalination installations according to the type of feed Low-pressure
pump Membrane modules Final
treatment Energy
recovery
turbine Desalted
water Fig. 2. Performance of desalination installations according to the type of feed (Lee et al. 2011). Active skin layer for membranes of new
generation contain nanomaterials, fi rst of all zeolites, nano-
-silica, carbon nanotubes, and graphene (Songa et al. 2018). Such membranes are characterized with higher permeability
compared to osmotic membranes used nowadays (Subramani
et al. 2011) at unchanged salt retention. In most RO desalination
installations, spiral wound membrane modules are used
(Al-Karaghouli and Kazmerski 2013, Bodzek and Konieczny
2011a). They characterize with better set of parameters like the
ratio of permeability to packing density, fouling control and
exploitation procedure, easy scaling up and others (Wilf 2007). There are four main companies which supply RO membrane
modules to industrial seawater desalination plants, i.e. DOW,
Toray, Hydranautics and Toyobo (Bodzek and Konieczny
2017, 2011a). overall energy demand to 2–4 kWh/m3 in the case of seawater
desalination, and to <1 kWh/m3 for brackish water treatment
systems (Fritzmann et al. 2007, Subramani et al. 2011). The permeate obtained from RO does not meet the standards
of drinking water, that is why it should be the treated. Water
dedicated to human consumption and to municipal purposes
is degassed (decarbonized), submitted to pH improvement
and disinfected before distribution or storage (Fritzmann et
al. 2007, Subramani et al. 2011). Another solution is water
remineralization using lime stone and alkalinity increase. One of the most crucial parameters of all desalination
technology is concentrate utilization or neutralization (Kim
2011, Perez-Gonzalez et al. 2012, Subramani and Jacangelo
2014), especially for plants localized far away from oceans. Brine may be deposited to oceans, surface water, deep wells
or special evaporation lagoons or may be introduced directly
to soil. Water desalination/demineralization The separate option is their treatment with the use of
RO, ED, thermal methods or their combination, membrane
distillation and FO (Bodzek and Konieczny 2011a, Kim 2011,
Perez-Gonzalez et al. 2012, Subramani and Jacangelo 2014). The main exploitation problem of RO installations is
fouling, which is the deposition of substances present in the
feed water on the membrane surface and/or inside membrane
pores. Fouling may be caused by microorganisms (biofouling),
precipitation of CaCO3, CaSO4 and BaSO4 (scaling) and
the formation of fi ltration cake by dissolved or suspended
substances (Bodzek and Konieczny 2011a, Gryta 2012). Fouling may have reversible character, if the deposit formed
on the membrane surface can be totally removed and the initial
capacity of the membrane can be restored. The irreversible
fouling is found when contamination of membrane takes
place inside membrane pores and thus the mechanical, but
also chemical cleaning is not suffi cient enough to recover the
initial effi ciency (Bodzek and Konieczny 2017, 2011a). In
order to limit the impact of fouling on the process effi ciency,
the pretreatment of feed is carried out. It may comprise all or
chosen operation units used in water treatment, i.e. coagulation
and fi ltration, adsorption, oxidation, fi nal fi ltration and so on
(Bodzek and Konieczny 2017, 2011b, 2005, El-Ghonemy
2014, Perez-Gonzalez et al. 2012). Recently, UF and MF
are reliable methods for removal of suspended substances,
some organic and microbiological contaminants, including
pathogens (Bodzek and Konieczny 2017, 2011b, Halpern et al. 2005). Modern desalination systems operated nowadays are
mostly confi gured into integrated (hybrid) units, among which
one can distinguish: (i) integration of RO with thermal methods,
(ii) use of NF for prevention of scaling, (iii) replacement of
conventional pretreatment with UF or MF, and (iv) combination
of RO with ion exchange or electro-deionization (EDI) to
production of deionized water (Ang et al. 2015, Bodzek and
Konieczny 2011b, Helal 2009). y
,
)
Desalination of water with the use of renewable energy is
already applied in some regions and may become more popular
in the nearest future. There are many combinations between
renewable energy sources and desalination processes (AbKadir et
al. 2010, Eltawil et al. 2009). In Figure 3 the possible desalination
systems with renewable energy are shown (Eltawil et al. 2009). Water desalination/demineralization The technological progress within the last 30–40 years
has decreased water desalination costs, due to the reduction
of materials and energy costs and energy consumption. The
detail comparison of different desalination plants is diffi cult
to make as actual costs depend on many variables, which are
specifi c for every investment. The costs of desalination mainly
depend on raw water quality, confi guration and capacity of the
installation, localization of the investment, accessibility and
price of electrical energy, concentrate utilization and others
(Bodzek and Konieczny 2011a, El-Ghonemy 2012, Ghaffour et
al. 2013). Table 3 presents costs of energy and water (average Desalination of water with the use of renewable energy is
already applied in some regions and may become more popular
in the nearest future. There are many combinations between
renewable energy sources and desalination processes (AbKadir et
al. 2010, Eltawil et al. 2009). In Figure 3 the possible desalination
systems with renewable energy are shown (Eltawil et al. 2009). The technological progress within the last 30–40 years
has decreased water desalination costs, due to the reduction
of materials and energy costs and energy consumption. The
detail comparison of different desalination plants is diffi cult
to make as actual costs depend on many variables, which are
specifi c for every investment. The costs of desalination mainly
depend on raw water quality, confi guration and capacity of the
installation, localization of the investment, accessibility and
price of electrical energy, concentrate utilization and others
(Bodzek and Konieczny 2011a, El-Ghonemy 2012, Ghaffour et
al. 2013). Table 3 presents costs of energy and water (average In modern installations for water desalination it is possible
to recover 30–40% of the energy from high pressure retentate
by means of special mechanical devices (Fritzmann et al. 2007,
Subramani et al. 2011). Special mechanical systems dedicated
to the transport of energy present in the retentate to the RO feed
water are used. The energy recovery systems can decrease the 2019-11-14 10:50:55
2019-11-14 10:50:55 Archives vol 45 no 4 a4 srodki_kor 1.indd 6
Archives vol 45 no 4 a4 srodki_kor 1.indd 6 Archives vol 45 no 4 a4 srodki_kor 1.indd 6
Archives vol 45 no 4 a4 srodki_kor 1.indd 6 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... 7 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... Water desalination/demineralization 7 multi-valent ions, as well as organics with a molecular weight
above 200–500 Da (Bodzek and Konieczny 2011a, 2006). Ion selectivity of NF membrane is based on the presence of
functional groups (mainly –COOH or –SO3H) with negative
charge on the surface or inside the pores of the membrane. Electrostatic interactions retain multivalent ions in retentate. This phenomenon is not observed for RO membranes. values) in commercial large scale desalination processes
(Bodzek and Konieczny 2017). Summing up, over the past several years, desalting
of seawater and brackish water became a method for the
production of water at a cost comparable to the cost of fresh
water production by other methods. The dominant techniques
used on a large scale are RO and MSF. Recently, research on
new desalination techniques has been carried out, especially
on membrane distillation and forward osmosis. Signifi cant
improvements in technology and process design, the availability
of alternative sources of energy, the possibility of pretreatment
and new materials used meant that the desalination process has
become ecologically-friendly source of fresh water in many
regions of the world, especially in those where their sources
are limited. After softening using NF, the salt concentration in permeate
is very low and, often, its remineralization is necessary. The
performed experiments confi rmed the applicability of NF
membranes for the softening of water (Bodzek 2002, Ghizellaoui
et al. 2005, Van der Bruggen et al. 2001, Wesolowska et al. 2002). They showed that it is possible to obtain soft waters and
low-hardness waters (total hardness ca.<200 mgCaCO3/L) from
very hard and hard waters (total hardness >300 mg CaCO3/L)
(Table 4) (Wesolowska et al. 2002). Also, high retention of
total hardness (>50%) and of carbonate hardness (>40%) as
well as calcium and magnesium ions were obtained, depending
on the type of membrane and raw water (Bodzek et al. 2002,
Wesolowska et al. 2002). Total hardness removal during NF
softening depends also on type of membrane used (Table 4),
so it is possible to choose the proper membrane for a given
type of water salinity and hardness (Anim-Mensah et al. 2008,
Homayoonfal et al. 2010, Van der Bruggen et al. 2001). Water softening Nanofi ltration process is an established and known method for
water softening, as an alternative to chemical and ion-exchange
(Bodzek 2002, Bodzek and Konieczny 2011a, Ghizellaoui
et al. 2005). The NF membranes are characterized with low
retention of monovalent ions and high retention of di- and Fig. 3. Distribution of renewable energy
in desalination process (PV – pervaporation,
MSF – multi-stage fl ash distillation, MED – multi-effect
distillation, MCVC – mechanical vapor compression) Izadpanah and Javidnia (2012) performed the experiments
with commercial spiral-wound NF membrane (pressure
range of 4–10 bar) to remove hardness and ions from diluted
seawater. The results show that this membrane is capable of
retaining 96–98% of the total hardness, and 79–89% of the
total dissolved solid (TDS). At the present time, NF is rather seen as a combinatory
process capable of removing hardness and a wide range of
other components in one step. Gorenfl o et al. (2002) examined
the NF-200B membrane for the treatment of groundwater with
high hardness and natural organic matter (NOM) content. The
results showed almost complete rejection of NOM (>95%)
and 74% and >86% rejection of Ca2+ and Mg2+ respectively. Also Orecki et al. (2004) studied surface water treatment by
NF using AFC30 membrane with cross-fl ow mode operation
and transmembrane pressure between 10–25 bars. They also Fig. 3. Distribution of renewable energy in desalination process (PV – pervaporation,
MSF – multi-stage fl ash distillation, MED – multi-effect
distillation, MCVC – mechanical vapor compression) Table 3. The cost of energy and water (average values) in commercial large scale desalination processes
Process
Heat
kWh/m3
Electricity
kWh/m3
Total energy
kWh/m3
Investment costs
USD/m3/d
Total cost of water
USD/m3
MSF
MED
SWRO
BWRO
7.5–12
4–7
–
–
2.5–4
1.5–2
(3–4)b
0.5–2.5
10–16
5.5–9
3–4
0.5–2.5
1200–2500
900–2000
900–2500
300–1200
(0.8–1.5)a
0.7–1.2
0.5–1.2
0.2–0.4
a Including grants (fuel price); b Including energy recovery system. Table 3. The cost of energy and water (average values) in commercial large scale desalination processes Table 4. Water softening results using NF membranes with different compactness
Membrane
NF-70
Filmtec
NF-45
Filmtec
UTC-20
Toray
UTC-60
Toray
Permeate hardness, mgCaCO3/L
14
114
14
59
Raw water hardness, mgCaCO3/L
280
280
280
280
Retention coeffi cient, %
95
59
95
79 Table 4. Inorganic micropollutants A number of inorganic anions (nitrate(V), chlorate(VII),
(V) and (III), bromate(V), arsenates(III) and (V), borate and
fl uoride) and heavy metals at harmful concentrations in natural
water sources and wastewaters can have adverse infl uence
on human health (Bodzek and Konieczny 2011a, b). The
permissible levels of these compounds, in drinking water and
wastewaters discharged to the environment, set by the WHO
and a number of countries are very low (in the range of μg/L to
a few mg/L). Thus, the majority of them can be considered as
charged micropollutants. Costs of membrane softening do not differ much from those
of the chemical softening, but membrane softening is more
benefi cial in view of the environment protection. Shahmansouri
and Bellona (2015) compared the costs of different options
for water softening and color removal. Unit water costs were
calculated for three alternative processes, i.e. lime and soda
ash softening, lime and soda ash softening + ozone injection
+ granular-activated carbon (GAC for color removal), and
softening using NF membranes. Results showed that for smaller
treatment plants (<150,000 m3/day), NF is a more cost-effective
method (water costs about 0.25 USD/m3), compared to lime and
soda ash softening system. For large softening facilities (i.e. systems with capacity>200,000 m3/day) water costs are smaller
(<0.25 USD/m3) and similar to other softening methods. Other
advantages of NF over lime softening include small footprint,
reduced chemical requirements, reduced chemical storage,
increased organic matter removal, and no sludge production. For highly colours water, NF membrane systems produce water
at a cheaper price compared to installations with lime soda,
ozonation, and GAC processes. Several treatment technologies, such as coagulation/
chemical precipitation – sedimentation – fi ltration, adsorption,
ion exchange, classical solvent extraction, evaporation and
biological methods, which are normally used for the removal of
inorganic micropollutants from natural waters or wastewaters,
represent a lot of exploitation problems. Membrane processes,
i.e. RO, NF, UF and MF in hybrid systems, and ED as well
as in liquid membranes and bioreactors, are also used for
the removal of micropollutamts (Bodzek 2015, Bodzek and
Konieczny 2011a, b, Crespo et al. 2004, Velizarov et al. 2004). In many cases, one membrane process can be integrated with
another system to produce high quality water. However, in
physical membrane processes, inorganic compounds are not
destroyed but concentrated. Therefore, post-treatment of the
concentrate stream can be diffi cult and costly in many cases. M. Bodzek 8 obtained complete removal of total organic carbon (TOC) and
reduced the SO4
2- content by approximately 90–99%, carbonate
by 82% and total hardness by 85.2%. Inorganic micropollutants The softening of water by means of UF or MF enhanced
with polymers consists in the complexing of metal ions and
polymers dissolved in water. The obtained complexes are
retained by UF membrane compared to unbounded metal
ions. The process was applied in the removal of metals from
diluted solutions. Natural and synthetic polyelectrolytes with
high content of carboxylic and/or amino groups are used as
complexing polymers, e.g. chitosan, poly(ethylamine) (PEI),
poly(diallyldimethylammonium)
chloride
(PDADMAC),
sodium salt of polyacrylic acid and others (Fatin-Rouge et al. 2006). Water softening Water softening results using NF membranes with different compactness Archives vol 45 no 4 a4 srodki_kor 1.indd 7
Archives vol 45 no 4 a4 srodki_kor 1.indd 7 2019-11-14 10:50:55
2019-11-14 10:50:55 M. Bodzek Removal of anions The RO process is highly effi cient in direct removal of
inorganic anions during drinking water treatment. However,
during the RO complete desalination is obtained, that is why
remineralization process of permeate is required (Bodzek
2015, Bodzek and Konieczny 2011a, b). NF enables the
partial desalination because it separates polyvalent from
monovalent ions with higher capacity at lower transmembrane
pressures (TMP) in comparison with RO process. Many
studies considering the removal of anions from natural waters
and purifi ed wastewaters by means of RO and NF have been
performed and promising results were obtained (Table 5)
(Bodzek 2015, Bodzek and Konieczny 2011a, b, Velizarov et
al. 2004). There is also the possibility of the application of ED and
EDI methods to eliminate the hardness from water (Bodzek
and Konieczny 2011a). A characteristic feature of the ED and
EDI processes is the ability to obtain a high degree of water
recovery. In addition, the EDI system is characterized by
a lower energy consumption and higher economic effi ciency
compared to the ED system. Table 5. RO and NF for removal of inorganic anions in the production of drinking water
Process, membrane and manufacturer
Anion
Feed
RO 4040-LHA-CPA2 (Hydranautics)
NO3
-
Natural water-188 mgNO3
-/L (South Africa)
RO, NF – different membranes (Osmonics)
NO3
-
Tap water (Poland)
RO, NF, UF – different membranes and manufacturer
As(V)
Pilot studies at various sites in USA
NF ES-10 (Nitto-Denko)
As(V)
Ground water – 0.6 mg As/L (Japan)
NF – different membranes (Dow Chemical and Nitto-Denko)
As(V)
Model water
NF Nanomax 50 (Millipore)
NO3
-
Model water
NF – different membranes (Nitto-Denko)
NO3
-
Surface water after MF (Japan)
NF Filmtec NF-70 (Dow Chemical)
NO3
-
Ground water (Belgium)
NF NF-300 (Osmonics)
NO3
-, F-
Ground water (California, USA)
NF Filmtec NF-45 and NF-70 (Dow Chemical)
F-
Model water
NF TFCS (Fluid Systems)
Cr(VI)
Model water Table 5. RO and NF for removal of inorganic anions in the production of drinking water 2019-11-14 10:52:26
2019-11-14 10:52:26 Archives vol 45 no 4 a4 srodki_kor 1.indd 8
Archives vol 45 no 4 a4 srodki_kor 1.indd 8 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... 9 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... Removal of anions 9 The use of MF and UF in the removal of inorganic
micropollutants is possible in integrated systems with:
coagulation, adsorption, complexing with polymers or surfactants
and biological reactions (Bodzek 2015, Bodzek and Konieczny
2011a, b). The interesting solution is the hybrid process of
sorption-membrane separation used for boron removal from
seawater or water after seawater desalination with RO. Boron is
removed by ion exchange resins (e.g. Dowex XUS 43594 – Dow
Chemicals, Diaion CRB01 – Mitsubishi or others) of very small
grain size (20 μm). Next resin is separated by means of MF. The
small size of grains of the resin allows to effectively decrease
the boron content from 2 to 0.243–0.124 mg/L, depending on
ion exchanger dose (0.25 to 1.0 g/L) (Dilek et al. 2002). Studies
have also been performed on the removal of boron from water
solutions using UF enhanced with polymers (PEUF), usually
with poly(vinyl alcohol) (PVA) or other polymers (Dilek et al. 2002). The process consists of the complexation of boron with
a polymer and the separation of complexes by UF membrane. The
retention depends also on pH, boron and polymer concentration
in the feed. MF and UF can be used for arsenic removal from
water by integrated systems with coagulation (Dilek et al. 2002). For example, from the water of As content equal to
40 μg/L, the water containing less than 2 μg/L can be obtained,
using ferric coagulants and membranes of pore size 0.22 and
1.22 μm (Dilek et al. 2002). In the integrated process, As is
adsorbed on coagulation fl ocks and next fl ocks are separated by
MF membrane. In such a case the removal of As(III) is more
effective than that of As(V) and preliminary oxidation of As(III)
to As(V) is required. obtained water has high quality, e.g. the concentration of Fe
and Mn in treated water is <0.1 and <0.05 mg/L, respectively
(Bodzek and Konieczny 2011a). The removal of Mn by means
of membrane fi ltration cannot be performed without oxidation
of dissolved Mn(II) ions to Mn(IV). The oxidants used are
KMnO4, O3, Cl2, ClO2, sodium hypochlorite or catalytic
bed covered with manganese compounds (AWWA 2005,
Koltuniewicz and Drioli 2008). The permissible concentration of heavy metals is
established in country’s regulations at very low level. Removal of anions Conventional methods such as precipitation, extraction or
ion exchange have many disadvantages, especially when
large amount of water with low concentration of metal ions is
treated. Membrane techniques (RO, NF, UF and ED) are often
applied to remove heavy metals from water and wastewaters on
industrial scale (Bodzek 2015, 2012, Bodzek and Konieczny
2011a, b). Bakalár et al. (2009) presented the results of the removal
of Cu, Ni and Zn using RO technology with composite
polyamide membrane TW3-1812-50 (Dow Filmtec). They
determined effect of the concentration of cations and TMP on
the separation effi ciency. In turn, Qdais and Moussa (2004)
tested the removal of Cu2+ and Cd2+ ions by means of RO
and NF. The RO removal effi ciency was high and amounted
to 98% for Cu and 99% for Cd, while for NF it was above
90%. NF is a better process for heavy metals removal, due to
ease of operation, reliability and comparatively low energy
consumption as well as high effi ciency (Fu and Wang 2011). Murthy and Chaudhari (2008, 2009) reported the application
of a thin-fi lm composite polyamide NF membrane for the
rejection of Ni ions from aqueous wastewater. The observed
rejection was 98% and 92% for an initial feed concentration
of 5 and 250 mg/L, respectively. For binary solution of Cd and
Ni, separation effi ciency for commercial NF membrane was
98.9% for Ni and 82.7%, for Cd ions. Retention of the cations
in the NF process strongly depends on the energy of hydration,
type and valence of co-ions passing through membrane as well
as the TMP and pH. Successful applications of ED and EDR include the
removal of various anions, e.g. nitrates (V), bromates (V),
chlorates (VII), arsenic (V), boron and fl uorides as well as
various heavy metals (Velizarov et al. 2004, Wisniewski 2001). Successful applications of ED and EDR include the
removal of various anions, e.g. nitrates (V), bromates (V),
chlorates (VII), arsenic (V), boron and fl uorides as well as
various heavy metals (Velizarov et al. 2004, Wisniewski 2001). During the RO, NF and ED processes the concentrate
with high concentration of anions is formed. Thus, the
use of membrane bioreactors (MBR) for the removal of
micropollutants from RO, NF and ED concentrates as well
as natural water and wastewaters, is proposed (Bodzek
2015, Bodzek and Konieczny 2011a, b, Crepso et al. 2004,
Velizarov et al. 2004, 2008). Removal of anions The biological degradation of
oxyanions (NO3
-, ClO4
-, BrO3
-) is based on their reduction to
harmless substances (N2, Cl-, Br-) at anaerobic condition and
the presence of microorganism (heterotrophic or autotrophic
bacteria) and proper electron donors (ethanol, methanol and
acetates for heterotrophic conditions and sulphur compounds
and hydrogen for autotrophic ones) (Crepso et al. 2004,
Velizarov et al. 2004). Autotrophic degradation gives lower
amount of the excess sludge, but the rate of process is low
(Kołtuniewicz and Drioli 2008), compared to heterotrophic
ones (Velizarov et al. 2004). Studies have shown full reduction
of nitrates (V), bromates (V) and chlorates(VII) to nitrogen,
bromides and chlorides by the same bacterial cultures which
are used for nitrates (V) reduction (Wang et al. 2008). The application of RO to the removal of heavy metals from
wastewaters can be presented on the example of electroplating
industry. Most frequently such effl uents contain Cr, Cu, Cd,
Zn, Ni, Pb and Ag ions, with concentration of 0.025 to 1 mg/L
(Bodzek 1999). The RO process allows recovering of water
of very high purity which can be returned to the technological
process without additional treatment. The concentrated
retentate may be reused in the electroplating bath (Bodzek
1999). An interesting solution of the removal of heavy metals
from aqueous solutions is the polymer enhanced ultrafi ltration
(PEUF) process (Korus 2012). It combines UF with metal
complexation using water-soluble polymers, such as polyvinyl
alcohol (PVA) and polyethyleneimine (PEI), and formed
complexes is retained by UF membrane. The permeate is free of
metal ions and retentate is subjected to regeneration in order to
recover both, the metal and polymer. The process was applied
for deactivation of radioactive liquid waste containing metal
ions, i.e. Cs, Co, Sr, Sb and Te isotopes, as well as lanthanides
(140La, 152Eu and 169Y) (Zakrzewska-Trznadel 2003). Korus
(2012) conducted studies on the removal of Ni, Cu and Zn
from synthetic and galvanic wastewater with the application Chromium Chromium most commonly occurs as cation (Cr+3) and Cr(VI)
in the form of anions (HCrO4
- and Cr2O7
2- ions, while at pH>6
as CrO4
2-). Cr(VI) is a strong oxidant, which is easily reduced
to Cr(III). Cr(III) is naturally present in the environment, as an
essential nutrient, while Cr(VI) is formed in industrial processes. The traditional way of removing Cr from water and wastewater
is reduction of Cr(VI) to the Cr(III), precipitation of Cr(OH)3
and fi ltration of suspension (Owlad et al. 2009). Several other
methods are also proposed, such as: adsorption and biosorption,
ion exchange (used on an industrial scale), solvent extraction
and electrochemical methods (Owlad et al. 2009). Chromium most commonly occurs as cation (Cr+3) and Cr(VI)
in the form of anions (HCrO4
- and Cr2O7
2- ions, while at pH>6
as CrO4
2-). Cr(VI) is a strong oxidant, which is easily reduced
to Cr(III). Cr(III) is naturally present in the environment, as an
essential nutrient, while Cr(VI) is formed in industrial processes. The traditional way of removing Cr from water and wastewater
is reduction of Cr(VI) to the Cr(III), precipitation of Cr(OH)3
and fi ltration of suspension (Owlad et al. 2009). Several other
methods are also proposed, such as: adsorption and biosorption,
ion exchange (used on an industrial scale), solvent extraction
and electrochemical methods (Owlad et al. 2009). (
)
The recovery and disposal of Cr can be made by high-
-pressure and low pressure membrane processes, liquid
membranes, ED and EDI (Bodzek 2012, Bodzek and Konieczny
2011a, Koltuniewicz and Drioli 2008, Owlad et al. 2009). RO
and NF can be used to directly separate Cr compounds from
solutions. Studies carried out on Cr(VI) removal using RO
Osmonics (Sepa-S) and cellulose acetate (CA) membranes
show that retention coeffi cients amounted to 80–96% and
96%, respectively, depending on the membrane compactness
(Bodzek et al. 2011a, Koltuniewicz and Drioli 2008, Owlad
et al. 2009). The removal of Cr, using RO and NF, becomes
cost-effective only if Cr concentration does not exceed 1–2 g/L
(Bodzek 2012). The obtained fi ltrate is then devoid of Cr,
but contains a signifi cant amount of salt, which can be used
for the preparation of etching baths. From the concentrated
Cr solution hydroxide is precipitated, and then dehydrated
sludge is dissolved in sulphuric acid and the resulting solution
can be used directly, for example, in the process of tanning
(Religa and Gawroński 2006). Chromium Retention coeffi cients of Cr
in NF increase with the increase of pH for higher Cr(VI)
concentrations. In strongly acidic pH Cr(VI) non-dissociated
chromic acid (H2CrO4) is formed. The increase of pH to 6.5
causes the formation of hydrochromate ions (HCrO4
–), the
concentration of which increases with further pH increase. If
pH is <7, chromate ions (CrO4
2–) are formed of concentration
also dependent on pH. Dichromate ions are also present in the
solution and their concentration depends on pH and chromium
content in the feed. Usually, this ion is dominant when the
concentration of chromium is high and pH is in the range of
1–7 and its concentration can be decreased by the increase of
pH (Bodzek and Konieczny 2011a, b). Recently, ED is frequently applied for the recovery of Au,
Pt, Ni, Ag, Pd, Cd, Zn and Sn/Pb metals from wastewaters,
fi rst of all to treat washery effl uents and wastewaters from
electroplating plants (Bodzek and Konieczny 2011a, b). The
solution of metal salt can be concentrated to the level that
corresponds to the electroplating bath, e.g. for Ni from 1 g/L
to 60 g/L (Bodzek 1999), which is much greater than with the
application of RO. The obtained retentate is used for fi lling up
the electroplating bath, whereas the dialysate is returned to the
washing installation. Hence, practically the whole quantity of
water and salts present in washery effl uents can be utilized. Liquid
membrane
processes,
especially
supported
liquid membranes (SLMs), have been suggested for heavy
metal removal due to their high specifi city, high intensity,
productivity and low energy requirements (Bodzek and
Konieczny 2011a). SLMs are very effective for the removal
and recovery of metals from wastewaters and process streams
since they combine extraction and stripping, into one step. In
the transport of metal ions in a SLM, the ion in the aqueous
feed solution forms a complex with the extractant HA in the
organic membrane phase at the interface between these two
bulk phases as follows (Bodzek and Konieczny 2011a): In the case of UF, the following possibilities of Cr removal
can be used: the pretreatment by UF before further purifi cation
with conventional methods or high-pressure membrane
processes, and application of PEUF or MEUF processes
(Bodzek 2012, Koltuniewicz and Drioli 2008, Owlad et al. 2009). M. Bodzek of the PEUF process. PVA, PEI, polyacrylic acid and sodium
polyacrylate as complexing agents were used in connection
with polysulphone and polyamide membranes. The obtained
effi ciency was 85–97% for polyamide membrane depending
on the polymer to the metal ratio, the pH and the kind of
metal. Sodium poly(styrene sulphonate), a water-soluble
anionic polymer with strong cation-exchange groups was
used as a complexing agent for lead ions complexing (Korus,
2010). The high rate of metal removal (85–99%) depended on
ratio of metals to polymer, pH of the solution and operating
UF conditions. It was possible to obtain the retentate with
a concentration of Pb 20-times higher than the concentration
of the feed solution which contained 50 mg Pb/L. Molinari et
al. (2008) used PEI to study the PEUF process in the selective
removal of Cu(II) from Ni(II) from solution. Preliminary tests
showed that optimal chemical conditions for Cu(II) and Ni(II)
complexation by the PEI were pH>6.0 and 8.0, respectively,
and polymer/metal weight ratio of 3.0 and 6.0, respectively. Aroua et al. (2007) investigated the removal of chromium
species from aqueous dilute solutions using PEUF process by
three water-soluble polymers, namely chitosan, PEI and pectin. High rejections approaching 100% for Cr(III) were obtained at
pH higher than 7 for the three tested polymers. Metal removal Iron and manganese can be removed from underground waters
by combining air oxidation with MF, particularly when the
concentrations of these metals are high and changing (Bodzek
and Konieczny 2011a). This hybrid method is similar to the
classical one, but instead of depth fi ltration, MF is applied. The 2019-11-14 10:52:26
2019-11-14 10:52:26 Archives vol 45 no 4 a4 srodki_kor 1.indd 9
Archives vol 45 no 4 a4 srodki_kor 1.indd 9 Archives vol 45 no 4 a4 srodki_kor 1.indd 9
Archives vol 45 no 4 a4 srodki_kor 1.indd 9 10 M. Bodzek Dispersed substances and microorganisms The turbidity of water is caused by the presence of suspended
mineral and organic molecules of different sizes (colloids,
coarse and fi ne suspensions). Usually, MF or UF are applied
to decrease water turbidity to 1 NTU, which corresponds to
drinking water level. There is little research in the literature on
the turbidity removal with membrane processes, but it follows
them clearly that the water of turbidity below 1 NTU can
be obtained from water of the initial turbidity 100 NTU and
greater (Taylor and Wiesner 2000). UF and MF are used for
water clarifi cation (Bodzek and Konieczny 2005). The study of
application of UF “Aquasource” module (France) showed that
drinking water of turbidity 0.03–0.04 NTU can be obtained
from waters with turbidity from 0.1 to 11.5–24.8 NTU (Taylor
and Wiesner 2000). Similar results were obtained for various
membrane modules (Taylor and Wiesner 2000). When water
contains colloidal fraction, membrane fi ltration is integrated
with coagulation in order to obtain fl ocks of greater size (Bodzek
and Konieczny 2005). Table 6 summarizes the percentage
removal of turbidity and natural organic matter (NOM) by
various membrane techniques (Bodzek and Konieczny 2006). Fig. 4. Removal of microorganisms by means of UF Practice has shown, however, that the UF membranes are
not always able to completely eliminate bacteria and viruses
from water. This is primarily connected with imperfections
in the membranes and membrane modules and the secondary
development of bacteria in water during its transport to the
fi nal recipient of water. In commercial membranes skin layer
can have discontinuity through which micro-organisms can
pass, as well as seals of the raw water from the permeate
streams is not always appropriate. The most effi cient in the
disinfection are the capillary modules in which isolation of
raw water from the permeate is easier than in the spiral wound
modules (Bodzek and Konieczny 2005). Furthermore, it was
found that cells of microorganisms can penetrate membrane
pores with diameters much smaller than the dimensions of the
cells (Sosnowski et al. 2004). Water can become naturally biologically contaminated
neededgustrial purposes, gically during its treatment or transport
in pipelines (Bodzek et al. 2019). Chromium M2+ + 2(HA)2 → MA2 (HA)2 + 2H+ Then, the metal-extractant complex diffuses from this
interface across the SLM to the interface between the organic
phase and the aqueous strip solution, where the metal ion is
stripped. The aqueous strip solution contains a strong acid, e.g. sulfuric acid. The stripping reaction is as follows: Both the ED and EDI processes can also be applied for the
removal and separation of Cr ions and their mixtures. Alvarado
et al. (2009) studied the ability of EDI and ED for the removal
of Cr(VI) from synthetic solutions containing 100 mg/L of
Cr(VI), using anion-exchange membranes by Neosepta. In EDI
process, a chamber with diluted solution was fi lled with mixed
ion exchange resin. In the ED the removal of Cr amounted to
98% at energy consumption of 1.2 kWh/m3, whereas in the EDI
process with the use of mixed bed 99.8% removal of Cr(VI)
was reached at energy consumption of 0.167 kWh/m3. MA2 (HA)2 + 2H+ → M2+ + 2(HA)2 This stripping reaction also regenerates the extractant at
this interface, which diffuses across the SLM back to the feed-
-membrane interface to complete the facilitated transport cycle. Archives vol 45 no 4 a4 srodki_kor 1.indd 10
Archives vol 45 no 4 a4 srodki_kor 1.indd 10 2019-11-14 10:52:26
2019-11-14 10:52:26 Archives vol 45 no 4 a4 srodki_kor 1.indd 10
Archives vol 45 no 4 a4 srodki_kor 1.indd 10 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... 11 Removal of organic pollutants 0
1
2
3
4
5
6
7
8
Reduction, log
Viruses
6.3
s
Bacte
7
7.2
eria
Gi
.1
7.4
iardia
4.7
5.1
Water I
Water I
Fig. 4. Removal of microorganisms by means of UF Dispersed substances and microorganisms Water, which contains viruses,
bacteria and protozoa as well as other microorganisms (fungi,
algae, snails, worms and crustaceans), if dedicated to potable
purposes, may seriously harm human health (Taylor and Wiesner
2000).There are several methods which can be used for water
disinfection, but some of them have a number of advantages and
disadvantages. It is also valid to treated and untreated wastewater
discharge to natural water source and sewage systems. Low-pressure membrane fi ltration can be applied to water
disinfection because it practically removes viruses, bacteria and
protozoa. The size of viruses’ cells is of a range of 20–80 nm,
while pore size of UF membranes is below 10 nm, hence,
theoretically, those cells should be completely rejected. The
sizes of bacteria (0.5–10 μm), cysts and oocysts (3–15 μm) are
larger, thus UF and MF can practically completely remove them
(Bodzek and Konieczny 2005, Taylor and Wiesner 2000). The
comparison of pore sizes of UF and MF membranes with sizes
of microorganisms indicates that UF process can be successfully
used for water disinfection (Bodzek and Konieczny 2005). In
Figure 4, the reduction of viruses, bacteria and protozoa in water
for different UF membranes is shown (Chen and Chen 2016,
Kosiol et al. 2017, Taylor and Wiesner 2000). The obtained
removal for all types of microorganisms was greater than 4 log,
i.e. 99.99%. Disinfection by-products )
The application of NF or RO enables total removal of
NOM, including DBP precursors from water, however it is very
often limited by high concentration of colloids and suspensions
in surface waters (Bodzek and Konieczny 2007). UF and MF
alone can be directly used to remove greater fractions of
NOM, including part of high molecular weight (MW) DBP
precursors, whereas medium and low MW compounds can be
eliminated in integrated systems (Urbanowska and Kabsch-
-Korbutowicz 2016). The removal of humic substances from
water by direct UF is possible only with modules with dense
membranes (MWCO 1000 Da) or hybrid systems of UF or
MF with coagulation, activated carbon adsorption or oxidation
(ozonation, photocatalysis) (Bodzek et al. 2011, Urbanowska and
Kabsch-Korbutowicz 2016). Recently, ion exchange – UF/MF
integrated process has been proposed for NOM removal,
especially MIEX resins (Kabsch-Korbutowicz et al. 2006,
Rajca 2012). Such solution is better than coagulation-UF
system. In Table 7 the comparison of effectiveness of NF, UF
and MF processes in NOM content control in natural waters is
shown. DBPs is a group of substances, which are formed during
the reaction of disinfectants with water pollutants. The most
important of DBPs are trihalomethanes (THMs) and haloacetic
acids (HAAs) (Bodzek 2015, Bodzek and Konieczny 2018). RO and NF are most often applied to remove THMs,
HAAs and other halogenated hydrocarbons from water. The
study showed that retention of RO and NF with Osmonic
membranes depended on the membrane permeability, i.e. the higher permeate fl ux the lower retention (Bodzek 2015,
Bodzek and Konieczny 2018). It was found that the MW
increase of THMs resulted in the retention increase according
to the following series: CHCl3<CHBrCl2<CHBr3<CHBr2Cl. The removal rate of chloroform varied from 67 to 87%,
bromodichloromethane 65–96.5%, dibromo-chloromethane
57–90.5% and tribromomethane 61–92% depending on the
applied membrane type (Bodzek 2015). In another study,
Uyak et al. (2008) showed that for NF200 and DS5 Osmonics
membranes operating pressure did not affect THM retention,
whereas initial concentration of THM had noticeable infl uence
on fl ux and retention. NF process is also suggested for the removal of HAAs
(chloro-, dichloro- and trichloroacetic acid; bromo- and
dibromoacetic acid) from water. The high reduction of HAAs
(90–100%), caused fi rst of all by repulsion forces, can be obtained
for dense negatively charged membranes, e.g. ES10 aromatic
polyamide membrane (Nitto-Denko Co. Ltd.) (Chalatip et al. 2009). Wang et al. Disinfection by-products (2018) obtained retention of HAAs amounted
to >75% with a fi ve-stage RO process during production of
drinking water. Environmental and operational variables like pH,
operating pressure, water matrix, and membrane age also played
important roles in HAAs removal. Increasing pH from 6.5 to 8.5
and membrane age apparently enhanced HAA rejections. Organic micropollutants In waters and wastewaters the following main groups of
micropollutants are present (Bodzek 2015, Bodzek and
Konieczny 2018): disinfection by-products (DBPs), endocrine
disrupting compounds (EDCs) and pharmaceutical active
compounds (PhACs). Some DBPs and PhACs in the aquatic
environment may also have estrogenic biological activity. Organic micropollutants (OM) have strong carcinogenic and
mutagenic properties. The presence of NOM in water may change the chemical
properties of organic micropollutants, i.e. increase solubility of
non-polar compounds, cause hydrolysis and photo-degradation,
and restrict the bioaccessibility by aquatic organisms (Bodzek
2015). The removal of OM during water treatment is usually
performed using adsorption on activated carbon or advanced
oxidation processes (AOPs) (Bodzek and Konieczny 2018). The fi rst method is unattractive when the amount of NOM in
water is high, whereas during AOPs process there is a possibility
of formation of DBPs with biological activity (Bodzek and
Konieczny 2018). Application of membrane processes seems
to be a good solution. They can be carried out as independent
processes as well as a part of integrated/hybrid systems with There are also studies on the removal of HAAs from
water in bioreactor with immobilized enzymatic ultrafi ltration
membranes (Kowalska et al. 2011). Enzymes isolated from
activated sludge strains of bacteria were used. 37% of the
monochloroacetic acid, 35% of monobromoacetic acid and
48.4% of dichloroacetic acid degradation were obtained
(Kowalska et al. 2011). M. Bodzek coagulation, adsorption or in membrane bioreactors (Bodzek
2015, Bodzek and Konieczny 2018). and Konieczny 2005). Effectiveness of NOM removal depends
on properties of membranes, thanks to a very wide molecular
size distribution of NOM (from 1 nm to 0.45 μm) (Bodzek
2015). Natural organic matter NOM in water ecosystems is the mixture of many compounds
with various properties and chemical structure, fi rst of all
soluble in water fulvic acids (MW≤2000 Da), hydrophobic
humic acids (MW≤2000–5000 Da) and insoluble humins
(Bodzek 2015). Humic substances are usually dissolved
in water, colloids and non-dissolved admixtures, and the
presence of a given form depends on water pH. The dissolved
NOM fraction in natural water is equal to ca. 80–90% of
total NOM. Humic substances cause intensive color of water
from brown to black. Additionally, complexation reactions of
humic substances with heavy metals or adsorption onto toxic
organics cause many health hazards. Humic substances are
also precursors of disinfection by-product (DBP) (Bodzek
et al. 2019). The removal of NOM is an important processes in
water treatment technology. The application of membrane
techniques in water treatment can remove DBP precursors
including part of NOM (Wilf 2010). Application of chloride
to water disinfection results in the formation of DBPs,
including trihalomethanes (THMs), halogenated acetic acids,
and other halogenated compounds (Bodzek 2015, Bodzek and
Konieczny 2005). Pressure-driven membrane processes allow
for controlling the formation of DBP, because membranes
retain NOM including DBP precursors (Van der Bruggen and
Vandecasteele 2003). The removal of NOM also decreases
the amount of chlorine required for disinfection, what is
necessary in the distribution system (Bodzek 2015, Bodzek Table 6. Turbidity and NOM removal using membrane
techniques
Process
Pressure,
kPa
Turbidity
removal,
%
NOM
removal,
%
Microfi ltration
<100
>97
<2
Ultrafi ltration
<100
>99
<10
Nanofi ltration
<500
>99
>90 Table 6. Turbidity and NOM removal using membrane
techniques 2019-11-14 10:52:26
2019-11-14 10:52:26 Archives vol 45 no 4 a4 srodki_kor 1.indd 11
Archives vol 45 no 4 a4 srodki_kor 1.indd 11 12
M. Bodzek 12 M. Bodzek Endocrine Disrupting Compounds – plant protection products (pesticides, herbicides and
insecticides), – phthalates, and phenols compounds (alkylphenols,
bisphenols), – synthetic hormones. Major sources of EDCs are food and drinking water, to
which these MPs are introduced with the rain, landfi ll leachate
and industrial wastewater. EDCs are present in natural waters
in the concentration ranging from ng/L to μg/L. Phytoestrogens and mycoestrogenes can be removed by
RO and NF processes with effectiveness amounting to 70–93%,
depending on the type of compounds and membranes (Dudziak
and Bodzek 2010). Dudziak (2013) studied the effi ciency of the
removal of selected mycoestrogens from water in coagulation,
sorption on activated carbon and NF processes. He found that
the coagulation removed mycoestrogens in 34%, adsorption
with PAC>80%, whereas the NF from 70 to 88%. p y
y
The presence of phthalates in the environment is caused
by massive production of plastics, mainly PVC, in which
they are used as plasticizers. According to their negative
effects on living organisms, the concentration of phthalates in
different parts of the environment, especially in water, should
be controlled. Polish regulations establish the permissible
concentration of di-n-butyl phthalate at the level of 20 μg/L. The surprisingly high retention of phthalates was observed
during both, the RO and NF processes (initial concentration
40 μg/L) (Bodzek 2005, Bodzek et al. 2004). Retention rates
achieved for diethyl phthalate, di-n-butyl and di-2-ethylhexyl
were very high and amounted from 89.7% (UF) to 99.9% (RO
and NF). The results obtained during the removal of phthalates
of MW 222–391 Da revealed that the MW of a compound did
not infl uence the removal effectiveness. ,
One of the main anthropogenic MPs in drinking water
sources are polycyclic aromatic hydrocarbons (PAHs). PAHs are harmful to human health, and most of them are very
carcinogenic, e.g. benzo(a)phyrene (permissible concentration
in drinking water – 0.010 μg/L and the sum of benzo(b)
fl uoranthene,
benzo(k)fl uoranthene,
benzo(g,h,i)perylene,
ideno(1,2,3-cd)pyrene – 0.1 mg/L). The retention coeffi cient
of PAHs, by means of RO and NF, was in the range from
85.9% to 99%, independently of MW of a compound and
concentration (Bodzek 2015, Bodzek and Konieczny 2019,
Luks-Betlej et al. 2001). Also UF membranes removed
PAHs to a high level, despite the fact that the MW of these
compounds are smaller than the cut-off of the UF membrane
(Bodzek 2015, Luks-Betlej et al. 2001). Endocrine Disrupting Compounds Recent investigations showed that EDCs appear more often
in natural waters and wastewaters, even biologically purifi ed Table 7. Comparative assessment of MF, UF and NF processes for NOM removal (Bodzek and Konieczny 2017)
Parameters
MF
UF
NF
NOM removal
<10%
0±30%
>80%
Suspensions and colloids removal
20±40%
70±90%
>95%
DBP removal
No
50% THM; 32% HAA
>80%
Requirements for cleaning
Backwashing
Cyclical cleaning
Cyclical cleaning
Performance problems
Moderate fouling
Fouling
Fouling, clogging
Pretreatment
In-line coagulation
or other process
In-line coagulation
or other process
No e assessment of MF, UF and NF processes for NOM removal (Bodzek and Konieczny 2017) Table 7. Comparative assessment of MF, UF and NF processes for NOM removal (Bodzek and Kon 2019-11-14 10:53:24
2019-11-14 10:53:24 Archives vol 45 no 4 a4 srodki_kor 1.indd 12
Archives vol 45 no 4 a4 srodki_kor 1.indd 12 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... 13 13 (Bodzek 2015). EDCs includes endogenic hormones, natural
organic compounds produced by fungi (mycoestrogenes) and
plants (phytoestrogenes), and a wide range of anthropogenic
MP among which the most important are (Bodzek and
Konieczny 2018): the concentration of the SAA (Bodzek 2015). When the
concentration is below critical micelle concentration (cmc) the
application of NF, eventually RO membranes, are suggested. In the case when the concentration of SAAs in water is greater
than cmc, UF process can be used (Kowalska 2012). y
)
– polycyclic
aromatic
hydrocarbons
(PAHs)
and
surfactants, Plant
protection
products
(pesticides,
herbicides
and insecticides) can be effectively removed from surface
and ground waters by NF or integrated systems of MF- or
NF-activated carbon adsorption. NF membranes eliminate
pesticides of MW above 190 Da to the amount below the limit
of detection, and, generally, the retention coeffi cient varies
from 50 to 100%, depending on MW and concentration MPs in
water (Bodzek 2015, Taylor and Weisner 2000). Research on
the application of UF process to the separation of atrazine from
water was carried out by several scientists (Majewska-Nowak
et al. 2001, Sarkar et al. 2007). The effi ciency of the process
depends on the type of membrane material and membrane
compactness. The best separation shows membranes with cut-
off about 1–2 kDa (retention rate – 60%) (Majewska-Nowak
et al. 2001). The increase of the effi ciency up to 85–95% can
be obtained during UF at the presence of NOM and/or cationic
polyelectrolyte. Endocrine Disrupting Compounds The cause of this
phenomenon was the adsorption of PAHs on the surface of
the UF membranes (Bodzek 2015). Studies on the removal
of PAHs from municipal landfi ll leachates and industrial
wastewaters were carried out by Smol et al. (2014a). The total
concentration of PAHs in permeates after RO was in the range
from 3.9 to 5.1 μg/L, at initial concentration of PAHs in the raw
landfi ll leachates amounting to 17.5–30.4 μg/L. The rate of the
removal of PAHs in RO was 71% (for individual hydrocarbons
were in the range 19–100%), and after initial treatment on sand
bed it was equal to 89%. The removal of PAHs by means of
NF and RO for biologically pretreated wastewater generated at
coke plant was also high (Smol et al. 2014b). It was found that
in the wastewater after the coagulation, the concentrations of
PAHs gradually lowered to the value 58.9 μg/L, at the initial
concentration equal 94.7 μg/L. The total content of 16 PAHs
after NF reached 18.7 μg/L, and after RO 5.94 μg/L. The
results show that a more effective PAHs removal is achieved
using the integrated system: coagulation – reverse osmosis
(Smol et al. 2014b). Phenolic xenoestrogenes (octylphenol, nonylphenol,
bisphenol A and bisphenol F) can be effectively removed from
water by means of NF. The retention coeffi cient of phenolic
xenoestrogenes strongly depends on a type of removed
compound and a membrane type (Bodzek 2015, Dudziak
and Bodzek 2008). The high retention of octylphenol and
nonylphenol in the range from 61 to 73% was observed for
SF-10 and DS-5-DK membranes, while in the case of bisphenol
A retention was higher (for membranes DS-5-DK – 69% and
for MQ – 16–75%) (Dudziak and Bodzek 2008). Archives vol 45 no 4 a4 srodki_kor 1.indd 13
Archives vol 45 no 4 a4 srodki_kor 1.indd 13 Pharmaceutical and Personal Care Products A novel hybrid membrane confi guration,
consisting of UF/NF and polymer-based activated carbon, was
developed and investigated for the removal of natural hormones
with a particular focus on estradiol (E2) (Tagliavin i and Schäfer
2018). Retention coeffi cients amounted to 60–80% depending
on membrane type (UF or NF). Removal of hormones from
wastewaters is also proposed by membrane bioreactors,
equipped
with
polyvinylidene
difl uoride
membrane. Comparing the obtained results with the conventional activated
sludge installation working in a full-scale (Zuehlke et al. 2006)
it was stated that in the case of MBR, estradiol and estrone
were removed in 99%, and ethinyloestradiol in 95%, but in
conventional treatment installation more than 90%. (
)
Pressure-driven membrane processes (MF, UF, NF
and RO) were also studied for the removal of EDCs,
pharmaceuticals and personal care products in a pilot scale
and industrial installations (Heberrer et al. 2008, Snyder et
al. 2007). Heberrer et al. (2008) performed investigation with
three-stage installation of capacity equal to 10,000 L/h which
included duplex bag fi lters with a particle separation <0.5 μm,
ultrafi ltration and reverse osmosis performed as one or two
stage process. The substrate of the study was water from
Teltow Channel (Berlin) and treated wastewater from Ruhleben
municipal wastewater treatment plant (Berlin). The presence of
PhAC in treated water from Teltow Channel was not observed,
whereas in RO permeate obtained during wastewater treatment
the amount of pharmaceutical was below 10 ng/L for both, one
and two stage confi gurations (Table 8) (Heberrer et al. 2008). NF and RO processes, used for the purifi cation of
wastewater and natural waters, can also be used for the removal
of residues of PPCPs (Bodzek 2015, Heberer and Feldmann
2008). The retention coeffi cients of polar and low volatile
PhACs and with low hydrophobicity amounted only to 40%. For polar organic compounds retention depends on the dipole
moment and pH (Kimura et al. 2005). Negatively charged
PhACs such as diclofenac is removed in more than 90–95%,
thanks to electrostatic repulsion (Bodzek 2015). For neutral It was found that MBR installations used in wastewater
treatment improved the effi ciency of the removal of PPCPs
in reference to conventional wastewater treatment plant. It
was caused not only by the extension of retention time and
increasing age of biomass, but also by increase of concentration Table 8. Pharmaceutical and Personal Care Products Pharmaceutical and Personal Care Products
One of the most important and quite specifi c and anthropogenic
MPs affecting the environment are compounds called
Pharmaceutical and Personal Care Products (PPCPs). This group includes compounds with pharmaceutical
activity, and substances used by people to maintain personal
hygiene (Heberer and Feldmann 2008). PPCPs enter quite
signifi cantly to surface water and groundwater as well
as wastewaters. Main sources of aqueous environment
pollution with pharmaceuticals are households and hospitals,
diagnostic units, pharmaceutical plants and livestock farms. Studies on effi ciency of the removal of PPCPs in wastewater Pressure driven membrane techniques are alternative
methods for the removal of surfactants (SAAs) from water,
while the effectiveness and the type of process depends on 2019-11-14 10:53:24
2019-11-14 10:53:24 14
M. Bodzek M. Bodzek 14 compounds, retention takes place according to molecular
exclusion and adsorption. For example, the neutrally charged
antipyretic phenacetin or anti-infl ammatory and antirheumatic
ibuprofen show retention >20%, because they are adsorbed
on membranes with relatively high hydrophobicity (Bodzek
2015). On the other hand, another neutral antipyretic
medicament, piramidon, is always retained in more than
70%, suggesting that its retention is affected also by other
phenomena. The retention of pharmaceuticals in the RO and
NF processes is also depended by membrane material (Heberer
and Feldmann 2008). For example, polyamide membranes had
higher effi ciency (57–91%) than cellulose acetate membranes. The concentration of PhAC also infl uences the retention rate
(Radjenović et al. 2009). For example, at a concentration of
100 ng/L retention was lower (14–72% for NF and for 50–80%
RO) compared to higher concentration amounted to 100 μg/L
(19–93% for NF and 71–95% for RO). treatment plants have shown that biological methods are not
always suffi cient (Bodzek and Konieczny 2018). The other
methods of PPCPs removal from water and wastewater
are advanced oxidation, activated carbon adsorption and
membrane processes (NF and RO) (Snyder et al. 2007), and,
in the case of wastewater, membrane bioreactors (Clara et al. 2005, Heberer and Feldmann 2008). )
Recently, an increase in concentrations of the synthetic
hormones (α-ethinylestradiol, mestranol and diethylstibelstrol)
resulting from discharges of these pharmaceuticals from
households, hospitals and pharmaceutical plants has been
observed. It was shown that this type of pollutants can be
eliminated by means of RO or NF. RO membranes totally
eliminate particular hormones while retention coeffi cients
obtained for NF and UF membranes were lower (Dudziak
and Bodzek 2008). Concluding remarks Membrane processes, such as RO, NF, UF, MF, ED and MBR,
can be used for inorganic and organic pollutants removal from
natural waters and wastewaters. Introduction and development of membrane techniques
in the production of drinking water and purifi cation of
wastewaters, in the last 40 years, was important stage in the
fi eld of water treatment effectiveness. Desalination of sea
and brackish water by means of RO is an established way for
drinking water production. Signifi cant improvements in design
of RO, the application of alternative energy sources, modern
pretreatment and new materials have contributed to the success
of the process. At the moment investment costs for large
desalting installations are similar to the modern conventional
water treatment plants. NF is the method of water softening,
because NF membranes can retain di- and multivalent ions,
but not monovalent. Drinking water containing viruses,
bacteria and protozoa, as well as other microorganisms, can
be disinfected by means of UF. Viruses are retained by UF
membranes, whereas bacteria and protozoa using both UF
and MF membranes. For the removal of NOM it is possible
to use direct NF or integrated systems combining UF or MF
with coagulation, adsorption and oxidation. NOM is regarded
as precursors of disinfection by-products, so its removal from
water is very important. y
p
Inorganic and organic micropollutants are found in
dangerous concentrations in water sources. Various classical
treatment technologies which are used for their removal from
water environment represent serious exploitation problems. The use of NF, RO and ED, in the treatment of water containing
micropollutants for drinking and industrial purposes can provide
more or less selective removal of the pollutants. Very important
are disinfection by-products, residue of pharmaceuticals and Mamo et al. (2018) compared the removal of selected
PhACs by means of MBR-RO and MBR-NF processes from
wastewater. RO membranes showed almost complete removal
of PhACs (>99%) at different process conditions, whereas for
NF, retention was lower (>90%). The choice between using
a RO versus NF membrane depends on the permeate reuse, the
discharge limits and the membrane MW cut-off and surface
morphology. Also process parameters (membrane recovery Table 9. Pharmaceutical and Personal Care Products Thus, the use of bioreactors
allows to receive treated wastewater of a very high quality, so
that the load of the MPs introduced into surface waters is lower
than that in the case of wastewaters coming from conventional
treatment plants. The results presented in table 9 (Barceló et al. 2009) show a distinct advantage of the membrane bioreactors
over the processes in typical wastewater treatment plants. and average permeate fl ux) and membrane condition have
a signifi cant effect on the removal of PhACs. and adsorption of MPs on the fl ocks of activated sludge. MBRs
with immersed UF modules have been checked for removal of
selected pharmaceuticals (clofi bric acid, diclofenac, ibuprofen,
ketoprofen, mefenamic acid and naproxsen) (Kimura et al. 2005). In the experiments higher removal rates of ketoprofen
and naproxen for MBR than for conventional treatment
system have been observed. Removal rates of phenazone,
propyphenazone, and formylamino-antipyrine were also
lower in conventional activated sludge installation (15%) than
in the MBR (60–70%) (Zuehlke et al. 2006). The removal of
pharmaceuticals using the MBR depended on the structure of
the PhAC molecules, e.g. the number of aromatic rings and pH,
i.e. higher removal of PhAC was observed at low pH conditions
due to adsorption on activated sludge fl ocks. Radjenović
et al. (2009) observed that in MBRs there is a signifi cant
improvement in the removal effi ciency of regulators of lipids
and cholesterol, statin drugs (gemfi brozil, bezafi brat, clofi bric
acid and pravastatin), β-blockers (atenolol and metoprolol),
antibiotics (ofl oxacin and erythromycin) and some painkillers
and anti-infl ammatory drugs. Other studies indicate a very
high removal rate of clofi bric acid and diclofenac in plants
equipped with the MBR, compared to conventional wastewater
treatment (Radjenović et al. 2009). Thus, the use of bioreactors
allows to receive treated wastewater of a very high quality, so
that the load of the MPs introduced into surface waters is lower
than that in the case of wastewaters coming from conventional
treatment plants. The results presented in table 9 (Barceló et al. 2009) show a distinct advantage of the membrane bioreactors
over the processes in typical wastewater treatment plants. Pharmaceutical and Personal Care Products The average concentrations of pharmaceuticals in biological treated wastewater and permeate after treatment
with two-stage RO system
Compounds
Concentration, ng/L
Retention coeffi cient, %
Raw water
Permeate
After preliminary
fi ltration and UF
After 1st RO stage
After 2nd RO stage
Benzafi brate
257
<5
7
96.0
>99.9
Carbamazepine
2282
<1
13
>99.9
>99.9
Clofi bric acid
178
<1
20
>99.4
>99.4
Diclofenac
869
<1
44
>99.9
>99.9
Fenofi bric acid
705
<1
22
97.0
>99.9
Ibuprofen
87
<1
12
98.5
>98.9
Indometacyn
46
<1
0
92.0
>97.8
Ketoprofen
99
<1
20
>99.0
>99.0
Naproxen
224
<1
0
98.2
>99.5
Oxazepam
153
<5
0
>99.3
>99.3
Primidone
734
<1
0
>99.9
>99.9
Propyphenazone
309
<1
46
99.3
>99.7 2019-11-14 10:53:24
2019-11-14 10:53:24 Archives vol 45 no 4 a4 srodki_kor 1.indd 14
Archives vol 45 no 4 a4 srodki_kor 1.indd 14 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... 15 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... 15 and adsorption of MPs on the fl ocks of activated sludge. MBRs
with immersed UF modules have been checked for removal of
selected pharmaceuticals (clofi bric acid, diclofenac, ibuprofen,
ketoprofen, mefenamic acid and naproxsen) (Kimura et al. 2005). In the experiments higher removal rates of ketoprofen
and naproxen for MBR than for conventional treatment
system have been observed. Removal rates of phenazone,
propyphenazone, and formylamino-antipyrine were also
lower in conventional activated sludge installation (15%) than
in the MBR (60–70%) (Zuehlke et al. 2006). The removal of
pharmaceuticals using the MBR depended on the structure of
the PhAC molecules, e.g. the number of aromatic rings and pH,
i.e. higher removal of PhAC was observed at low pH conditions
due to adsorption on activated sludge fl ocks. Radjenović
et al. (2009) observed that in MBRs there is a signifi cant
improvement in the removal effi ciency of regulators of lipids
and cholesterol, statin drugs (gemfi brozil, bezafi brat, clofi bric
acid and pravastatin), β-blockers (atenolol and metoprolol),
antibiotics (ofl oxacin and erythromycin) and some painkillers
and anti-infl ammatory drugs. Other studies indicate a very
high removal rate of clofi bric acid and diclofenac in plants
equipped with the MBR, compared to conventional wastewater
treatment (Radjenović et al. 2009). References Forward osmosis processes: Yesterday, today and tomorrow,
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chromium ions from aqueous solutions by polymer-enhanced
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(USA), Denver. Chung, T.-S., Zhang, S., Wang, Y.K., Su, J. & Ling, M.M. (2012). Concluding remarks The effi ciency of removal of pharmaceuticals in installations with MBR,
as well as in conventional wastewater treatment plants
Pharmaceutics
Effi ciency of the removal
with MBR [%]
Effi ciency of the removal in conventional
wastewater treatment plant [%]
Naproxen
99.3
85.1
Ketoprofen
91.9
51.5
Ibuprofen
99.8
82.5
Diclofenac
87.4
50.1
Acetaminophen
99.6
98.4
Propyfenazon
64.6
42.7
Sulfamethoxazole
60.5
55.6
Erythromycin
67.3
23.8
Hydrochlorothiazide
66.3
76.3
Glibenclamide
47.3
44.5
Gemfi brozil
89.6
38.8
Bezafi brat
95.8
48.4
Klofi bric acid
71.8
27.7 Table 9. The effi ciency of removal of pharmaceuticals in installations with MBR,
as well as in conventional wastewater treatment plants 2019-11-14 10:53:25
2019-11-14 10:53:25 Archives vol 45 no 4 a4 srodki_kor 1.indd 15
Archives vol 45 no 4 a4 srodki_kor 1.indd 15 M. Bodzek 16 endocrine disrupting compounds. For endocrine disrupting
compounds, special attention is paid onto polycyclic aromatic
hydrocarbons,
surface-active
substances,
chlorinated
pesticides, phthalates, alkylphenols, polychlorinated biphenyls,
hormones, synthetic pharmaceuticals and other substances
disposed to the environment. The application of MF and UF
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by polymer-based spherical activated carbon (PBSAC) assisted 2019-11-14 10:53:25
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Archives vol 45 no 4 a4 srodki_kor 1.indd 18 Archives vol 45 no 4 a4 srodki_kor 1.indd 18
Archives vol 45 no 4 a4 srodki_kor 1.indd 18 19 Membrane separation techniques – removal of inorganic and organic admixtures and impurities from water... Membranowe techniki separacji – usuwanie domieszek
i zanieczyszczeń nieorganicznych i organicznych ze środowiska wodnego Streszczenie: Zanieczyszczenia występujące w ujmowanych wodach, powodują, że skuteczne oczyszczanie jest
kłopotliwe, a układ (schemat technologiczny) oczyszczania powinien być opracowywany indywidualnie dla danej
wody na podstawie badań technologicznych. Aby zapewnić wymaganą jakość wody do picia bezpiecznych dla
zdrowia i życia konsumentów, często niezbędne jest stosowanie niekonwencjonalnych i wysokoefektywnych
procesów, mimo podwyższenia kosztów i potrzeby bardzo starannej i profesjonalnej eksploatacji układu
oczyszczania wody. Ponadto, niedogodności związane z tradycyjnym oczyszczaniem wód naturalnych oraz
zmieniające się podejście, co do koncepcji uzdatniania wód dla celów konsumpcyjnych, przede wszystkim
wzrastające wymagania odnośnie jakości wody do picia, stwarzają możliwości zastosowania nowych technik
separacji, wśród których metody membranowe mają największe zalety i możliwości i są obecnie brane pod uwagę
jako procesy alternatywne. W uzdatnianiu wody i oczyszczaniu ścieków stosuje się przede wszystkim techniki
membranowe, których siłą napędową jest różnica ciśnień po obu stronach membrany, ale brane są pod uwagę też
inne procesy jak elektrodializa, perwaporacja, destylacja membranowa i membrany ciekłe. Wybór odpowiedniego
procesu membranowego zależy od zakresu wielkości występujących i usuwanych z wody zanieczyszczeń
i domieszek. Techniki membranowe mogą być stosowane do usuwania zanieczyszczeń z wody jako procesy
samodzielne, lub w połączeniu z uzupełniającymi procesami jednostkowymi, tworząc systemy hybrydowe. W pracy omówiono możliwości wykorzystania technik membranowych w uzdatnianiu wód naturalnych. Odwrócona osmoza zatrzymuje jony jednowartościowe i większość związków organicznych małocząsteczkowych
i jest stosowana do odsalania wód oraz do usuwania jonów azotanowych i mikrozanieczyszczeń organicznych. Membrany nanofi ltracyjne zatrzymują koloidy, szereg związków organicznych małocząsteczkowych oraz jony
dwuwartościowe; można je zatem zastosować do zmiękczania wody i usuwania mikrozanieczyszczeń organicznych. Ultrafi ltracja i mikrofi ltracja stanowią barierę dla substancji rozproszonych i mikroorganizmów i dlatego można
je stosować do klarowania i dezynfekcji wody oraz jako metoda usuwania mętności wody. Procesy hybrydowe
obejmujące techniki membranowe stosuje się do uzdatniania wody do picia w połączeniu z ozonowaniem,
koagulacją, adsorpcją na węglu aktywnym do usuwania niżej cząsteczkowych związków organicznych lub
w bioreaktorach do usuwania azotanów. 2019-11-14 10:53:25
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Archives vol 45 no 4 a4 srodki_kor 1.indd 19
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https://www.scielo.br/j/jbchs/a/rNVYxsFH7s8z5nzvtxmdtXb/?lang=en&format=pdf
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English
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Use of a Digital Image in Flow Analysis: Determination of Nitrite and Nitrate in Natural Waters
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Journal of the Brazilian Chemical Society
| 2,015
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cc-by
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aInstituto de Química, Universidade Federal da Bahia, Salvador-BA, Brazil
bUniversidade Federal do Oeste da Bahia, Barreiras-BA, Brazil
cInstituto Federal de Educação, Ciência e Tecnologia da Bahia, Salvador-BA, Brazil
dEscola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba-SP, Brazil aInstituto de Química, Universidade Federal da Bahia, Salvador-BA, Brazil
bUniversidade Federal do Oeste da Bahia, Barreiras-BA, Brazil
cInstituto Federal de Educação, Ciência e Tecnologia da Bahia, Salvador-BA, Brazil
dEscola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba-SP, Brazil aInstituto de Química, Universidade Federal da Bahia, Salvador-BA, Brazil
bUniversidade Federal do Oeste da Bahia, Barreiras-BA, Brazil
cInstituto Federal de Educação, Ciência e Tecnologia da Bahia, Salvador-BA, Brazil
dEscola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba-SP, Brazil A webcam is proposed as the detector in a flow system with multicommutation, and the
feasibility of the approach is demonstrated in the determination of nitrate and nitrite in natural
waters. The typical transient signal inherent to the flow system was obtained using a digital video
and quantified by ImageJ software. The linear dynamics range for nitrite and nitrate determinations
were 0.2 to 2.0 mg L-1 NO2
− and 1.0 to 10.0 mg L-1 NO3
−, with relative standard deviation < 2% for
both analytes. The limits of detection were 0.01 and 0.04 mg L-1 for nitrite for nitrate, respectively,
and the sampling rate were 80 and 103 h-1 for nitrite and nitrate, respectively. The use of webcams
has a high potential for analysis in the visible region of the electromagnetic spectrum, and the
proposed strategy constitutes a promising alternative to traditional absorbance measurements
that depend on conventional equipment. The webcam detection system is attractive, especially in
relation to field analysis. Keywords: webcam, flow analysis, video digital, transient signal, nitrate, groundwater *e-mail: oldair@ufob.edu.br http://dx.doi.org/10.5935/0103-5053.20150245 http://dx.doi.org/10.5935/0103-5053.20150245 A J. Braz. Chem. Soc., Vol. 27, No. 1, 70-76, 2016. Printed in Brazil - ©2016 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00 J. Braz. Chem. Soc., Vol. 27, No. 1, 70-76, 2016. Printed in Brazil - ©2016 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00 Article Use of a Digital Image in Flow Analysis: Determination of Nitrite and Nitrate in
Natural Waters Jorge Luís O. Santos,a Oldair D. Leite,*,b Anete D. M. Vieira,b Djane S. Jesusc and
Marcos Y. Kamogawad Introduction both reagent consumption and waste generation,8-10 a
portable setup may even be developed.11,12 The interest in the nitrite and nitrate determination in
waters is due to the toxicological effects of these ions. Excessive ingestion by infants may result in the oxidation
of hemoglobin to methemoglobin, reducing oxygen
transport to the tissues.1 Nitrites are also precursors of
N-nitrosamines, compounds known for their potential
carcinogenic and teratogenic actions.2 The interest in the nitrite and nitrate determination in
waters is due to the toxicological effects of these ions. For spectrophotometric flow-based determinations of
nitrite and nitrate in conventional spectrophotometers,
portable spectrophotometers13 and LED-based photometers
have been used.14 Excessive ingestion by infants may result in the oxidation
of hemoglobin to methemoglobin, reducing oxygen
transport to the tissues.1 Nitrites are also precursors of
N-nitrosamines, compounds known for their potential
carcinogenic and teratogenic actions.2 Recently, colorimetric methods using digital images
have been reported. In most of these methods, the images
are captured by digital devices such as digital cameras,
webcams or scanners, and the digital images are treated
by a custom-built software.15-17 These species should be monitored, and several
analytical methods have been developed highlighting the
spectrophotometric methods with the Griess reaction. These methods are mostly chosen due to their simplicity,
ruggedness and detection limits.3 To minimize sample
and reagent consumption, increase the sampling rate and
improve analytical precision, analytical flow systems have
been proposed.4-7 In this regard, the following method was proposed:
determining the ascorbic acid concentration using a scanner
for image acquisition and color parameters (RGB) to
calculate the analytical response,15 the relation (equation 1)
was used, where I0 and I are the intensities of the blue
component (B) of the digital images obtained from the
blank solution and the standard solution, respectively. The
standard or sample solutions were placed into transparent
bottles positioned on the scanner. Flow analysis systems exploiting multicommutation
provide a powerful alternative to enhance the versatility of
flow-based procedures, with the advantage of minimizing (1) A = log(I0/I) Vol. 27, No. 1, 2016 Santos et al. 71 Barreiras-BA, Brazil. The samples were filtered through
0.45 µm cellulose membrane filters before analysis.20 Andrade et al.17 propose a digital image-based
flow‑batch analyzer for determination of AlIII and CrVI
in natural waters, using a webcam as a detector and
RGB parameters to calculate the analytical response. The multicommuted flow analyzer In flow systems, the data acquisition of the analytic
signal usually has a transient peak shape. Evaluating the
shape of the recorded transient signal is important for system
optimization and monitoring analysis, and evaluating the
transient peak shape of the flow systems is important for
system optimization, as it provides relevant information,
including the dispersion coefficient, mixing conditions,
mean residence time, carry over and the presence of spurious
signals (e.g., bubbles or the Schlieren effect).18 The multicommuted flow system comprised a model
IPC-4 peristaltic pump (Ismatec, Switzerland) equipped
with TygonTM pumping tubes, three-way 161T031
solenoid valves (NResearch, USA), polyethylene
tubing (0.8 mm i.d.) and acrylic confluence connectors. System control and data acquisition were performed
with a Pentium 2.1 GHz microcomputer equipped with
a commercial electronic interface (National Instruments,
USA) and a lab-made electronic circuit similar to that
previously described.22 The software was developed in
Labview 7.0 and the Windows XP operating system. The aim of this work was to develop a multicommuted
flow system that used a webcam as a photometric detector,
allowing the acquisition of the transient analytical signal. This system was applied to nitrite and nitrate determination
in groundwater. The multicommuted flow system (Figure 1) was
operated according to the valve-switching course in Table 1. The manifold comprised 04 three-way solenoid valves: one
valve for each managed solution (V1, V2, and V4) and one
for nitrite or nitrate determination (V3). Introduction The analytical figures of merit were similar to those of
conventional procedures, demonstrating the webcam’s
potential as a detector. In both applications, the images were
acquired with the static solutions inside a flask or chamber. l The reference method was the one proposed by the
American Public Health Association (APHA),21 which is
based on the Griess reaction; it was performed in batches,
using the 800 XI model Femto spectrophotometer (São
Paulo, Brazil). Reagents and solutions Figure 1. Flow diagram of the system for nitrite and nitrate determination. V1-V4: solenoid valves; C: buffer solution (pH = 7.2) (4.3 mL min-1);
S: sample (4.3 mL min-1); R: Griess reagent (2.1 mL min-1); CR: Cd
reduction mini-column (3 mm i.d. × 6 cm); B1 (0.8 mm i.d. × 60 cm) and
B2 (0.8 mm i.d. × 90 cm): coiled reactors; x and y: confluence sites; FL:
fluorescent lamp; webcam: detector. All solutions were prepared with double-distilled
and deionized water (18.0 MΩ cm) and chemicals with
analytical grade quality. The 1000 mg L-1 tartrazine (λ = 422 nm), Porceau 4R
(λ = 507 nm) and bright blue (λ = 603 nm) were prepared
by direct dissolution of the clean dried substances (BASF,
Germany) in water. Reference solutions (0.025-1.0 mg L-1 NO2
− and
0.10-5.0 mg L-1 NO3
−) were prepared by dilution of
1000 mg L-1 stock solutions prepared from NaNO2 and
NaNO3 (Merck, USA). The nitrite stock solution was
treated with a few drops of chloroform and standardized
against potassium permanganate.4 The reagent (R)
was 2.0% (m/v) sulphanilamide plus 0.1% (m/v) N-1-
naphthylethylenediamine dihydrochloride (NED) solution,
also 0.5 mol L-1 in phosphoric acid. The carrier stream was
0.5% (m/v) Na2B4O7 plus 0.3% (m/v) Na2EDTA buffer
solution with pH adjusted to 7.25 with HCl. Cadmium
fillings were copperized19 and packed into a glass tube (6 cm
long, 3 mm internal diameter, i.d.) retained by glass wool. Figure 1. Flow diagram of the system for nitrite and nitrate determination. V1-V4: solenoid valves; C: buffer solution (pH = 7.2) (4.3 mL min-1);
S: sample (4.3 mL min-1); R: Griess reagent (2.1 mL min-1); CR: Cd
reduction mini-column (3 mm i.d. × 6 cm); B1 (0.8 mm i.d. × 60 cm) and
B2 (0.8 mm i.d. × 90 cm): coiled reactors; x and y: confluence sites; FL:
fluorescent lamp; webcam: detector. The system operation started with all valves switched
off, and the carrier solution was pumped through V1, V2
and V3 towards the flow cell, while S was recycled by
V2. For nitrite determination, the V1 and V2 valves were
simultaneously switched on for 5 s, and the B1 reactor
was filled with the sample solution (Step 1). In the next
step, the V4 valve was switched on, and the sample zone
received the Griess reagent at the y confluence point; Sample preparation and reference method Groundwater samples were collected from wells in Use of a Digital Image in Flow Analysis J. Braz. Chem. Soc. 72 Table 1. Valve switching course Table 1. Valve switching course
Step
V1
a
V2
a
V3
a
V4
a
time / s
Description
Nitrite
1
1
1
0
0
5
fill B1
b with sample
2c
1
0
0
1
1
sampling Rd
3c
0
0
0
0
2
sampling Se
4
0
0
0
0
15
measure signal
Nitrate + Nitrite
5
0
0
1
0
10
fill B1
b and CR
f with buffer solution
6
1
1
1
0
10
fill B1
b and CR
f with sample
7c
0
0
1
1
1
sampling Rd
8c
0
0
1
0
2
sampling Se
9
0
0
0
0
15
measure signal
aV1-V4: solenoid valves, 0 and 1, solenoid valves switched off or on, respectively; bB1 (0.8 mm i.d. × 60 cm); crepeated steps (5 sampling cycles); dR: Griess
reagent (2.1 mL min-1); eS: sample (4.3 mL min-1); fCR: Cd reduction mini-column (3 mm i.d. × 6 cm). aV1-V4: solenoid valves, 0 and 1, solenoid valves switched off or on, respectively; bB1 (0.8 mm i.d. × 60 cm); crepeated steps (5 sampling cycles); dR: Griess
reagent (2.1 mL min-1); eS: sample (4.3 mL min-1); fCR: Cd reduction mini-column (3 mm i.d. × 6 cm). capture the digital images. The webcam was configured to
capture 24-bit digital images (16.7 million colors) at a rate
of 30 frames s-1 and 640 × 480 pixels of spatial resolution.23
The images were captured and stored as AVI files using the
software provided by the webcam manufacturer. sample aliquots were intercalated with the reagent, as
described in Steps 2 and 3 (Table 1). This sequence was
repeated until the B2 reactor was filled (0.45 mL) with the
binary string (5 sampling cycles). In the final step, all valves
were switched off, and the sample zone was transported to
the flow cell, allowing the nitrite determination (Step 4). For digital image acquisition, the flow cell (70 µL
inner volume) was positioned in front of the webcam. Sample preparation and reference method To avoid the influence of stray environmental light, the
webcam and flow cell were placed inside a polystyrene
box (21 × 30 × 18 cm) and illuminated with a white 5 W
fluorescent lamp (Figure 1), as suggested elsewhere.24 For nitrite and nitrate determination, the V3 valve was
switched on for 10 s, directing the flowing sample through
the cadmium column (Step 5). In the meantime, the V1,
V2 and V3 valves were switched on for 10 s, and the B1
reactor and cadmium column (CR) were filled with the
sample solution (Step 6). During this step, the nitrate ions
were converted to nitrite. Steps 7 and 8 were analogous to
the determination of nitrite (in Steps 2-4), and the obtained
signal (Step 9) was proportional to the concentration of
nitrate plus nitrite. All measurements were based on digital
image analysis and carried out in triplicate. For each determination, a video file with 90 s of
recording was created, and, using JPG Converter® software,
one image was extracted for every second of the video file,
meaning 90 image files were automatically saved in JPEG
format. These files were subsequently regrouped with
ImageJ software,25 using the stack image tool. With the
images in a stack, the oval tool was used to select an area
of 9500 pixel2 in the observation window of the flow cell
(Figure 2). Automatically, the RGB values in the selected
area were acquired for all images in the stack. In this
process, it was necessary to use a plugin developed in Java. Parameters such as sample and reagent volume, carrier
and sample flow rate, mixing coil length and number of
sampling cycles were evaluated using a single-variable
optimization procedure. The NED and sulphanilamide
concentrations were evaluated by a central composite
design (Table 2). A mathematical approach aiming at a linear relationship
between the proposed RGB-based value and the analyte
concentration was already described.26 This model used the
concept of an RGB-based value associated with a vector (ν)
of the color value. The RGB-based value was calculated by
equation 2, where R2
s-b, G2
s-b and B2
s-b are the differences Digital images sensor A Leadership© (Brazil) brand webcam (5.0 Mega model)
with a (charge-coupled device) CCD sensor was used to Table 2. Central composite design. NED and sulphanilamide concentration p
g
p
Factor
Level
NEDa / (%, m/v)
0.03
0.05
0.10
0.15
0.17
Sulphanilamide / (%, m/v)
1.3
1.5
2.0
2.5
2.7
Coded values
−1.41
−1
0
1
1.41
aN-1-Naphthylethylenediamine dihydrochloride (NED). al. 73
0
100
200
300
400
500
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
100
200
300
400
500
0
50
100
150
200
250
300
Absorbance
time / s
600.0 mg L
-1
600.0 mg L
-1
400.0 mg L
-1
400.0 mg L
-1
200.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
50.0 mg L
-1
50.0 mg L
-1
100.0 mg L
-1
Blue solution
RGB-based value / a.u. 0
100
200
300
400
500
600
0.0
0.2
0.4
0.6
0.8
1.0
0
100
200
300
400
500
600
0
20
40
60
80
100
120
140
800.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
50.0 mg L
-1
800.0 mg L
-1
600.0 mg L
-1
600.0 mg L
-1
400.0 mg L
-1
400.0 mg L
-1
200.0 mg L
-1
50.0 mg L
-1
100.0 mg L
-1
Absorbance
time / s
Green solution
RGB-based value / a.u. 0
100
200
300
400
500
0.0
0.5
1.0
1.5
0
100
200
300
400
500
0
50
100
150
200
600.0 mg L
-1
400.0 mg L
-1
400.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
600.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
50.0 mg L
-1
50.0 mg L
-1
Absorbance
time / s
Red solution
RGB-base value / a.u. Figure 3. Transient signals to increasing concentrations of the dyes red,
green and blue, obtained by the flow analysis system and detection by
webcam and spectrophotometer. Vol. 27, No. 1, 2016 73 Santos et al. Figure 2. Image acquisition of flow cell and area selected in ImageJ. Webcam detector optimization The dye solutions were deliberately used to evaluate
the performance of the webcam detector, as the maximum
wavelengths were distributed in the visible spectrum
(507 nm for red dye, 422 nm for green and 630 nm for blue). Figure 3 shows the transient signals recorded for
increasing concentrations of red, green and blue dyes, using
the spectrophotometer and the webcam. For both techniques,
a linear regression between the analytical responses and the
concentrations was observed, this is shown in Table 3. Figure 3. Transient signals to increasing concentrations of the dyes red,
green and blue, obtained by the flow analysis system and detection by
webcam and spectrophotometer. Analysis of Table 3 reveals that the webcam detection
method yielded analytical curves with high slopes and
good linear regression coefficients for all different colors
of solution. Coefficients of variation (n = 10) of 1.4%, 1.8%
and 1.6% were obtained for 400 mg L-1 solutions of red,
green and blue, respectively. and reagent volume, number of sampling cycles, reactor
lengths and flow rates were evaluated, and the tested range
and selected values are shown in Table 4. The best concentrations determined by the experimental
design of sulphalamide and NED were 2.3% and 0.12%,
respectively, and the regression coefficient was 0.9989,
showing the good mathematical model adjustment
(Figure 4). Digital images sensor 0
100
200
300
400
500
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
100
200
300
400
500
0
50
100
150
200
250
300
Absorbance
time / s
600.0 mg L
-1
600.0 mg L
-1
400.0 mg L
-1
400.0 mg L
-1
200.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
50.0 mg L
-1
50.0 mg L
-1
100.0 mg L
-1
Blue solution
RGB-based value / a.u. Figure 2. Image acquisition of flow cell and area selected in ImageJ. in the mean values of the RGB components between the
sample or standard solution and the blank solution. 0
100
200
300
400
500
600
0.0
0.2
0.4
0.6
0.8
1.0
0
100
200
300
400
500
600
0
20
40
60
80
100
120
140
800.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
50.0 mg L
-1
800.0 mg L
-1
600.0 mg L
-1
600.0 mg L
-1
400.0 mg L
-1
400.0 mg L
-1
200.0 mg L
-1
50.0 mg L
-1
100.0 mg L
-1
Absorbance
time / s
Green solution
RGB-based value / a.u. 2
2
2
s b
s b
s b
RGB based value =
(R
+ G
+ B
)
(2) (2) The detection and quantification limits were estimated
as LD = 3Sb/β and LQ = 10Sb/β, respectively, where Sb is
the standard deviation of the blank solution’s data and β is
the angular coefficient of the analytical curve. Analytical figures of merit, such as detection and
quantification limits, linearity and the correlation coefficient
of the analytical curve were considered for the dyes in
solutions containing red, green and blue components. This
was done in order to compare detector performances for
the webcam and the conventional detector, a model 800 XI
spectrophotometer (Femto, São Paulo, Brazil). 0
100
200
300
400
500
0.0
0.5
1.0
1.5
0
100
200
300
400
500
0
50
100
150
200
600.0 mg L
-1
400.0 mg L
-1
400.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
600.0 mg L
-1
200.0 mg L
-1
100.0 mg L
-1
50.0 mg L
-1
50.0 mg L
-1
Absorbance
time / s
Red solution
RGB-base value / a.u. Multicommuted flow system To optimize the flow system, parameters such as sample Use of a Digital Image in Flow Analysis 74 J. Braz. Chem. Soc. Table 3. Correlation between the measurements performed using the web
solutions with regression parameters (Y = mC + b)
Solution
Webcam
ma
bb
r
Red
0.33466
0.06906
0.9
Green
0.12916
0.09243
0.9
Blue
0.46874
0.04649
0.9
am = angular coefficient; bb = linear coefficient; cr = linear regression coef
Table 4. Optimized parameters of the multicommuted flow system for
nitrite and nitrate determination
Parameter
Interval
Selected value
Flow rate-peristaltic pump / (mL min-1)
3.1-6.6
4.3
B2 reactor length / cm
50-100
90
Sampling cycles
3-7
5
Sample volume / mL
35-70
70
Reagent volume / mL
35-70
35
pH of the buffer solution
6.0-8.5
7.2
Sulphalamide / (%, m/v)
1.3-2.7
2.3
NEDa / (%, m/v)
0.03-0.17
0.12
Phosphoric acid / (mol L-1)
0.3-1.5
0.5
aN-1-Naphthylethylenediamine dihydrochloride (NED). Figure 4. Central composite design of sulphanilamide and NED
concentration. RGB-based value = 49.67 + 2.19NED − 2.46(NED)2
+ 2.86(sulphanilamide) − 2.71(sulphanilamide)2 + 0.25(NED)
(sulphanilamide). Table 3. Correlation between the measurements performed using the webcam and absorbance values for different concentrations of red, green and blue
solutions with regression parameters (Y = mC + b)
Solution
Webcam
Absorbance
ma
bb
rc
ma
bb
rc
Red
0.33466
0.06906
0.9986
0.00216
−0.00149
0.9999
Green
0.12916
0.09243
0.9997
0.00101
−0.04723
0.9993
Blue
0.46874
0.04649
0.9997
0.00154
0.06323
0.9988
am = angular coefficient; bb = linear coefficient; cr = linear regression coefficient. Table 3. Correlation between the measurements performed using the webcam and absorbance values for different concentrations of red, green and blue
solutions with regression parameters (Y = mC + b) Table 4. Optimized parameters of the multicommuted flow system for
nitrite and nitrate determination nitrate. The optimized conditions are presented in Table 4. Under the proposed conditions, the dynamic ranges were
0.2 to 2.0 mg L-1 NO2
− and 1.0 to 10.0 mg L-1 NO3
−. The
linear behavior was described by the equations 3 and 4. Parameter
Interval
Selected value
Flow rate-peristaltic pump / (mL min-1)
3.1-6.6
4.3
B2 reactor length / cm
50-100
90
Sampling cycles
3-7
5
Sample volume / mL
35-70
70
Reagent volume / mL
35-70
35
pH of the buffer solution
6.0-8.5
7.2
Sulphalamide / (%, m/v)
1.3-2.7
2.3
NEDa / (%, m/v)
0.03-0.17
0.12
Phosphoric acid / (mol L-1)
0.3-1.5
0.5
aN-1-Naphthylethylenediamine dihydrochloride (NED). Multicommuted flow system RGB-based value = – 0.647 + 112 [NO2
−]
(R2 = 0.9956)
(3)
RGB-based value = –16.2 + 20.0 [NO3
−]
(R2 = 0.9944)
(4) (3) (4) The coefficients of variation (relative standard
deviation, RSD) were estimated at 0.62% and 1.34% for
10 measures corresponding to solutions of 0.6 mg L-1 NO2
−
and 6.0 mg L-1 NO3
−, respectively. At a 99.5% confidence
level, the detection limits were estimated as 0.01 mg L-1
NO2
− and 0.04 mg L-1 NO3
−. Figure 4. Central composite design of sulphanilamide and NED
concentration. RGB-based value = 49.67 + 2.19NED − 2.46(NED)2
+ 2.86(sulphanilamide) − 2.71(sulphanilamide)2 + 0.25(NED)
(sulphanilamide). 0
100
200
300
400
500
0
50
100
150
200
250
0.0
0.4
0.8
1.2
1.6
2.0
(a)
RGB-based value / a.u. time / s
2.0 mg L
-1
1.2 mg L
-1
0.6 mg L
-1
0.4 mg L
-1
0.2 mg L
-1
(b)
Concentration of nitrite / (mg L
-1)
Figure 5. (a) Transient signals; (b) calibration curve for determination of
the 0.2-2.0 mg L-1 NO2
−, using detection by webcam. 0
100
200
300
400
500
0
50
100
150
200
250
0.0
0.4
0.8
1.2
1.6
2.0
(a)
RGB-based value / a.u. time / s
2.0 mg L
-1
1.2 mg L
-1
0.6 mg L
-1
0.4 mg L
-1
0.2 mg L
-1
(b)
Concentration of nitrite / (mg L
-1) Concentration of nitrite / (mg L
-1) Figure 4. Central composite design of sulphanilamide and NED
concentration. RGB-based value = 49.67 + 2.19NED − 2.46(NED)2
+ 2.86(sulphanilamide) − 2.71(sulphanilamide)2 + 0.25(NED)
(sulphanilamide). Figure 5. (a) Transient signals; (b) calibration curve for determination of
the 0.2-2.0 mg L-1 NO2
−, using detection by webcam. Groundwater samples were spiked with 0.6, 1.2 and
1.6 mg L-1 NO2
− and 3.0, 6.0 and 8.0 mg L-1 NO3
− and analyzed
by the proposed method. The results are shown in Table 5. Recoveries ranged from 96.9% to 109.2% for nitrite
and 100.0% to 103.2% for nitrate, similar values to those
obtained by reference methods NBR 1261927 and APHA
4500-NO3 I.21 Groundwater samples were spiked with 0.6, 1.2 and
1.6 mg L-1 NO2
− and 3.0, 6.0 and 8.0 mg L-1 NO3
− and analyzed
by the proposed method. The results are shown in Table 5. Multicommuted flow system The reagent volume per determination was 175 µL,
and the total waste generation was about 2.2 and 3.2 mL
for nitrite and nitrate, respectively. The sampling rate was
103 h-1 for nitrite and 80 h-1 for nitrate. Recoveries ranged from 96.9% to 109.2% for nitrite
and 100.0% to 103.2% for nitrate, similar values to those
obtained by reference methods NBR 1261927 and APHA
4500-NO3 I.21 Analytical application Figure 7 shows the results of the determination for
nitrite and nitrate in groundwater samples employing both
the proposed procedure and the reference. The excellent The transient signals and linear calibration graphics
are shown in Figures 5 and 6, respectively, for nitrite and 75 Vol. 27, No. 1, 2016 Santos et al. Table 5. Mean values and uncertainties (n = 3) for recovery of nitrite and nitrate in groundwater samples by the proposed and reference procedures
Compound added
in samples
Fortification /
(mg L-1)
Reference procedurea,b
Proposed Procedure
Error / %
Found / (mg L-1)
Recovery / %
Found / (mg L-1)
Recovery / %
Nitrite
1.60
1.56 ± 0.01
97.5
1.55 ± 0.03
96.9
-0.6
1.20
1.24 ± 0.01
103.3
1.31 ± 0.03
109.2
5.6
0.60
0.60 ± 0.01
99.5
0.60 ± 0.01
100.0
0
Nitrate
8.00
8.32 ± 0.12
104.0
8.25 ± 0.14
103.1
-0.8
6.00
6.00 ± 0.18
100.0
6.11 ± 0.11
101.8
1.8
3.00
2.98 ± 0.07
99.5
3.00 ± 0.16
100.0
0.7
aNBR 12619 method(for nitrite); bAPHA 4500-NO3
-I method (for nitrate). uncertainties (n = 3) for recovery of nitrite and nitrate in groundwater samples by the proposed and reference procedures aNBR 12619 method(for nitrite); bAPHA 4500-NO3
-I method (for nitrate). aNBR 12619 method(for nitrite); bAPHA 4500-NO3
-I method (for nitrate). 0
200
400
600
800
0
20
40
60
80
100
120
140
160
180
200
0
2
4
6
8
10
(b)
(a)
1.0 mg L
-1
3.0 mg L
-1
6.0 mg L
-1
8.0 mg L
-1
10.0 mg L
-1
RGB-based value / a.u. time / s
Concentration of nitrate / (mg L
-1)
Figure 6. (a) Transient signals; (b) calibration curve for determination of
the 1.0-10.0 mg L-1 NO3
−, using detection by webcam. 0
200
400
600
800
0
20
40
60
80
100
120
140
160
180
200
0
2
4
6
8
10
(b)
(a)
1.0 mg L
-1
3.0 mg L
-1
6.0 mg L
-1
8.0 mg L
-1
10.0 mg L
-1
RGB-based value / a.u. time / s
Concentration of nitrate / (mg L
-1) detect the visible region of the electromagnetic spectrum. In addition, since this characteristic can dispense a
wavelength selector, it could be exploited to reduce cost
and simplify instrumentation methods for measurements
in the visible region. References 1. Boylston, M.; Beer, D.; Study, M. A. C.; Crit. Care Nurse 2011,
22, 50. 2. Maekawa, A.; Ogiu, T.; Onodera, H.; Furuta, K.; Matsuoka, C.;
Ohno, Y.; Odashima, S.; Food Chem. Toxicol. 1982, 20, 25. Proposed procedure 3. Moorcroft, M.; Talanta 2001, 54, 785. 3. Moorcroft, M.; Talanta 2001, 54, 785. Figure 7. Comparison of webcam detector and conventional method in
groundwater samples; (a) determination of nitrite; (b) determination of nitrate. 4. Giné, M. F.; Bergamin, F. H.; Zagatto, E. A. G.; Reis, B. F.;
Anal. Chim. Acta 1980, 114, 191. 4. Giné, M. F.; Bergamin, F. H.; Zagatto, E. A. G.; Reis, B. F.;
Anal. Chim. Acta 1980, 114, 191. correlation demonstrates the accuracy of the new procedure
and proves the viability the webcam detector. 5. Ahmed, M. J.; Stalikas, C. D.; Tzouwara-Karayanni, S. M.;
Karayannis, M. I.; Talanta 1996, 43, 1009. 5. Ahmed, M. J.; Stalikas, C. D.; Tzouwara-Karayanni, S. M.;
Karayannis, M. I.; Talanta 1996, 43, 1009. 6. Zhi-Qi, Z.; Lou-Jun, G.; Han-Ying, Z.; Qian-Guang, L.; Anal. Chim. Acta 1998, 370, 59. Analytical application The procedure was implemented
with inexpensive instrumentation by exploiting the
multicommutation approach and using a webcam as
an analytical detector. Low reagent consumption and
minimal waste generation offer additional advantages. The method is simple, rapid and inexpensive, and it was
successfully applied for the determination of nitrite and
nitrate in water samples. Concentration of nitrate / (mg L
-1) Concentration of nitrate / (mg L
-1) Figure 6. (a) Transient signals; (b) calibration curve for determination of
the 1.0-10.0 mg L-1 NO3
−, using detection by webcam. Figure 6. (a) Transient signals; (b) calibration curve for determination of
the 1.0-10.0 mg L-1 NO3
−, using detection by webcam. Acknowledgements 0.1
0.2
0.03
0.06
0.09
0.12
0.15
0.18
0.21
(b)
Determination of nitrate
m = angular coef.= 0.9999
b = linear coef. = -0.0166
R = 0.99
(a)
Determination of nitrite
m = angular coef.= 0.9817
b = linear coef. = -0.0038
R = 0.95
Reference procedure
Proposed procedure
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Reference procedure
Proposed procedure
Figure 7. Comparison of webcam detector and conventional method in
groundwater samples; (a) determination of nitrite; (b) determination of nitrate. 0.1
0.2
0.03
0.06
0.09
0.12
0.15
0.18
0.21
(b)
Determination of nitrate
m = angular coef.= 0.9999
b = linear coef. = -0.0166
R = 0.99
(a)
Determination of nitrite
m = angular coef.= 0.9817
b = linear coef. = -0.0038
R = 0.95
Reference procedure
Proposed procedure
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Reference procedure
Proposed procedure The authors are grateful to E. A. G. Zagatto for critical
comments and to Fundação de Amparo à Pesquisa do
Estado da Bahia (PRONEX AGUA and PPP 049/2011),
Fundação de Amparo à Pesquisa do Estado de São Paulo
for it financial support (2005/00878-0; 2011/23498-9),
CNPq and CAPES. Conclusions 7. Yue, X.-F.; Zhang, Z.-Q.; Yan, H.-T.; Talanta 2004, 62, 97. 7. Yue, X.-F.; Zhang, Z.-Q.; Yan, H.-T.; Talanta 2004, 62, 97. The proposed flow procedure is robust and very easy
to operate, and it has a great potential in analysis to 8. Feres, M. A.; Reis, B. F.; Talanta 2005, 68, 422. 9. Rocha, F. R.; Reis, B. F.; Anal. Chim. Acta 2000, 409, 227. Use of a Digital Image in Flow Analysis J. Braz. Chem. Soc. 76 Sedimento, Comunidades Aquáticas e Efluentes Líquidos;
CETESB: São Paulo, SP, Brasil, 2011. 10. Pons, C.; Santos, J. L. M.; Lima, J. L. F. C.; Forteza, R.;
Cerdà, V.; Microchim. Acta 2008, 161, 73. 21. American Public Health Association (APHA), American Water
Works Association (AWWA), Water Environment Federation
(WEF); Standard Methods for the Examination of Water and
Wastewater, 20th ed.; American Public Health Association:
Washington D.C., 1998. 11. Ródenas-Torralba, E.; Rocha, F. R. P.; Reis, B. F.; Morales-
Rubio, A.; de la Guardia, M.; J. Autom. Methods Manage. Chem. 2006, 2006, 20384. 12. Mankasingh, U.; Worsfold, J. P.; Instrum. Sci. Technol. 2010,
38, 187. 13. http://www.oceanoptics.com/Products/spectrometers.asp,
accessed in September, 2015. 22. Reis, B. F.; Giné, M. F.; Zagatto, E. A. G.; Lima, J. L. F. C.;
Lapa, R. A.; Anal. Chim. Acta 1994, 293, 129. 14. Crispino, C. C.; Reis, B. F.; Anal. Methods 2014, 6, 302. 23. http://www.leadership.com.br, accessed in September 2015. 15. Gomes, M. S.; Trevizan, L. C.; Nóbrega, J. A.; Kamogawa,
M. Y.; Quim. Nova 2008, 31, 1577. 24. Lima, M. B.; Andrade, S. I. E.; Barreto, I. S.; Almeida, L. F.;
Araújo, M. C. U.; Microchem. J. 2013, 106, 238. 16. Wongwilai, W.; Lapanantnoppakhun, S.; Grudpan, S.;
Grudpan, K.; Talanta 2010, 81, 1137. 25. http://rsb.info.nih.gov/ij/, accessed in September, 2015. 26. Lyra, W. S.; dos Santos, V. B.; Dionízio, A. G. G.; Martins,
V. L.; Almeida, L. F.; Gaião, E. N.; Diniz, P. H. G. D.; Silva,
E. C.; Araújo, M. C. U.; Talanta 2009, 77, 1584. 17. Andrade, S. I. E.; Lima, M. B.; Barreto, I. S.; Lyra, W. S.;
Almeida, L. F.; Araújo, M. C. U.; Silva, E. C.; Microchem. J. 2013, 109, 106. 27. ABNT NBR 12619, ABNT: São Paulo, 1995. 27. ABNT NBR 12619, ABNT: São Paulo, 1995. 18. Dias, A. C. B.; Borges, E. P.; Zagatto, E. A. G.; Worsfold, P. J.;
Talanta 2006, 68, 1076. Conclusions Submitted: July 15, 2015
Published online: September 22, 2015 9. Henriksen, A.; Selmer-Olsen, A. R.; Analyst 1970, 95, 5 20. Environmental Control Agency of São Paulo (CETESB);
Guia Nacional de Coleta e Preservação de Amostras: Água, FAPESP has sponsored the publication of this article.
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English
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Nal-IRI with 5-fluorouracil (5-FU) and leucovorin or gemcitabine plus cisplatin in advanced biliary tract cancer - the NIFE trial (AIO-YMO HEP-0315) an open label, non-comparative, randomized, multicenter phase II study
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BMC cancer
| 2,019
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cc-by
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Abstract Patients are allocated in two arms: Arm A
(experimental intervention) nal-IRI mg/m2, 46 h infusion)/5-FU (2400 mg/m2, 46 h infusion)/leucovorin (400 mg/m2, 0.5
h infusion) d1 on 14 day-cycles; Arm B (standard of care) cisplatin (25 mg/m2, 1 h infusion)/gemcitabine (1000 mg/m2,
0.5 h infusion) d1 and d8 on 21 day-cycles. The randomization (1:1) is stratified for tumor site (intrahepatic vs. extrahepatic biliary tract), disease stage (advanced vs. metastatic), age (≤70 vs. > 70 years), sex (male vs. female) and
WHO performance score (ECOG 0 vs. ECOG 1). Primary endpoint of the study is the progression free survival (PFS) rate
at 4 months after randomization by an intention-to-treat analysis in each of the groups. Secondary endpoints are the Methods: NIFE is an interventional, prospective, randomized, controlled, open label, two-sided phase II study. Within
the study, 2 × 46 patients with locally advanced, non-resectable or metastatic BTC are to be enrolled by two stage
design of Simon. Data analysis will be done unconnected for both arms. Patients are allocated in two arms: Arm A
(experimental intervention) nal-IRI mg/m2, 46 h infusion)/5-FU (2400 mg/m2, 46 h infusion)/leucovorin (400 mg/m2, 0.5
h infusion) d1 on 14 day-cycles; Arm B (standard of care) cisplatin (25 mg/m2, 1 h infusion)/gemcitabine (1000 mg/m2,
0.5 h infusion) d1 and d8 on 21 day-cycles. The randomization (1:1) is stratified for tumor site (intrahepatic vs. extrahepatic biliary tract) disease stage (advanced vs metastatic) age (≤70 vs > 70 years) sex (male vs female) and 0.5 h infusion) d1 and d8 on 21 day-cycles. The randomization (1:1) is stratified for tumor site (intrahepatic vs. extrahepatic biliary tract), disease stage (advanced vs. metastatic), age (≤70 vs. > 70 years), sex (male vs. female) and
WHO performance score (ECOG 0 vs. ECOG 1). Primary endpoint of the study is the progression free survival (PFS) rate
at 4 months after randomization by an intention-to-treat analysis in each of the groups. Secondary endpoints are the
overall PFS rate, the 3-year overall survival rate, the disease control rate after 2 months, safety and patient related
outcome with quality of life. The initial assessment of tumor resectability for locally advanced BTCs is planned to be
reviewed retrospectively by a central surgical board. Exploratory objectives aim at establishing novel biomarkers and
molecular signatures to predict response. The study was initiated January 2018 in Germany. Abstract Background: Biliary tract cancer (BTC) has a high mortality. Primary diagnosis is frequently delayed due to mostly
unspecific symptoms, resulting in a high number of advanced cases at the time of diagnosis. Advanced BTCs are in
principle chemotherapy sensitive as determined by improved disease control, survival and quality of life (QoL). However, median OS does not exceed 11.7 months with the current standard of care gemcitabine plus cisplatin. Thereby, novel drug formulations like nanoliposomal-irinotecan (nal-IRI) in combination with 5- fluorouracil (5-FU)/
leucovorin may have the potential to improve therapeutic outcomes in this disease. Methods: NIFE is an interventional, prospective, randomized, controlled, open label, two-sided phase II study. Within
the study, 2 × 46 patients with locally advanced, non-resectable or metastatic BTC are to be enrolled by two stage
design of Simon. Data analysis will be done unconnected for both arms. Patients are allocated in two arms: Arm A
(experimental intervention) nal-IRI mg/m2, 46 h infusion)/5-FU (2400 mg/m2, 46 h infusion)/leucovorin (400 mg/m2, 0.5
h infusion) d1 on 14 day-cycles; Arm B (standard of care) cisplatin (25 mg/m2, 1 h infusion)/gemcitabine (1000 mg/m2,
0.5 h infusion) d1 and d8 on 21 day-cycles. The randomization (1:1) is stratified for tumor site (intrahepatic vs. extrahepatic biliary tract), disease stage (advanced vs. metastatic), age (≤70 vs. > 70 years), sex (male vs. female) and
WHO performance score (ECOG 0 vs. ECOG 1). Primary endpoint of the study is the progression free survival (PFS) rate
at 4 months after randomization by an intention-to-treat analysis in each of the groups. Secondary endpoints are the
overall PFS rate, the 3-year overall survival rate, the disease control rate after 2 months, safety and patient related
outcome with quality of life. The initial assessment of tumor resectability for locally advanced BTCs is planned to be
reviewed retrospectively by a central surgical board. Exploratory objectives aim at establishing novel biomarkers and
molecular signatures to predict response. The study was initiated January 2018 in Germany. Methods: NIFE is an interventional, prospective, randomized, controlled, open label, two-sided phase II study. Within
the study, 2 × 46 patients with locally advanced, non-resectable or metastatic BTC are to be enrolled by two stage
design of Simon. Data analysis will be done unconnected for both arms. Perkhofer et al. BMC Cancer (2019) 19:990
https://doi.org/10.1186/s12885-019-6142-y Perkhofer et al. BMC Cancer (2019) 19:990
https://doi.org/10.1186/s12885-019-6142-y Open Access Nal-IRI with 5-fluorouracil (5-FU) and
leucovorin or gemcitabine plus cisplatin in
advanced biliary tract cancer - the NIFE trial
(AIO-YMO HEP-0315) an open label, non-
comparative, randomized, multicenter
phase II study Nal-IRI with 5-fluorouracil (5-FU) and
leucovorin or gemcitabine plus cisplatin in
advanced biliary tract cancer - the NIFE trial
(AIO-YMO HEP-0315) an open label, non-
comparative, randomized, multicenter
phase II study L. Perkhofer1*, A. W. Berger1,2, A. K. Beutel1, E. Gallmeier3, S. Angermeier4, L. Fischer von Weikersthal5, T. O. Goetze6,
R. Muche7, T. Seufferlein1 and T. J. Ettrich1 © 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.
* Correspondence: lukas.perkhofer@uniklinik-ulm.de
1Department of Internal Medicine I, Ulm University, Albert-Einstein-Allee 23,
89081 Ulm, Germany
Full list of author information is available at the end of the article Abstract Discussion: The NIFE trial evaluates the potential of a nanoliposomal-irinotecan/5-FU/leucovorin combination in the
first line therapy of advanced BTCs and additionally offers a unique chance for translational research. Trial registration: Clinicaltrials.gov NCT03044587. Registration Date February 7th 2017. Keywords: Biliary tract cancer, Cholangiocarcinoma, Chemotherapy, Nanoliposomal-irinotecan, Palliative treatment Keywords: Biliary tract cancer, Cholangiocarcinoma, Chemotherapy, Nanoliposomal-irino * Correspondence: lukas.perkhofer@uniklinik-ulm.de
1Department of Internal Medicine I, Ulm University, Albert-Einstein-Allee 23,
89081 Ulm, Germany
Full list of author information is available at the end of the article Methods and study design g
Biliary tract cancer (BTC) is a rare type of cancer and
ranks beyond 10th in Western World tumor incidence
[1]. However, the incidence particularly of intrahepatic
BTC is rising, [2, 3] resulting BTC to be the 5th leading
cause of cancer related deaths [1]. The main reason for
the high mortality of BTCs can be found in the generally
advanced stage at primary diagnosis, due to often miss-
ing early symptoms [4]. 5-year overall survival rates do
not exceed 5% for patients with advanced or metastatic
disease [1]. Advanced BTCs respond to chemotherapy,
resulting in an improved disease control rate, survival
time and quality of life (QoL) [5–7]. However, overall
survival rates beyond 10 months remain rare in the pal-
liative setting. The current standard of care combines
conventional chemotherapeutic agents for patients who
are in a good performance status. Therapy is based on
the ABC-02 phase III trial that demonstrated a beneficial
progression-free (PFS) and overall survival (OS) for a
combination of gemcitabine plus cisplatin compared
to gemcitabine alone (Cis + Gem vs. Gem: OS 11.7 vs. 8.1 months; PFS 8.0 vs. 5.0 months) [6]. However, the
therapeutic landscape in oncology is steadily evolving
bringing novel compounds into daily clinical routine
in various cancer entities. Several antibodies and in-
hibitors like cetuximab or sorafenib were evaluated in
advanced BTC, but failed to improve outcome [5, 8]. Irinotecan combined with 5-FU showed promising re-
sults in the 1st- [9] and 2nd-line treatment [10] of
advanced BTC and is commonly used as therapeutic
option after failure of the 1st-line therapy with gemci-
tabine/cisplatin. Consequently, encapsulation of irino-
tecan in pegylated liposomes could be of value in
advanced BTC as efficacy and tolerability of this drug
are already proven in a number of solid tumors in-
cluding pancreatic [11], gastric [12] and colorectal
cancers
[13]. Nanoliposomal-irinotecan
(nal-IRI)
showed extended plasma half-life and increased intra-
tumoral concentrations compared to conventional iri-
notecan in preclinical models [14–16]. The NAPOLI-
1 trial transferred this to the patient and demon-
strated in a phase III setting a significantly prolonged
OS for 2nd-line therapy with nal-IRI/5-fluorouracil
(5-FU)/leucovorin (LV) in patients with metastatic
pancreatic cancer compared to 5-FU/LV only [11]. The superiority shown in the NAPOLI-1 trial pro-
vides compelling evidence for a potential efficacy in
advanced BTC. Methods and study design The toxicity profile of nal-IRI is simi-
lar to what has been described for irinotecan that is
routinely used in clinical practice by oncologists [12]. The NIFE phase II trial aims to challenge the current pal-
liative first-line therapy of BTC by use of nanoliposomal-
irinotecan/5-FU/leucovorin and to further establish specific
bi
k
i
t NIFE is an interventional, prospective, randomized, con-
trolled, open label, two-sided phase II study, using the
optimal two-stage design of Simon in each of the experi-
mental arms. Exploratory objectives: Exploratory objectives: Exploratory biomarkers analysis (cfDNA exome
sequencing, transcriptome, miRNA-arrays prior to
and after start of treatment, and on progression). Establishment of predictive/prognostic biomarker
profiles for advanced BTC Tumor evolution under chemotherapy Perkhofer et al. BMC Cancer (2019) 19:990 Perkhofer et al. BMC Cancer (2019) 19:990 Page 2 of 7 Page 2 of 7 Background Methods and study design Study objectives
Primary objective PFS rate at 4 months, defined as the proportion of
patients with non-progressive disease 4 months after
randomization by intention-to-treat analysis Secondary objectives: Secondary objectives: Overall progression-free survival 3-years overall survival Disease control rate according to RECIST 1.1 [17]
after 2 months Objective tumor response rate (ORR) according to
RECIST 1.1 [17] Toxicity/safety according to CTCAE-criteria version
4.03 (≥Grade 3/4) Patient-related outcome/quality of life/time to
definitive deterioration (TUDD) to be assessed with
the following tools: EORTC QLQ-BIL21, QLQ-C30
and HADS-D Tumor resectability in accordance with a
retrospective central surgical board compared to
local surgical review Radiological response according to RECIST 1.1 [17]
and volumetry determined by a retrospective central
radiological review Main inclusion and exclusion criteria
Inclusion criteria: Inclusion criteria: Clinically significant cardiovascular disease (incl. Myocardial infarction, unstable angina, symptomatic
congestive heart failure, serious uncontrolled cardiac
arrhythmia) within 6 months before enrollment Histologically confirmed, non-resectable, locally ad-
vanced or metastatic adenocarcinoma of the intrahe-
patic or extrahepatic biliary tract (not papillary
cancer or gallbladder cancer) Prior (< 3 years) or concurrent malignancy (other
than biliary-tract cancer) which either progresses or
requires active treatment. Exceptions are: basal cell
cancer of the skin, pre-invasive cancer of the cervix,
T1a or T1b prostate carcinoma, or superficial urin-
ary bladder tumor [Ta, Tis and T1]. g
Non-resectability has to be stated by the local
multidisciplinary tumor board Measurable or assessable disease according to
RECIST 1.1 [17] Known Gilbert-Meulengracht syndrome Known chronic hypoacusis, tinnitus or vertigo Previous enrollment or randomization in the present
study (does not include screening failure). If applicable, adequately treated biliary tract
obstruction before study entry with total bilirubin
concentration ≤2 x ULN Exclusion criteria: Exclusion criteria: Patient selection and randomization Approximately 120 patients have to be screened to get
92 randomized patients (46 patients per arm). In the
study, 30 participating centers are planned. The trial is
randomized in a 1:1 ratio to the experimental (Arm A)
or standard arm (Arm B) to get comparable sample sizes
by stratified permutated block randomization to avoid a
selection bias, see Fig. 1. The randomization (1:1) is
stratified for tumor site (intrahepatic vs. extrahepatic bil-
iary tract), disease stage (advanced vs. metastatic), age
(≤70 vs. > 70 years) [18], sex (male vs. female) and WHO
performance score (ECOG 0 vs. ECOG 1). The NIFE phase II trial aims to challenge the current pal-
liative first-line therapy of BTC by use of nanoliposomal-
irinotecan/5-FU/leucovorin and to further establish specific
biomarker signatures. Perkhofer et al. BMC Cancer (2019) 19:990 Page 3 of 7 Fig. 1 Flow diagram NIFE trial Main inclusion and exclusion criteria Main inclusion and exclusion criteria
Inclusion criteria: Treatment Treatment is planned in an outpatient setting for all
study drugs and will continue until there is evidence of
disease progression or occurrence of any other discon-
tinuation criterion. If nal-IRI or cisplatin have to be dis-
continued permanently under therapy for a reason other
than progressive disease, treatment should continue with
the remaining drug in the trial, with full adherence to all
protocol-related requirements. Within a therapy cycle,
treatment should continue on schedule, but a variance
of ±5 days may be allowed to accommodate holidays,
weekends or other justifiable events. Staging assessments Staging assessments Adequate blood count, liver-enzymes, and renal
function:
◦AST (SGOT)/ALT (SGPT) ≤5 x institutional
upper limit of normal
◦Serum Creatinine ≤1.5 x institutional ULN and a
calculated glomerular filtration rate ≥30 mL per
minute
◦Patients not receiving therapeutic anticoagulation
must have an INR < 1.5 ULN and PTT < 1.5 ULN
within 7 days prior to randomization Medical history and demographics including dates
and description of initial diagnosis of advanced
biliary tract cancer and relevant concurrent illness Complete physical examination including: weight,
height, BSA, vital signs (blood pressure, heart rate,
respiratory rate and oral body temperature) Residual symptoms/toxicities from previous
therapies should be recorded according to the NCI
Common Toxicity Criteria No prior palliative chemotherapy for biliary tract
cancer ECOG Performance Status ECOG Performance Status
Review of prior/concomitant medications Review of prior/concomitant medications No adjuvant treatment within 6 months prior to
study entry Tumor assessment according to RECIST 1.1 [17]
done by local investigator in the context of standard
care (contrast enhanced multislice CT of the Written informed consent including participation in
translational research Page 4 of 7 Perkhofer et al. BMC Cancer (2019) 19:990 Page 4 of 7 abdomen or abdominal MRI and an enhanced
multislice thoracic CT scan)
EORTC QLQ-BIL21, QLQ-C30 and HADS-D
questionnaire
Nutritional risk score
12-lead ECG
Hematological tests, Clinical chemistry
Serum Tumor Marker (Ca 19–9, CEA) abdomen or abdominal MRI and an enhanced
multislice thoracic CT scan) abdomen or abdominal MRI and an enhanced
multislice thoracic CT scan) software
[19]. H0:
less
than
40%
of
patients
are
progression-free by 4 months of nal-IRI plus 5-FU/leucov-
orin. Alternative
hypothesis:
≥60%
of
patients
are
progression-free by 4 months of nal-IRI plus 5-FU/leucov-
orin. If 7 or less of the first 18 patients assigned to nal-IRI
plus 5-FU/leucovorin have a tumor response or stable dis-
ease at 4 months, H0 will be accepted and the study will
be terminated. If 8 or more patients with tumor response
or stable disease are observed, another 28 patients in each
treatment group are to be included. At the final analysis,
H0 will be accepted if less than 23 of the total 46 patients
in the nal-IRI plus 5-FU/leucovorin group had a tumor re-
sponse or stable disease at 4 months. With this design,
alpha = 10% (significance level) and power = 90%. Quality of life assessment and time to definitive
deterioration Arm A (experimental arm): Arm A (experimental arm): Nanoliposomal-irinotecan 80 mg/m2 as 1.5 h
infusion Health related quality of life (HRQL) will be assessed by
using the EORTC QLQ-C30 questionnaire version 3.0. The questionnaire contains 5 functions (physical, role,
cognitive, emotional, and social), 9 symptoms (fatigue,
pain, nausea and vomiting, dyspnea, loss of appetite, in-
somnia, constipation, diarrhea and financial difficulties)
and the global health status/quality of life (GBH/QoL)
[20]. To further specify the assessment the module for
biliary tract cancer (QLQ-BIL21) with 21 items related
to disease symptoms, treatment side effects and emo-
tional issues in BTC is included [21]. A calculation of
the median time to definitive deterioration (TUDD)
using
the
EORTC QLQ-C30
questionnaire data
is
planned. The TUDD will be calculated in accordance to
Anota et al. and Bonnetain et al. and is defined as an on-
going deterioration of at least 5 points compared to the
baseline [22, 23]. The emotional and social impact of be-
ing diagnosed with BTC is highly relevant. To detect
anxiety and depression, which are the most common co-
morbidities of physical illness, the HADS-D question-
naire (Hospital Anxiety and Depression Scale – German
version) is used. The HADS-D has 14 items (7 anxiety, 7
depression) each with a 4-point verbal rating scale
scored from 0 to 3. The scale deliberately avoids physical
indicators of mental disorders (e.g., insomnia, weight
loss) and severe psychopathological symptoms allowing
high sensitivity with proven psychometric quality criteria
[24, 25]. 5-fluorouracil 2400 mg/m2 as 46 h infusion Leucovorin 400 mg/m2 as 0.5 h infusion Cycle q2w ± 5 days Arm B (standard arm):
2 Follow-up All subjects undergo follow-up for survival until the end
of the study irrespective of subsequent treatments, or
until the sponsor ends the study (follow-up extension
phase). Patient contact is to be established by telephone
interview or face-to-face, whichever prevails. The following procedures will be performed during
follow-up every 8 weeks: Assessment of survival status Anti-cancer treatments must be recorded during
follow up Reporting of all adverse events (AEs) and severe
adverse events (SAEs) within 4 weeks after the end
of treatment (EoT) visit Staging assessments As the
study will be analyzed as intention-to-treat analysis (ITT),
all patients will be analyzed (missing data will be consid-
ered as failure). Hence, a sample size of n = 46 per treat-
ment arm and a total N = 92 enrolled and randomized
patients is required. It is assumed that approx. 120 pa-
tients need to be screened for eligibility. EORTC QLQ-BIL21, QLQ-C30 and HADS-D
questionnaire Nutritional risk score
12-lead ECG
Hematological tests, Clinical chemistry Serum Tumor Marker (Ca 19–9, CEA) Arm B (standard arm):
2 Cisplatin 25 mg/m2 as 1 h infusion on day 1 and day
8 Gemcitabine 1000 mg/m2 as 0.5 h infusion on day 1
and day 8 Cycle q3w ± 5 days Translational research This trial provides the opportunity to systematically ob-
tain biologic material from therapy naive patients suffer-
ing from advanced BTC for comprehensive molecular
characterization. It allows to assess treatment associated
tumor evolution under 1st-line palliative chemotherapy
with different regimens. Consequently, we will collect tis-
sue samples obtained for initial diagnosis for exome se-
quencing best versus worst responders. We hypothesize
that exome sequencing of microdissected tumor cells from
initially taken core biopsies will identify important biologic
differences
between
tumors
responding to
cytotoxic
chemotherapy compared to those not responding to the
treatment
and
thereby
provide
potential
predictive
markers. In parallel, blood samples of each patient will be
taken prior to treatment, after 4–5 weeks of treatment,
thereafter in parallel to radiologic tumor assessments until
disease progression (radiologically confirmed). Circulating
cell-free tumor DNA will be extracted and analyzed by
targeted genotyping in order to verify the potential of li-
quid biopsy as a disease diagnosis and treatment monitor-
ing tool, as previously shown. Mutation profiles obtained
from tissue and blood will be compared to evaluate
whether tumor DNA analysis from blood yields a pattern
comparable to tumor tissue and could be used to establish
“easy to obtain” prognostic and predictive markers for nal-
IRI based treatment. yp
The knowledge on BTC biology is still limited com-
pared to other solid cancers. Recent sequencing studies
shed more light on the mutational landscape of BTC
and encourage the use of novel therapeutic targets [40–
42]. However, a synergistic chemotherapy backbone is
commonly needed like in other difficult to treat GI ma-
lignancies [43, 44]. Thereby, advanced BTC already
showed the limitations of such strategies with no effects
by adding cetuximab to standard chemotherapy in the
BINGO trial [5]. Anyhow the spectrum of BTC muta-
tions appears to lie within other gastrointestinal epithe-
lial cancers with similar oncogenic mutations [42, 45,
46]. As a consequence a proper definition of BTC sub-
types is paramount potentially guiding future treat-
ment
approaches. Therefore
an
expanded
liquid
biopsy program like that included in the NIFE trial
may allow new insights on stratification and especially
on
the
development
of
the
mutational
landscape
under therapy. Nutritional screening The nutritional risk score (NRS) questionnaire will be
used for the evaluation of nutritional anomalies. Malnu-
trition and weight loss are common problems in ad-
vanced BTC patients and contribute to morbidity and
mortality. Furthermore, tolerance of chemotherapy is
often worse in patients with severe malnutrition. The
NRS questionnaire is a simple tool to screen patients for
malnutrition [26]. The questionnaires will be completed
at time of screening, every 8 weeks under therapy and at
the EoT visit. Discussion Median overall survival in patients with advanced BTC
is still devastating, generally not exceeding 1 year with
the current therapeutic concepts. The results of the
ABC-02 6 and the BINGO trial [5] defined gemcitabine/
cisplatin (or oxaliplatin) as treatment of choice in ad-
vanced BTC first line therapy. Therefore the investiga-
tors reported a progression-free survival (PFS) rate of
54% at 4-months in the gemcitabine/oxaliplatin group. Irinotecan was evaluated in several combinations in ad-
vanced BTC as first-line treatment, [27–30] showing the
most promising results in combination with a thymidy-
late synthase inhibitor [31–33]. There is evidence that
the nanoliposomal formulation of irinotecan may confer
improved efficacy of the drug [14, 15, 34–37]. This en-
couraged us to try nal-IRI/5-FU/leucovorin in the first
line treatment of advanced BTC, particularly given the
positive data on safety and tolerability in both phase II
and III trials as well as in real-life data in PDAC [11, 38,
39]. The NIFE trial aims to update and widen the treat-
ment landscape in advanced BTC by using Nal-IRI/5-FU/
leucovorin. For the NIFE trial we assume that ≥60% of pa-
tients are progression-free after 4 months of nal-IRI/5-FU/
leucovorin. An interim analysis is planned after 18 pa-
tients have been enrolled to confirm the hypothesis. Sample size calculation and statistical analysis HRQL should be assessed at following time points: HRQL should be assessed at following time points: HRQL should be assessed at following time points: Simon’s optimal two-stage design was used for sample size
calculation for each group by OneArmPhaseTwoStudy At baseline, within 7 days prior to randomization At baseline, within 7 days prior to randomization Perkhofer et al. BMC Cancer (2019) 19:990 Page 5 of 7 Page 5 of 7 Before the beginning of each cycle of systemic
therapy Ulm University approved the NIFE-trial as leading ethics
committee for all German sites according to German regu-
lative laws for trials (Arzneimittelgesetz). In addition, local
ethics committees approved the participating sites. The trial
is registered with ClinicalTrials.gov (NCT0344587). At end of treatment visit Quality of life assessment should be performed even
when chemotherapy cannot be given at the
beginning of a cycle e.g. due to toxicity reasons. Competing interests (m)DFS: (median) disease free survival; (m)OS: (median) overall survival;
(m)PFS: (median) progression free survival; 5-FU: 5- fluorouracil; AE: Adverse
event; AIO: Arbeitsgemeinschaft Internistische Onkologie; BSA: Body surface
area; BTC: Biliary tract cancer; CBC: Complete blood count; cfDNA: Circulating
free DNA; Cis: Cisplatin; CT: Computed Tomography; CTC: Common toxicity
criteria; CTCAE: Common Terminology Criteria for Adverse Events;
DCR: Disease control rate; ECG: Electrocardiogram; ECOG: Eastern
Cooperative Oncology Group; EORTC: European Organization for Research
and Treatment of Cancer; EOT: End of treatmend; FOLFIRINOX: Fluorouracil
leucovorine, irinotecan, oxaliplatin; GBH: Global health status; G-
CSF: Granulocyte-colony stimulating factor; Gem: Gemcitabine; HADS-
D: Hospital Anxiety and Depression Scale; HR: Hazard ratio; HRQL: Health
related quality of life; ITT: Intention to treat; LV: Leucovorin; MRI: Magnetic
Resonance Imaging; Nab-Paclitaxel: Nano albumin bound Paclitaxel; Nal-
IRI: Nanoliposomal-Irinotecan; NCI: National Cancer Institute; NRS: Nutritional
risk score; ORR: Objective response rate; PDAC: Pancreatic ductal
adenocarcinoma; QLQ-C30: Quality of life questionnaire-core 30; QoL: Quality
of life; RECIST: Response Evaluation Criteria in Solid Tumors; SAE: Severe
adverse event; TUDD: Time until definitive deterioration; ULN: Upper limit of
normal; WHO: World health organization Nanoliposomal-Irinotecan, 5-FU and leucovorin are provided by Servier. LP and TJE
received travel grants from IPSEN Pharma, the other authors declare no conflicts of
interest. The trial is sponsored according to German regulatory laws by the AIO
Studien GmbH. The study was not externally reviewed. Availability of data and materials Not applicable. Data sharing is planned once the trial is completed Not applicable. Data sharing is planned once the trial is completed. 8. Moehler M, et al. Gemcitabine plus sorafenib versus gemcitabine alone in
advanced biliary tract cancer: a double-blind placebo-controlled multicentre
phase II AIO study with biomarker and serum programme. Eur J Cancer. 2014;50:3125–35. Authors’ contributions
TJE LP
h 3. Saha SK, Zhu AX, Fuchs CS, Brooks GA. Forty-year trends in
cholangiocarcinoma incidence in the U.S.: intrahepatic disease on the rise. Oncologist. 2016;21:594–9. https://doi.org/10.1634/theoncologist.2015-0446. TJE, LP wrote the protocol. RM conducted statistical trial planning. TJE and
AWB handled ethics and regulatory affairs. TJE and LP wrote the paper draft. AWB, AKB, GTO, EG, SA, LFvW, TS contributed in the trial design and
modifications and data collection. All authors have approved the final
version of the manuscript. 4. Vogel A, Wege H, Caca K, Nashan B, Neumann U. The diagnosis and
treatment of cholangiocarcinoma. Dtsch Arztebl Int. 2014;111:748–54. https://doi.org/10.3238/arztebl.2014.0748. 5. Malka D, et al. Gemcitabine and oxaliplatin with or without cetuximab in
advanced biliary-tract cancer (BINGO): a randomised, open-label, non-
comparative phase 2 trial. Lancet Oncol. 2014;15:819–28. https://doi.org/10. 1016/S1470-2045(14)70212-8. Funding
Th
i l i The trial is funded by an unrestricted grant by Servier. The study has not undergone
peer-review by the funding body. Data collection, analysis, interpretation of data and
manuscript writing are independent from Servier. The protocol hasn’t undergone
peer-review by the funding body. 6. Valle J, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract
cancer. N Engl J Med. 2010;362:1273–81. https://doi.org/10.1056/
NEJMoa0908721. 7. Eckel F, Schmid RM. Chemotherapy in advanced biliary tract carcinoma: a
pooled analysis of clinical trials. Br J Cancer. 2007;96:896–902. https://doi. org/10.1038/sj.bjc.6603648. References
S
l R The phase 2 trial started in 01/2018 and is still ongoing and recruiting
patients. Further study information is open-access available at clinicaltrials. gov (NCT03044587). Outlines of the protocol were presented as a poster at
ASCO 2018 and ESMO 2018. 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;
67:7–30. https://doi.org/10.3322/caac.21387. 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;
67:7–30. https://doi.org/10.3322/caac.21387. 2. von Hahn T, et al. Epidemiological trends in incidence and mortality of
hepatobiliary cancers in Germany. Scand J Gastroenterol. 2011;46:1092–8. https://doi.org/10.3109/00365521.2011.589472. Ethical aspects, trial registration All patients have to sign written informed consent includ-
ing participation in translational research and any locally-
required authorization (including EU Data Privacy Directive
in the EU, Declaration of Helsinki) obtained from the sub-
ject prior to performing any protocol-related procedures,
including screening evaluations. The ethics committee of To sum up, the NIFE trial evaluates the potential of
nanoliposomal-irinotecan/5-FU/leucovorin in the first
line therapy of advanced BTCs and additionally offers a
unique chance for translational research. Page 6 of 7 Perkhofer et al. BMC Cancer (2019) 19:990 Page 6 of 7 Perkhofer et al. BMC Cancer (2019) 19:990 Ethics approval and consent to participate
h
h
f
l
h kk The ethics committee of Ulm University (Ethikkommission der Universität
Ulm) approved the NIFE-trial as leading ethics committee for all German sites
(reference number 11/17). In addition, local ethics committees approved the
participating sites. The local ethics committees are as follows: Ethikkommis-
sion bei der Sächsischen Landesärztekammer, Ethikkommission der Bayer-
ischen Landesärztekammer, Ethikkommission des Fachbereichs Medizin der
Philipps-Universität Marburg, Ethikkommission der Landesärztekammer
Baden-Württemberg, Ethikkommission der Medizinischen Fakultät der HHU
Düsseldorf, Ethikkommission des Fachbereichs Medizin der Goethe-
Universität Frankfurt am Main, Ethikkommission der Ärztekammer
Mecklenburg-Vorpommern, Ethikkommission der Ärztekammer Westfalen-
Lippe und der Westfälischen Wilhelms-Universität Münster, Ethikkommission
der Ärztekammer Nordrhein, Geschäftsstelle der Ethikkommission des Landes
Berlin, Ethikkommission der Medizinischen Fakultät der Universität Duisburg-
Essen Universitätsklinikum Essen, Ethikkommission des Landes Sachsen-
Anhalt c/o Landesamt für Verbraucherschutz, Ethikkommission der FSU Jena,
Ethikkommission bei der Ärztekammer Niedersachsen, Ethikkommission der
Medizinischen Fakultät der Universität zu Köln, Ethikkommission bei der
Landesärztekammer Hessen. The trial is registered at Clinicaltrials.gov
(NCT03044587) and the European Clinical Trials Database (2016–002467-34). All patients signed informed consent according to GCP. 9. Feisthammel J, et al. Irinotecan with 5-FU/FA in advanced biliary tract
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1 1Department of Internal Medicine I, Ulm University, Albert-Einstein-Allee 23,
89081 Ulm, Germany. 2Department of Internal Medicine and
Gastroenterology, Klinikum im Friedrichshain, Landsberger Allee 49, 10249
Berlin, Germany. 3Department of Gastroenterology and Endocrinology,
University of Marburg, Baldingerstraße, 35043 Marburg, Germany. 4Internal
Medicine I, Klinikum Ludwigsburg, Posilipostraße 4, 71640 Ludwigsburg,
Germany. 5Gesundheitszentrum St. Marien, Mariahilfbergweg 7, 92224
Amberg, Germany. 6Institute of Clinical Cancer Research (IKF) at Krankenhaus
Nordwest, UCT-University Cancer Center, Steinbacher Hohl 2-26, 60488
Frankfurt, Germany. 7Institute of Epidemiology and Medical Biometry, Ulm
University, Schwabstraße 13, 89081 Ulm, Germany. Received: 5 April 2019 Accepted: 9 September 2019 Received: 5 April 2019 Accepted: 9 September 2019 Ethics approval and consent to participate
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h
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h kk 14. Drummond DC, et al. Development of a highly active nanoliposomal
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WAT1 (WALLS ARE THIN1) defines a novel auxin transporter in plants and integrates auxin signaling in secondary wall formation in Arabidopsis fibers
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© 2011 Ranocha 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. Background suggested that WAT1 encoded a putative transporter
belonging to the Plant Metabolite Exporter family [3]. WAT1:GFP fusion protein experiments localized WAT1
on the tonoplast, confirming the prediction that WAT1
is a membrane protein. Although WAT1 is plant-speci-
fic, it shares structural similarities with bacterial amino
acid transporters in that it consists of ten transmem-
brane domains encompassed within a tandem Domain
of Unknown Function 6 (DUF6). Our knowledge of signaling mechanisms involved in
secondary cell wall (SCW) formation is quite limited. To
discover novel markers of SCW, a genomics approach
using Zinnia elegans xylogenic cultures was undertaken
that identified hundreds of gene candidates expressed at
the onset of secondary wall formation [1]. Arabidopsis
homologs and the corresponding T-DNA mutants for
each Zinnia gene were identified and the panel of Ara-
bidopsis cell wall mutants was subjected to developmen-
tal and wall-related phenotyping. To characterize WAT1 function, we recently tested its
capacity to transport tryptophan and/or auxin in both
yeast and Xenopusoocytes. Neither WAT1-expressing
yeast cells nor Xenopus oocytes were able to facilitate
radiolabeled Trp import or export. However, we have
been able to demonstrate that WAT1 facilitates auxin
import in both expression systems. These results clearly
place WAT1 among the ranks, along with PINs, AUX/
LAXs andABCB/MDR/PGPs, as a novel, bona fide auxin
transporter in plants. ORAL PRESENTATION Open Access WAT1 (WALLS ARE THIN1) defines a novel auxin
transporter in plants and integrates auxin
signaling in secondary wall formation in
Arabidopsis fibers Philippe Ranocha1, Oana Dima2, Judith Felten3, Amandine Freydier1, Laurent Hoffmann1, Karin Ljung3,
Benoit Lacombe4, Claire Corratgé4, Jean-Baptiste Thibaud4, Björn Sundberg3, Wout Boerjan2, Deborah Goffner1* From IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery
Arraial d Ajuda, Bahia, Brazil. 26 June - 2 July 2011 * Correspondence: goffner@scsv.ups-tlse.fr
1Université de Toulouse; UPS; UMR 5546, Surfaces Cellulaires et Signalisation
chez les Végétaux; BP 42617, F-31326, Castanet-Tolosan, France
Full list of author information is available at the end of the article Ranocha et al. BMC Proceedings 2011, 5(Suppl 7):O24
http://www.biomedcentral.com/1753-6561/5/S7/O24 Ranocha et al. BMC Proceedings 2011, 5(Suppl 7):O24
http://www.biomedcentral.com/1753-6561/5/S7/O24 Results and conclusion Among the most interesting mutants was wat1 (walls
are thin1). The most conspicuous phenotypic feature of
wat1 was the severe reduction (sometimes to the extent
of being inexistent) of SCW in xylary and interfascular
stem fibers. Interestingly, xylem vessel wall thickness
and morphology were not modified by the mutation. In
addition to the SCW phenotype, wat1 was characterized
by 5-Me-tryptophan seedling toxicity, severely decreased
auxin transport and content in stems, and massive
down-regulation of auxin-related genes. These data led
us to the conclusion that WAT1 acts as an upstream
regulator of SCW deposition in fibers, presumably
through an auxin-mediated mechanism [2]. This study constitutes the first functional characteriza-
tion of any of the 46 members of the WAT1 gene family
in Arabidopsis and our hope is that this discovery will
help pave the way in identifying the functions of other
family members. Moreover, the wat1 mutant will be an
ideal tool to address the question as to how auxin sub-
cellular homeostasis plays a role in fiber SCW formation
in Arabidopsis. Our current efforts to understand poplar
WAT1-mediated auxin signaling in wood formation in
trees will also be discussed. Bioinformatic analysis of WAT1, annotated as ‘homo-
log to a Medicago truncatula nodulin gene, MtNOD21, * Correspondence: goffner@scsv.ups-tlse.fr
1Université de Toulouse; UPS; UMR 5546, Surfaces Cellulaires et Signalisation
chez les Végétaux; BP 42617, F-31326, Castanet-Tolosan, France
Full list of author information is available at the end of the article Page 2 of 2 Page 2 of 2 Ranocha et al. BMC Proceedings 2011, 5(Suppl 7):O24
http://www.biomedcentral.com/1753-6561/5/S7/O24 Published: 13 September 2011 Published: 13 September 2011 Author details
1 1Université de Toulouse; UPS; UMR 5546, Surfaces Cellulaires et Signalisation
chez les Végétaux; BP 42617, F-31326, Castanet-Tolosan, France. 2VIB
Department of Plant Systems Biology, UGent Department of Plant
Biotechnology and Genetics, 9052 Gent, Belgium. 3Umeå Plant Science
Center, Department of Forest Genetics and Plant Physiology, Swedish
University of Agricultural Sciences, 90183 Umeå, Sweden. 4Biochimie et
Physiologie Moléculaire des Plantes, CNRS UMR 5004, Institut National de la
Recherche Agronomique U386, Montpellier SupAgro, Université Montpellier
II, Place Viala, 34060 Montpellier Cedex, France. References References
1. Pesquet E, Ranocha P, Legay S, Digonnet C, Barbier O, Pichon M, Goffner D:
Novel markers of xylogenesis in Zinnia elegansare differentially regulated
by auxin and cytokinin. Plant Physiol 2005, 139:1821-1839. 2. Ranocha Ph, Denancé N, Vanholme R, Freydier A, Martinez Y, Hoffmann L,
Köhler L, Pouzet C, Renou JP, Sundberg B, Boerjan W, Goffner D: Walls Are
Thin1 (WAT1), an Arabidopsis homolog of Medicago truncatula
NODULIN21, regulates auxin homeostasis and is required for secondary
wall formation in fibers. Plant J 2010, 63:468-483. 3. Saier MH, Tran CV, Barabote RD: TCDB: the Transporter Classification
Database for membrane transport protein analyses and information. Nucleic Acids Res 2006, 34(Database issue):D181-186. doi:10.1186/1753-6561-5-S7-O24
Cite this article as: Ranocha et al.: WAT1 (WALLS ARE THIN1) defines a
novel auxin transporter in plants and integrates auxin signaling in
secondary wall formation in Arabidopsis fibers. BMC Proceedings 2011 5
(Suppl 7):O24. References
1. Pesquet E, Ranocha P, Legay S, Digonnet C, Barbier O, Pichon M, Goffner D:
Novel markers of xylogenesis in Zinnia elegansare differentially regulated
by auxin and cytokinin. Plant Physiol 2005, 139:1821-1839. 2. Ranocha Ph, Denancé N, Vanholme R, Freydier A, Martinez Y, Hoffmann L,
Köhler L, Pouzet C, Renou JP, Sundberg B, Boerjan W, Goffner D: Walls Are
Thin1 (WAT1), an Arabidopsis homolog of Medicago truncatula
NODULIN21, regulates auxin homeostasis and is required for secondary
wall formation in fibers. Plant J 2010, 63:468-483. 3. Saier MH, Tran CV, Barabote RD: TCDB: the Transporter Classification
Database for membrane transport protein analyses and information. Nucleic Acids Res 2006, 34(Database issue):D181-186. doi:10.1186/1753-6561-5-S7-O24
Cite this article as: Ranocha et al.: WAT1 (WALLS ARE THIN1) defines a
novel auxin transporter in plants and integrates auxin signaling in
secondary wall formation in Arabidopsis fibers. BMC Proceedings 2011 5
(Suppl 7):O24. 1. Pesquet E, Ranocha P, Legay S, Digonnet C, Barbier O, Pichon M, Goffner D:
Novel markers of xylogenesis in Zinnia elegansare differentially regulated
by auxin and cytokinin. Plant Physiol 2005, 139:1821-1839. 2. Ranocha Ph, Denancé N, Vanholme R, Freydier A, Martinez Y, Hoffmann L,
Köhler L, Pouzet C, Renou JP, Sundberg B, Boerjan W, Goffner D: Walls Are
Thin1 (WAT1), an Arabidopsis homolog of Medicago truncatula
NODULIN21, regulates auxin homeostasis and is required for secondary
wall formation in fibers. Plant J 2010, 63:468-483. 3. Saier MH, Tran CV, Barabote RD: TCDB: the Transporter Classification
Database for membrane transport protein analyses and information. References Nucleic Acids Res 2006, 34(Database issue):D181-186. doi:10.1186/1753-6561-5-S7-O24
Cite this article as: Ranocha et al.: WAT1 (WALLS ARE THIN1) defines a
novel auxin transporter in plants and integrates auxin signaling in
secondary wall formation in Arabidopsis fibers. BMC Proceedings 2011 5
(Suppl 7):O24. doi:10.1186/1753-6561-5-S7-O24
Cite this article as: Ranocha et al.: WAT1 (WALLS ARE THIN1) defines a
novel auxin transporter in plants and integrates auxin signaling in
secondary wall formation in Arabidopsis fibers. BMC Proceedings 2011 5
(Suppl 7):O24. Submit your next manuscript to BioMed Central
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The impact of socioeconomic deprivation on the risk of atrial fibrillation in patients with diabetes mellitus: a nationwide population-based study
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Citation for published version (APA):
Han, M., Lee, S.-R., Choi, E.-K., Park, S.-H., Lee, H., Chung, J., Choi, J., Han, K.-D., Oh, S., & Lip, G. Y. H.
(2022). The impact of socioeconomic deprivation on the risk of atrial fibrillation in patients with diabetes mellitus:
A nationwide population-based study. Frontiers in Cardiovascular Medicine, 9, Article 1008340.
https://doi.org/10.3389/fcvm.2022.1008340 The impact of socioeconomic deprivation on the risk of atrial fibrillation in patients
with diabetes mellitus
A nationwide population-based study DOI (link to publication from Publisher):
10.3389/fcvm.2022.1008340 DOI (link to publication from Publisher):
10.3389/fcvm.2022.1008340 Document Version
Publisher's PDF, also known as Version of record Citation for published version (APA):
Han, M., Lee, S.-R., Choi, E.-K., Park, S.-H., Lee, H., Chung, J., Choi, J., Han, K.-D., Oh, S., & Lip, G. Y. H. (2022). The impact of socioeconomic deprivation on the risk of atrial fibrillation in patients with diabetes mellitus:
A nationwide population-based study. Frontiers in Cardiovascular Medicine, 9, Article 1008340. https://doi.org/10.3389/fcvm.2022.1008340 General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners
and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Aalborg Universitet Aalborg Universitet The impact of socioeconomic deprivation on the risk of
with diabetes mellitus
A nationwide population-based study
Han, Minju; Lee, So-Ryoung; Choi, Eue-Keun; Park, Sang-H
Jaewook; Choi, JungMin; Han, Kyung-Do; Oh, Seil; Lip, Gre
Published in:
Frontiers in Cardiovascular Medicine
DOI (link to publication from Publisher):
10.3389/fcvm.2022.1008340
Creative Commons License
CC BY 4.0
Publication date:
2022
Document Version
Publisher's PDF, also known as Version of record
Link to publication from Aalborg University
Citation for published version (APA):
Han, M., Lee, S.-R., Choi, E.-K., Park, S.-H., Lee, H., Chung, J., Choi, J
(2022). The impact of socioeconomic deprivation on the risk of atrial fibri
A nationwide population-based study. Frontiers in Cardiovascular Medic
https://doi.org/10.3389/fcvm.2022.1008340 The impact of socioeconomic deprivation on the risk of atrial fibrillation in patients
with diabetes mellitus
A nationwide population-based study
Han, Minju; Lee, So-Ryoung; Choi, Eue-Keun; Park, Sang-Hyeon; Lee, HuiJin; Chung,
Jaewook; Choi, JungMin; Han, Kyung-Do; Oh, Seil; Lip, Gregory Y. H. Published in:
Frontiers in Cardiovascular Medicine The impact of socioeconomic
deprivation on the risk of atrial
fibrillation in patients with
diabetes mellitus: A nationwide
population-based study OPEN ACCESS
EDITED BY
Yongjun Jiang,
The Second Affiliated Hospital
of Guangzhou Medical University,
China Minju Han1†, So-Ryoung Lee1†, Eue-Keun Choi1,2*,
Sang-Hyeon Park1, HuiJin Lee1, Jaewook Chung1,
JungMin Choi1, Kyung-Do Han3, Seil Oh1,2 and
Gregory Y. H. Lip1,2,4,5 1Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea,
2Department of Internal Medicine, Seoul National University College of Medicine, Seoul,
South Korea, 3Statistics and Actuarial Science, Soongsil University, Seoul, South Korea, 4Liverpool
Centre for Cardiovascular Science, Liverpool Heart and Chest Hospital, University of Liverpool,
Liverpool, United Kingdom, 5Department of Clinical Medicine, Aalborg University, Aalborg, Denmark Objective: To evaluate the relationship between socioeconomic status and
the risk of atrial fibrillation (AF) in patients with diabetes mellitus (DM). Han M, Lee S-R, Choi E-K, Park S-H,
Lee H, Chung J, Choi J, Han K-D,
Oh S and Lip GYH (2022) The impact
of socioeconomic deprivation on
the risk of atrial fibrillation in patients
with diabetes mellitus: A nationwide
population-based study. Research design and methods: From the National Health Insurance Service
(NHIS) database, we identified 2,429,610 diabetic patients who underwent
national health check-ups between 2009 and 2012. Tracing back the subjects
for 5 years from the date of health check-up, we determined the subjects’
income and whether they received medical aid (MA) during the past 5 years. Subjects were divided into six groups according to the number of years
of receiving (MA groups 0 through 5) and into four groups according to
socioeconomic status change during the past 5 years. We estimated the risk
of AF for each group using the Cox proportional-hazards model. Front. Cardiovasc. Med. 9:1008340. doi: 10.3389/fcvm.2022.1008340 © 2022 Han, Lee, Choi, Park, Lee,
Chung, Choi, Han, Oh and Lip. 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. © 2022 Han, Lee, Choi, Park, Lee,
Chung, Choi, Han, Oh and Lip. This is
an open-access article distributed
nde the te ms of the C eati e Results: During a median follow-up of 7.2 ± 1.7 years, 80,257 were newly
identified as AF. General rights
C
i h
d - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study
You may not further distribute the material or use it for any profit making activity or commercial gain You may not further distribute the material or use it for any profit making activity o
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the work immediately and investigate your claim. y
this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to
ely and investigate your claim. TYPE Original Research
PUBLISHED 16 November 2022
DOI 10.3389/fcvm.2022.1008340 TYPE Original Research
PUBLISHED 16 November 2022
DOI 10.3389/fcvm.2022.1008340 Data source and study population The
prevalence
of
atrial
fibrillation
(AF)
and
AF-
related morbidity and mortality is increasing globally. It
is associated with an increasingly older adult population,
more prevalent comorbidities, and unhealthy lifestyles (1,
2). For example, the Framingham study identified aging and
cardiovascular comorbidities such as hypertension, diabetes
mellitus (DM), coronary artery disease, and valvular heart
disease as the most potential risk factors for the development
of AF (3). We used data from the National Health Insurance Service
(NHIS) database. The Korean NHIS is a compulsory public
medical assistance system with over 51 million Koreans
currently participating. In addition, the Korean National Health
Insurance Corporation provides annual or biennial national
health examinations for people over the age of 20, and these data
are linked to the NHIS database. We
identified
2,429,610
subjects
with
DM
and
without prevalent AF, who underwent a national health
examination at least once between 2009 and 2012. Patients
younger than 20 years of age, those with missing values
among covariates, and those diagnosed with AF within
1
year
after
the
health
examination
were
excluded. Figure 1 shows the flowchart of study enrolment. This
study was exempt from review by the Seoul National
University
Hospital
Institutional
Review
Board
(E-
2105-141-1220). Diabetes mellitus is known to increase the risk of AF by
28% (4, 5) and is an important factor in the prognosis of
AF. In addition, the risk of AF is increased in patients with
poorly controlled DM and macrovascular and microvascular
complications (6). Prevention of incident AF and appropriate
management of the prevalent AF in patients with diabetes
would lower the risk of morbidity and mortality. Indeed,
this holistic or integrated care approach to AF care is being
increasingly promoted (7, 8), given that adherence to holistic
management is associated with improved clinical outcomes
(9, 10). Among
the
non-disease
risk
factors
for
AF,
many
studies have been conducted on whether the risk of AF
changes according to health inequalities and socioeconomic
status (11–16). Although socioeconomic status shows an
inverse
relationship
with
overall
morbidity,
mortality,
and cardiovascular diseases (17, 18), the results on the
relationship between socioeconomic status and AF remain
controversial. Previous studies have shown that a lower
income is associated with a higher risk of AF (11, 12). However, a weak relationship between a low socioeconomic
status and the risk of AF has also been reported (13). The impact of socioeconomic
deprivation on the risk of atrial
fibrillation in patients with
diabetes mellitus: A nationwide
population-based study The MA groups showed a higher risk of AF than the non-
MA group with the hazard ratios (HRs) and 95% confidence interval (CI) 1.32
(1.2–1.44), 1.33 (1.22–1.45), 1.23 (1.13–1.34), 1.28 (1.16–1.4), and 1.50 (1.39–
1.63) for MA groups 1 through 5, respectively. Dividing subjects according
to socioeconomic condition change, those who experienced worsening
socioeconomic status (non-MA to MA) showed higher risk compared to the
persistent non-MA group (HR 1.54; 95% CI 1.38–1.73). Frontiers in Cardiovascular Medicine frontiersin.org 01 Han et al. 10.3389/fcvm.2022.1008340 10.3389/fcvm.2022.1008340 Conclusion: Low socioeconomic status was associated with the risk of AF in
patients with diabetes. More attention should be directed at alleviating health
inequalities, targeting individuals with socioeconomic deprivation to provide
timely management for AF. socioeconomic status, atrial fibrillation, social medicine, diabetes mellitus, medical
aid beneficiaries Data source and study population Nevertheless,
low
socioeconomic
status
notably
elevates
the prevalence of various diseases, including DM, obesity, and
depression (19, 20), and these diseases are risk factors for AF
(3, 21). Assessment of covariates completed as part of the national health examination (26). Alcohol consumption of less than 30 g per day was defined as
mild drinking, and 30 g or more was defined as heavy drinking. Regular physical activity was defined as performing moderate-
intensity exercise more than five times a week or vigorous-
intensity exercise more than three times a week (26). Detailed
definitions of the diagnoses, including AF and comorbidities,
such as hypertension and dyslipidaemia, are presented in
Supplementary Table 1 (24). Covariates included age, sex, smoking status, drinking
status, regular exercise, comorbidities of hypertension and
dyslipidaemia, body mass index (BMI), waist circumference,
systolic blood pressure (SBP), diastolic blood pressure (DBP),
fasting blood glucose level, total cholesterol, and high and low-
density lipoprotein (HDL and LDL) cholesterol (24). The details
of the subjects’ DM were analyzed to determine whether they
had been diagnosed with DM 5 years or more, whether the
subject was taking DM medication, and whether the number
of diabetic medications was three or more (25). Smoking
status (never, ex, or current smoker), drinking status, and
physical activity were assessed using a self-report questionnaire Definition of medical aid beneficiary Medical aid (MA) is a public assistance system that the
state guarantees for the medical problems of low-income people
who cannot sustain life or have difficulties in living (22). People
who are unable to work and homeless are usually recipients of
the MA; the recipient household’s income must be less than
40% of the median national household income to benefit from
the MA (23). By tracing back 5 years from the index date of the
subject’s health examination, we obtained the participants’
household income status and categorized the study population
into six groups according to the number of years the
subject was a beneficiary of the MA program (0 through 5;
named non-MA, MA 1, MA 2, MA 3, MA 4, and MA 5
groups, respectively). The detailed study design is illustrated in
Figure 1. We investigated the association between socioeconomic
status and AF risk in patients with diabetes using a population-
based nationwide cohort study. Frontiers in Cardiovascular Medicine frontiersin.org 02 Han et al. 10.3389/fcvm.2022.1008340 FIGURE 1
Study design and flowchart of enrolment. Study outcome and follow-up The primary outcome was the occurrence of incident AF
during the follow-up period. AF was defined as at least one Frontiers in Cardiovascular Medicine frontiersin.org 03 10.3389/fcvm.2022.1008340 Han et al. classified into four groups: initial MA recipients and MA
recipients
later
(persistent
MA
group),
initial
non-MA
recipients
but
later
becoming
MA
recipients
(non-MA
to MA group), initial MA recipients but later becoming
non-MA recipients (MA to non-MA group), and initial
non-MA recipients and non-MA recipients later (persistent
non-MA group). In these three complementary analyses,
the multivariable-adjusted HRs of incident AF among the
groups were estimated. hospitalization or at least two outpatient clinic visits with
diagnostic codes of AF (I480-I484 and I489), according to the
International Classification of Disease, 10th Revision (ICD-10)
(6, 27, 28). Subjects were followed up from the index date of
the national health examination until the occurrence of AF,
death, or the end of the study period (31 December 2018),
whichever came first. Subgroup analyses Baseline characteristics were described across groups with
different numbers of years of receiving MA. Continuous
variables were expressed as mean ± standard deviation,
and categorical variables were presented as numbers and
percentages. Differences among the groups were examined
using analysis of variance (ANOVA). The crude incidence
rate (IR) of incident AF was calculated as the number of
events per 1,000 person-years (PY). To analyze the association
between the number of years of receiving MA and the risk
of incident AF, we used univariate and multivariate Cox
proportional hazard regression models. The outcomes for
the groups were presented as hazard ratios (HRs) and 95%
confidence intervals (CIs). Adjustments were made for the
covariates of age, sex, hypertension, dyslipidaemia, BMI, fasting
blood glucose level, smoking, drinking status, and regular
physical activity. We performed subgroup analyses and interaction tests to
evaluate the potential impact of age, sex, duration of DM,
insulin use, use of three or more antidiabetic medications, and
comorbidities, including hypertension and dyslipidaemia, on
the relationship between socioeconomic status and the risk of
AF. P for interaction less than 0.1 was considered significant. Results Among a total of 2,429,610 subjects (mean age 56.9 ± 12.4,
60% men), 2,364,792 did not have a history of MA (97.3%, non-
MA group), and 10,697 subjects had a history of MA at least
for 1 year (0.4%, MA 1 group), 11,005 for 2 years (0.5%, MA 2
group), 12,431 for 3 years (0.5%, MA 3 group), 10,689 for 4 years
(0.4%, MA 4 group), and 19,996 subjects benefited from MA
for 5 years (0.8%, MA 5 group) (Figure 1). Table 1 shows the
baseline characteristics of each group. The level of significance was set at 0.05 and all analyses were
two-sided. Statistical analyses were conducted using SAS version
9.4 (SAS Institute, Cary, NC, USA). Compared with the non-MA group, the MA group had
a higher prevalence of hypertension and dyslipidaemia and a
lower prevalence of regular physical activity. The proportion
of obese people with BMI ≥25 kg/m2 was significantly
higher in the MA 5 group (48.6% in the non-MA group,
46.6% in the MA 1, 47.2% in the MA 2 group, 47.3% in
the MA 3 group, 47.3% in the MA 4 group, and 50.2% in
the MA 5 group; p < 0.0001). The proportion of patients
with a DM duration of 5 years or longer was significantly
higher in the MA group than in the non-MA group (29.9
vs. 32.9 to 45.4% in the MA ≥1 group, p < 0.0001). The
proportion of subjects taking three or more antidiabetic drugs
was higher in the MA group than in the non-MA group (18.6
to 24.7% in the MA group vs. 13.7% in the non-MA group,
p < 0.0001). Complementary analyses We further conducted multiple complementary analyses
to investigate the associations between various indicators
of SES and the risk of incident AF. First, to check whether
there
is
a
relationship
between
various
income
levels
and AF risk, participants were divided into 21 groups
according
to
health
insurance
premiums
paid
in
the
index year (the year of national health examination): the
MA group and income level 1 through 20 groups, with
higher
numbers
indicating
higher
insurance
premiums. We cross-sectionally analyzed the effect of income level
on the risk of AF in the index year. Second, to check
whether low income–applying a more lenient definition
than MA beneficiary–defined as paying the bottom 20% of
health insurance premiums also affects the risk of AF, we
divided the participants into six groups according to the
number of years of low income. Lastly, to check whether
changes in socioeconomic status affect AF risk, whether
the subjects received MA in the index year and 4 years
ago from the index year were investigated. Subjects were The baseline characteristics based on the occurrence of AF
during the follow-up period are summarized in Supplementary
Table 2. Patients who developed new AF during follow-up
(AF group) were older than those who did not develop the
arrhythmia (non-AF group) (65.1 ± 10.32 years in AF group
vs. 56.59 ± 12.39 years in non-AF group, p < 0.0001), while
the sex ratio between groups was similar (men 59.93% in AF Frontiers in Cardiovascular Medicine frontiersin.org 04 Han et al. 10.3389/fcvm.2022.1008340 TABLE 1 Baseline characteristics of the subjects grouped by the number of times receiving medical aid. Complementary analyses Cumulative medical aid burden
0
1
2
3
4
5
P-value
n = 2,364,792
n = 10,697
n = 11,005
n = 12,431
n = 10,689
n = 19,997
Age
56.82 ± 12.44
58.5 ± 13.18
59.82 ± 13.11
60.52 ± 12.39
60.1 ± 12.41
56.49 ± 8.73
<0.0001
<40
199,126 (8.42%)
705 (6.59%)
545 (4.95%)
417 (3.35%)
376 (3.52%)
326 (1.63%)
<0.0001
40–64
1,486,221 (62.85%)
6,042 (56.48%)
6,091 (55.35%)
7,022 (56.49%)
6,049 (56.59%)
14,616 (73.09%)
≥65
679,445 (28.73%)
3,950 (36.93%)
4,369 (39.7%)
4,992 (40.16%)
4,264 (39.89%)
5,054 (25.28%)
Male sex
1,429,887 (60.47%)
4,938 (46.16%)
4,642 (42.18%)
5,068 (40.77%)
4,341 (40.61%)
9,195 (45.98%)
<0.0001
Smoking
<0.0001
Never
1,305,493 (55.21%)
6,633 (62.01%)
7,133 (64.82%)
8,220 (66.13%)
7,104 (66.46%)
11,920 (59.61%)
Former
428,469 (18.12%)
1,260 (11.78%)
1,250 (11.36%)
1,355 (10.9%)
1,106 (10.35%)
2,332 (11.66%)
Current
630,830 (26.68%)
2,804 (26.21%)
2,622 (23.83%)
2,856 (22.97%)
2,479 (23.19%)
5,744 (28.73%)
Drinking
Non-MA
1,333,833 (56.4%)
7,390 (69.08%)
7,965 (72.38%)
9,132 (73.46%)
7,866 (73.59%)
14,878 (74.4%)
Mild
787,339 (33.29%)
2,464 (23.03%)
2,341 (21.27%)
2,476 (19.92%)
2,158 (20.19%)
3,813 (19.07%)
Heavy
243,620 (10.3%)
843 (7.88%)
699 (6.35%)
823 (6.62%)
665 (6.22%)
1,305 (6.53%)
Regular exercise
488,724 (20.67%)
1,645 (15.38%)
1,706 (15.5%)
1,922 (15.46%)
1,627 (15.22%)
3,159 (15.8%)
<0.0001
Hypertension
1,295,148 (54.77%)
6,502 (60.78%)
7,033 (63.91%)
8,184 (65.84%)
6,970 (65.21%)
12,848 (64.25%)
<0.0001
Dyslipidemia
935,703 (39.57%)
4,720 (44.12%)
5,081 (46.17%)
5,980 (48.11%)
5,306 (49.64%)
10,978 (54.9%)
<0.0001
BMI, kg/m2
25.06 ± 3.88
24.92 ± 3.79
24.96 ± 3.88
25.01 ± 3.87
24.98 ± 3.91
25.29 ± 4.13
<0.0001
BMI < 18.5
36,462 (1.54%)
323 (3.02%)
309 (2.81%)
354 (2.85%)
303 (2.83%)
616 (3.08%)
18.5 ≤BMI < 23
588,690 (24.89%)
2,941 (27.49%)
3,124 (28.39%)
3,441 (27.68%)
3,039 (28.43%)
5,231 (26.16%)
23 ≤BMI < 25
589,915 (24.95%)
2,442 (22.83%)
2,383 (21.65%)
2,758 (22.19%)
2,295 (21.47%)
4,119 (20.6%)
25 ≤BMI < 30
917,367 (41.08%)
4,001 (37.4%)
4,147 (37.68%)
4,667 (37.54%)
4,023 (37.64%)
7,582 (37.92%)
30 ≤BMI
178,358 (7.54%)
990 (9.25%)
1,042 (9.47%)
1,211 (9.74%)
1,029 (9.63%)
2,448 (12.24%)
Waist circumference, cm
85.34 ± 8.88
84.93 ± 9.43
85.16 ± 12
85.1 ± 9.53
85.02 ± 12.14
85.85 ± 10.03
<0.0001
SBP, mmHg
129.01 ± 15.87
128.42 ± 16.65
128.51 ± 16.57
128.59 ± 16.76
128.26 ± 16.57
126.42 ± 16.37
<0.0001
DBP, mmHg
79.11 ± 10.29
78.49 ± 10.49
78.32 ± 10.33
78.38 ± 10.4
78.2 ± 10.4
77.73 ± 10.42
<0.0001
Glucose, mg/dL
137.98 ± 47.89
141.09 ± 54.27
141.33 ± 54.79
140.62 ± 54.06
141.22 ± 55.33
141.23 ± 54.78
<0.0001
Total cholesterol, mg/dL
197.53 ± 46.02
195.32 ± 44.51
193.52 ± 49.97
192.12 ± 45.62
192.77 ± 49.37
187.7 ± 44.84
<0.0001
HDL, mg/dL
52.49 ± 31.91
52.46 ± 28.36
52.01 ± 25.33
52.03 ± 47.87
51.99 ± 24.56
51.21 ± 39.87
<0.0001
LDL, mg/dL
113.81 ± 92.14
111.24 ± 63.33
110.58 ± 62.65
110.49 ± 132.27
108.6 ± 51.75
104.61 ± 43.59
<0.0001
Diabetes mellitus
<0.0001
Disease duration ≥5 year
706,448 (29.87%)
3,522 (32.93%)
4,140 (37.62%)
5,021 (40.39%)
3,787 (35.43%)
9,069 (45.35%)
<0.0001
OnDM medication
1,280,126 (54.13%)
7,054 (65.94%)
7,661 (69.61%)
9,240 (74.33%)
7,835 (73.3%)
16,345 (81.74%)
<0.0001
≥3DM medications
323,077 (13.66%)
1,988 (18.58%)
2,211 (20.09%)
2,762 (22.22%)
2,393 (22.39%)
4,931 (24.66%)
<0.0001 The number of years of receiving
medical aid and the risk of incident
atrial fibrillation group vs. 60.02% in non-AF group, p = 0.6272). The incidence
of underlying hypertension and dyslipidaemia was higher in
the AF group (hypertension 72.34% in AF group vs. 54.42%,
p < 0.0001; dyslipidaemia 42.21% in AF group vs. 39.75%,
p < 0.0001). Finally, the proportion of patients who were
diagnosed with diabetes more than 5 years ago and who used
three or more antidiabetics was also higher in the AF group
(duration of diabetes ≥5 years 39.31% in the AF group vs. 29.81% in the non-AF group, p < 0.0001; ≥three antidiabetics
16.52% in the AF group vs. 13.8% in the non-AF group,
p < 0.0001). During the mean of 7.2 ± 1.7 years of follow-up (17,436,758
PY), AF was newly diagnosed in 80,257 patients (3.30%). The
crude IR and the unadjusted and adjusted HRs for each group
are summarized in Figure 2. The risk of AF was higher by 23% to
50% in the MA groups than in the non-MA group: the adjusted
HRs (95% CI) in the MA 1 group 1.32 (1.20–1.44); 1.33 (1.22–
1.45) in the MA 2 group; 1.23 (1.13–1.34) in the MA 3 group; Frontiers in Cardiovascular Medicine frontiersin.org 05 Han et al. 10.3389/fcvm.2022.1008340 1.28 (1.16–1.4) in the MA 4 group, and 1.50 (1.39–1.63) in the
MA 5 group. Notably, the MA 5 group showed the highest risk
of AF compared to the non-MA group. received MA in the index year and 4 years before the index
year (Figure 3). Subjects who were not MA beneficiaries
both in the index year and 4 years before the index year
(persistent non-MA group) were regarded as the reference
group. Discussion Patients with low income for 2, 3, 4, and 5 years were
associated with a higher risk of AF compared to subjects
without low income for 5 years: the adjusted HRs (95% CI)
in patients with low income for 2 years 1.04 (1.01–1.07),
for 3 years 1.07 (1.03–1.10), for 4 years 1.04 (1.01–1.08),
and for 5 years 1.09 (1.06–1.12), whereas those with low
income for 1 year did not show a significant difference. The
association between the number of years with low income
and incident AF was significantly attenuated compared with
the association between the number of years with MA,
and the risk of AF. In this study, we investigated the effect of SES on the risk of
incident AF. The main findings of the study were as follows: (1)
the MA group showed a 23–50% higher risk of AF compared to
the non-MA group; (2) the cumulative burden of MA exposure
up to five times showed the highest risk of AF compared to
the non-MA group; (3) the risk of incident AF increased by
1.57 times in the MA group compared to the group with the
highest income; and (4) those who became non-MA group
during follow-up still had an increased risk of AF compared to
the non-MA group, suggesting a legacy effect of MA on the risk
of AF. To the best of our knowledge, this is the first study to report
the impact of the cumulative burden of MA exposure on AF
risk, especially in patients with diabetes. Our study supports
the alleviation of health inequalities by targeting individuals
with socioeconomic deprivation in order to provide timely
management of AF. Subgroup analyses Compared with group 20, the group of subjects estimated
to have the highest income, the adjusted HRs of groups 1 to 19
showed an increasing trend of AF risk, whereas the MA group
showed a 57% higher risk of AF (adjusted HR 1.57, 95% CI,
1.47–1.68, Supplementary Figure 1). Subgroup analyses were performed for age, sex, diabetes
duration, insulin use, three or more antidiabetic drugs,
hypertension, and dyslipidaemia (Supplementary Table 3). There was no significant interaction between the subgroups for
each item, except for age (p for interaction <0.001). Among
the age groups classified as <40, 40–64, ≥65 years, the 40–64-
year age group and ≥65-year group showed the same trend as
the main result, whereas the group under 40-year-old of age
did not show an association between the risk of AF and the
number of MA history. According to MA, the increase in AF risk
was slightly attenuated in those aged ≥65 years and was most
pronounced in the 40–64-year-old group. Income levels at the index year and the
risk of incident atrial fibrillation The AF risk was higher in subjects who became the new
MA group in the index year or who continuously belonged to
MA groups (non-MA to MA group, adjusted HR 1.54, 95%
CI 1.38–1.73; and persistent MA group 1.51, 95% CI 1.40–
1.63). Subjects in the non-MA group at index year (MA to
non-MA group) still had a 20% higher AF risk than those
who were persistently in the non-MA group (adjusted HR 1.22,
95% CI 1.16–1.30). To understand the relationship between income level and
the risk of AF, we divided the participants who did not receive
MA as of the index year (the year of the national health
examination) into 20 groups according to their income level. The income level was estimated using the amount of health
insurance premiums paid. We analyzed the risk of AF among
21 groups, comprising the MA beneficiary group (MA group)
and income level 1–20 groups. The number of years with low income
and the risk of incident atrial fibrillation To check whether low income, which is defined as patients
with income levels of less than 20% of the entire Korean
population, also affects the risk of AF, we divided the total
population into six groups in the same way according to the
number of years of low income (Supplementary Figure 2). This applies a more lenient definition than that of the MA
beneficiary criterion. Changes of socioeconomic status
assessed with or without receiving
medical aid and the risk of atrial
fibrillation Diabetes is associated with worsening of AF prognosis
and is a potent risk factor for AF (29, 30). Patients with
AF and underlying DM also showed a higher risk of stroke To determine whether changes in socioeconomic status
affected the risk of AF, we investigated whether the subject Frontiers in Cardiovascular Medicine frontiersin.org 06 Han et al. 10.3389/fcvm.2022.1008340 FIGURE 2
The risk of atrial fibrillation according to the cumulative burden of medical aid. Adjusted model corrected for age, sex, body mass index, blood
glucose level, smoking, drinking, regular physical activity, hypertension, dyslipidemia, insulin use, ≥3 antidiabetic medication, and ≥5 years of
diabetes mellitus duration. AF, atrial fibrillation; IR, incidence rate; PY, person-year; HR, hazard ratio; CI, confidence interval. FIGURE 3
Changes in socioeconomic status and the risk of atrial fibrillation. (A) Changes of socioeconomic status. (B) Hazard ratios according to the
changes of socioeconomic status. FIGURE 2
The risk of atrial fibrillation according to the cumulative burden of medical aid. Adjusted model corrected for age, sex, body mass index, blood
glucose level, smoking, drinking, regular physical activity, hypertension, dyslipidemia, insulin use, ≥3 antidiabetic medication, and ≥5 years of
diabetes mellitus duration. AF, atrial fibrillation; IR, incidence rate; PY, person-year; HR, hazard ratio; CI, confidence interval. FIGURE 2
The risk of atrial fibrillation according to the cumulative burden of medical aid. Adjusted model corrected for age, sex, body mass index, blood
glucose level, smoking, drinking, regular physical activity, hypertension, dyslipidemia, insulin use, ≥3 antidiabetic medication, and ≥5 years of
diabetes mellitus duration. AF, atrial fibrillation; IR, incidence rate; PY, person-year; HR, hazard ratio; CI, confidence interval. FIGURE 2
The risk of atrial fibrillation according to the cumulative burden of medical aid. Adjusted model corrected for age, sex, body mass index, blood
glucose level, smoking, drinking, regular physical activity, hypertension, dyslipidemia, insulin use, ≥3 antidiabetic medication, and ≥5 years of
diabetes mellitus duration. AF, atrial fibrillation; IR, incidence rate; PY, person-year; HR, hazard ratio; CI, confidence interval. FIGURE 3
Changes in socioeconomic status and the risk of atrial fibrillation. (A) Changes of socioeconomic status. (B) Hazard ratios according to the
changes of socioeconomic status. FIGURE 3
Changes in socioeconomic status and the risk of atrial fibrillation. (A) Changes of socioeconomic status. (B) Hazard ratios according to the
changes of socioeconomic status. FIGURE 3
Changes in socioeconomic status and the risk of atrial fibrillation. (A) Changes of socioeconomic status. Strengths and limitations Our study defined the cumulative burden of MA, enabling
us to longitudinally identify the subjects’ socioeconomic status
in the previous 5 years. Our study, which more comprehensively
determines the subjects’ socioeconomic status using the concept
of cumulative MA burden, confirms once again that there
is an inverse correlation between low socioeconomic status
and the risk of AF. In our study, the socioeconomic status of the subjects was
identified longitudinally by screening for 5 years, not at any
single time point. This method has the advantage of being able
to grasp the burden of the low socioeconomic status experienced
by the subjects during the period, and further being able to
determine whether there was a change in the socioeconomic
status during the period, so that the situation of the subjects
could be evaluated in a more diversified way. In addition, we
limited the subjects of this study to patients with diabetes. Since
DM is one of the notable risk factors for AF, setting this risk
factor as the subject’s prerequisite allowed us to focus more on
the influence of socioeconomic status. In addition, one of the
strengths of our study is that we identified the socioeconomic
status of the participants using the NHIS database (24). Instead
of collecting information on income through self-questionnaires
reported by participants, we improved the reliability of the
results by using a database that records the exact amount
of health insurance premium payments. The NHIS database
holds information on all citizens residing in Korea in all
age groups, reducing the possibility of selection bias, and the
resident registration number jointly recorded further ensures
the accuracy of information. Becoming a beneficiary of MA is accepting the status
of the socially underprivileged. The stigmatization of welfare
beneficiaries has been studied sociologically for decades. Previous studies have reported an association between living
on welfare, increased mental stress, and negative emotions (33,
34). The problem of poor self-care with health in people of
low socioeconomic status is also a frequently studied topic
(31, 41). The individual’s psychological stress and neglect
of healthcare accompanying the process of accepting a new
status as underprivileged might have increased the risk of AF. In addition, we found that an unhealthy lifestyle was more
prevalent in the MA group than in the non-MA group. Patients
in the MA group had a higher prevalence of current smoking
and non-regular exercise than those in the non-MA group. Changes of socioeconomic status
assessed with or without receiving
medical aid and the risk of atrial
fibrillation The group of patients who experienced an
improvement in socioeconomic status (MA to non-MA group)
still showed a 22% higher AF risk than the persistent non-MA
group. This suggests a prophylactic effect of MA on the risk
of AF, emphasizing that SES still has a considerable impact on
clinical outcomes in patients with diabetes. Changes of socioeconomic status
assessed with or without receiving
medical aid and the risk of atrial
fibrillation (B) Hazard ratios according to the
changes of socioeconomic status. The literature shows that many studies on SES and various
cardiovascular outcomes have shown an inverse correlation. A previous study reported an inverse relationship between
socioeconomic status and almost all risk factors for CVD,
including diabetes, obesity, smoking, and physical activity (36). More recently, in a population-based cohort study using data
from the US National Health and Nutrition Examination Survey
(US NHANES) and UK Biobank, adults with low socioeconomic
status and no healthy lifestyle factors showed a higher risk of
all-cause mortality and incident CVD higher (3.53 times and
2.09 times, respectively) than those with high socioeconomic
status and healthy lifestyle factors (37). This study also reported
that the influence of unhealthy lifestyle was smaller than that of
socioeconomic inequity. than those without diabetes (29, 30). Thus, the diagnosis and
appropriate management of AF in patients with diabetes are
important for improving clinical outcomes. Health inequalities
and socioeconomic status are also important factors influencing
the outcome of patients with diabetes. Socioeconomic status
has been associated with knowledge of diabetes, self-care, and
clinical outcomes in patients with type 2 DM (31). In particular,
diabetes outcomes comprising multiple components, such as
HbA1c level, LDL level, blood pressure, and the physical
and mental components of the QOL score, are correlated
with socioeconomic factors (32–35). In our study, we further
investigated the association between SES and the risk of AF in
patients with diabetes and found a strong correlation between
low SES and the risk of AF. Frontiers in Cardiovascular Medicine frontiersin.org 07 Han et al. Han et al. Han et al. 10.3389/fcvm.2022.1008340 10.3389/fcvm.2022.1008340 However, regarding the association between socioeconomic
status and AF risk, previous studies have reported controversial
results (11, 12, 38–40). Lower family income was associated
with a higher risk of AF (11). Residents of lower socioeconomic
status also had a higher risk of incident AF (12) and higher
mortality when hospitalized for AF (38). Regarding studies that
reported conflicting results, AF-related mortality was higher
in European countries with higher GDP (39). The inverse
relationship between socioeconomic status and AF risk was not
evident in older adult individuals with the highest prevalence
of AF (40). year showed different risks of AF according to their previous
socioeconomic status. Strengths and limitations This
is consistent with the study results that socioeconomic inequity
in various health outcomes is highly associated with lifestyle
factors, such as smoking, alcohol consumption, physical activity,
and diet (42–44). A previous study reported that smoking,
alcohol use, and physical activity were significantly associated
with new-onset AF (42). These findings imply that lifestyle
factors may have a therapeutic value in patients with diabetes. For example, abstinence from alcohol is associated with a lower
risk of developing AF in patients with newly diagnosed T2DM
(43). In patients with newly diagnosed AF, current alcohol intake
is associated with an increased risk of ischemic stroke, whereas
alcohol abstinence after AF diagnosis is associated with a lower
risk of ischemic stroke (44). This study had some limitations. As mentioned, the subjects
were limited to patients with diabetes at enrolment to focus
more on the impact of socioeconomic status on incident AF. Therefore, this study alone cannot explain whether the results
can be equally applied to the non-diabetic population. However,
since all subjects had diabetes, the correction for diabetes was
more reliable than when the entire population was enrolled. We
believe that even when conducting further research on whether
socioeconomic status affects the increase in AF risk among
non-diabetic individuals, only non-diabetics should be included
as subjects, since DM is a clear risk factor for AF (3, 5, 34)
and the correction of its impact might not be complete in the
coexistence of subjects with diabetes and those without diabetes
in the analysis. Interestingly, we found that AF risk differed according
to socioeconomic status change. First, the risk of AF was
similarly higher in those who received MA at the index year,
regardless of a previous history of MA. Current SES had a
more significant impact on the risk of AF than past SES. A worsening socioeconomic status would have resulted in
greater psychological pressure on the subjects, increasing the
risk of AF. Second, patients in the non-MA group at the index Low socioeconomic status is associated with the risk
of AF in patients with diabetes. More attention should
be directed at alleviating health inequalities and targeting
individuals with socioeconomic deprivation to provide timely
management of AF. Frontiers in Cardiovascular Medicine frontiersin.org 08 Han et al. 10.3389/fcvm.2022.1008340 Ethics statement Author E-KC research grants or speaking fees from Bayer,
BMS/Pfizer, Biosense Webster, Chong Kun Dang, Daiichi-
Sankyo, Dreamtech Co., Ltd., Medtronic, Samjinpharm, Sanofi-
Aventis, Seers Technology, and Skylabs. Author GL consultant
and speaker for BMS/Pfizer, Boehringer Ingelheim, and Daiichi-
Sankyo. No fees are received personally. This study was exempt from review by the Seoul National
University Hospital Institutional Review Board (E- 2105-141-
1220). Written informed consent was not required for this
study in accordance with the local legislation and institutional
requirements. The remaining 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. Data availability statement 1711138358, and KMDF_PR_20200901_0173). The funding
sources took part in data collection and analyses, but had no role
in the design of this study and its execution, interpretation of the
data, or decision to submit results. This study used publicly available datasets from Korean
National Health Insurance Service Database https://nhiss.nhis. or.kr/bd/ab/bdaba021eng.do. Author contributions MH, S-RL, E-KC, SO, and GL initially conceptualized the
subject of this study. S-RL and E-KC designed the study. MH,
S-HP, HL, JC, and JMC performed the literature research and
data collection. MH, S-RL, and K-DH analyzed the data. MH
and S-RL were major contributors on writing the manuscript. All authors read and approved the final manuscript. Supplementary material The Supplementary Material for this article can be
found online at: https://www.frontiersin.org/articles/10.3389/
fcvm.2022.1008340/full#supplementary-material Funding This research was supported by a grant from the Patient-
Centered Clinical Research Coordinating Center (PACEN)
funded by the Ministry of Health and Welfare, South Korea
(grant number: HC21C0028), and by the Korea Medical Device
Development Fund grant funded by the Korea government (the
Ministry of Science and ICT, the Ministry of Trade, Industry and
Energy, the Ministry of Health and Welfare, and the Ministry
of Food and Drug Safety) (Project numbers: HI20C1662, 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
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clustering of unhealthy lifestyle factors and risk of new-onset atrial fibrillation: a
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020-75822-y 44. Lee SR, Choi EK, Jung JH, Han KD, Oh S, Lip GYH. Lower risk of stroke
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eurheartj/ehab315 Frontiers in Cardiovascular Medicine frontiersin.org 10
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Tolerogenic Dendritic Cells That Inhibit Autoimmune Arthritis Can Be Induced by a Combination of Carvacrol and Thermal Stress
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Abstract * E-mail: f.broere@uu.nl Competing Interests: The authors have declared that no competing interests exist. * E-mail: f.broere@uu.nl ¤ Current address: Singapore Immunology Network (SIgN), Singapore, Singapore ¤ Current address: Singapore Immunology Network (SIgN), Singapore, Singapore Tolerogenic Dendritic Cells That Inhibit Autoimmune
Arthritis Can Be Induced by a Combination of Carvacrol
and Thermal Stress Rachel Spiering1, Ruurd van der Zee1, Jose´e Wagenaar1, Dimos Kapetis2, Francesca Zolezzi2¤, Willem van
Eden1, Femke Broere1* ses and Immunology, Utrecht University, Utrecht, The Netherlands, 2 Department of Biotechnology and Bioscience, Genopolis, Universit 1 Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands, 2 Department of Biotechnology and Bioscience, Genopolis, University
of Milano-Bicocca, Milan, Italy Abstract Tolerogenic dendritic cells (DCs) can induce regulatory T cells and dampen pathogenic T cell responses. Therefore, they are
possible therapeutic targets in autoimmune diseases. In this study we investigated whether mouse tolerogenic DCs are
induced by the phytonutrient carvacrol, a molecule with known anti-inflammatory properties, in combination with a
physiological stress. We show that treatment of DCs with carvacrol and thermal stress led to the mRNA expression of both
pro- and anti-inflammatory mediators. Interestingly, treated DCs with this mixed gene expression profile had a reduced
ability to activate pro-inflammatory T cells. Furthermore, these DCs increased the proportion of FoxP3+ regulatory T cells. In
vivo, prophylactic injection of carvacrol-thermal stress treated DCs pulsed with the disease inducing antigen was able to
suppress disease in a mouse model of arthritis. These findings suggest that treatment of mouse bone marrow derived DCs
with carvacrol and thermal stress induce a functionally tolerogenic DC that can suppress autoimmune arthritis. Herewith
carvacrol seems to offer novel opportunities for the development of a dietary based intervention in chronic inflammatory
diseases. Citation: Spiering R, van der Zee R, Wagenaar J, Kapetis D, Zolezzi F, et al. (2012) Tolerogenic Dendritic Cells That Inhibit Autoimmune Arthritis Can Be Induced by
a Combination of Carvacrol and Thermal Stress. PLoS ONE 7(9): e46336. doi:10.1371/journal.pone.0046336 Editor: Song Guo Zheng, University of Southern California, United States of America Editor: Song Guo Zheng, University of Southern California, United States of America Editor: Song Guo Zheng, University of Southern California, United States of America Received April 7, 2012; Accepted August 31, 2012; Published September 25, 2012 Received April 7, 2012; Accepted August 31, 2012; Published September 25, 2012 ering et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Spiering et al. This is an open-access article distributed under the terms of the Creative Commons Attribut
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Innovatiegerichte Onderzoeksprogramma (IOP) Genomics: IGE07004 (http://www.agentschapnl.nl/en) European Union FP7 TOLERAGE: HEALTH-F4-
2008-20215 (http://www.tolerage.eu/) Dutch Arthritis Association (http://www.reumafonds.nl/informatie-voor-doelgroepen/professionals/research-funding). 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. PLOS ONE | www.plosone.org Carvacrol/thermal stress treatment of BMDC Bone marrow derived dendritic cells (BMDC) were isolated
from the bone marrow of 10–15 week old BALB/c mice and
cultured in IMDM (Gibco) supplemented with 10% FBS (Lonza),
100 units/ml penicillin, 100 mg/ml streptomycin and 561025 M
b-mercaptoethanol
in
the
presence
of
20 ng/ml
GM-CSF
(Cytogen) [17]. On day 8 BMDC were seeded in 6 wells plates
at 16106 cells/ml. The next day, cells were treated with 0.1 mM
carvacrol (Sigma) dissolved at 100 mM in ethanol. Control
cultures were incubated with medium alone or 0.1% ethanol. After two hours at 37uC plates were sealed and placed in a 42.5uC
preheated water bath for one hour (TS treatment). Cells were able
to recover at 37uC for different time points. With this treatment
protocol we have obtained optimal stimulation of Hsp70 in
previous experiments [18]. Carvacrol Treated DCs Inhibit Autoimmune Arthritis carvacrol also directly inhibited the production of prostaglandin E2
(PGE2) [16]. synthesis, double strand cDNA was used in an in vitro transcription
reaction to generate biotinylated cRNA. After purification and
fragmentation and relative quality control checks by Agilent
Bioanalyzer, biotinylated cRNA was used for hybridization. Fragmented cRNAs were hybridized to the standard arrays for
16 hours at 45uC; the arrays were then washed and stained using
the GeneChip Fluidics Station 450 and scanned using GeneChip
Scanner 3000. The images were analyzed using Affymetrix GCOS
software version 1.4. For several reasons, oral administration of carvacrol may not be
the best approach. For the application for humans, the amount of
carvacrol needed to be effective may be too high and because of its
anti-bacterial properties high dosages of carvacrol could negatively
affect the intestinal flora. Therefore, we explored the effect of
carvacrol in the induction of a tolerogenic phenotype in DCs in
the PGIA model. We found that both TS and carvacrol in
combination with TS induced some of the phenotypic character-
istics of a tolerogenic DC. However, functionally only carvacrol-
TS treated DCs were able to suppress autoimmune arthritis in the
PGIA model, possibly by the induction of high levels of stress
related immuno-regulatory molecules. FoxP3 BMDC were treated as described above. After overnight
recovery cells were stained with APC-anti-CD11c (HL3) plus
one of the following antibodies: PE-anti-CD40 (3/23), FITC-anti-
CD86 (GL1) or PE-anti-I-Ad/I-Ed (M5/114) (BD Biosciences). To
analyze HSP70 expression, cells were stained with APC-anti-
CD11c followed by intracellular HSP70 staining [8]. Single cell
suspensions of spleen and mLN were stained with pacific-blue
labeled anti-CD4 (RM4-5) and APC-anti-KJ1.26 (OVA-TCR)
(BD Biosciences). Additionally, FoxP3 staining was carried out
with a FoxP3 (FJK-16S; PE labeled) staining kit as instructed
(eBioscience). Flow-cytometry was performed on a FACSCanto
(BD Biosciences). RNA isolation for microarray BMDC were treated as described above. Cells were harvested
0.5 hour after carvacrol addition or TS treatment. Total RNA was
extracted from 26106 BMDC using the double extraction
protocol: first by acid guanidinium thiocyanate-phenol-chloroform
extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-
up procedure. Microarray expression profiling Three independent (n = 3) replicates were performed for each
experimental condition (controls, carvacrol, TS and carvacrol-TS). Total RNA was processed for the use onGeneChipH Mouse
Genome 430A 2.0 Array (Affymetrix, Santa Clara, CA) containing
22.000 murine probe sets which interrogates approximately
14.000 transcripts. RNA quality controls, hybridization, washing,
staining, and scanning procedures were performed by Genopolis
(University of Milano-Bicocca). In brief, RNA purity was assessed
by spectrophotometer
(Nanodrop) and integrity by Agilent
Bioanalyzer. 0.3 mg RNA for each sample were used in a reverse
transcription reaction (MessageAmp III RNA Amplification kit,
Ambion) to generate first-strand cDNA. After second-strand Analysis of array data y
y
Data handling was done using Bioconductor software [19]. The
Robust Multi-array Analysis [20] method was employed to
calculate probe set intensity. After checks of sample data to verify
the quality of replicates, hierarchical clustering based on Pearson
correlation coefficients was performed. The identification of
differentially expressed genes was addressed using a linear
modelling approach (Limma) and empirical Bayes methods [21]
together with false discovery rate correction of the p-value [22]. Differentially expressed genes with p-values of #0.05 were
selected. Probe sets were annotated following Affymetrix annota-
tion files. Primary data are available in the public domain through
ArrayExpress at http://www.ebi.ac.uk/arrayexpress under the
accession number E-MEXP-3489. For data analysis Database for
Annotation, Visualisation and Integrated Discovery (DAVID) was
used. Ethics statement The animal studies and experiments were approved by the
Utrecht University Animal Experimental Committee. In vivo effect of carvacrol treated BMDC on antigen
specific T cells CD4+ pOVA-specific T cells were isolated from spleens of
DO11.10 mice and labeled with 5,6-carboxy-succinimidyl-fluor-
esceine-ester (CFSE) as described before [23]. Acceptor BALB/c
mice received intravenously 16107 CD4+ CFSE-labeled cells in
100 ml PBS. The subsequent day BMDC were treated as above,
allowed to recover for four hours and pulsed for another two hours
with 20 mg/ml pOVA prior to intraperitoneally transfer of 16106
BMDC to acceptor mice. Three days later mice were sacrificed
and spleen and mesenteric lymph nodes (mLN) were isolated. Mice Female BALB/c mice were purchased from Charles River
Laboratories. DO11.10 mice, transgenic for the pOVA (ovalbu-
min 323–339) specific T cell receptor (TCR), were bred and kept
under standard conditions and received water and food ad libitum. Introduction mechanisms. In contrast, antigen specific therapies are expected to
locally suppress immune responses and this approach could
obviate these severe side effects. Fully matured dendritic cells (DC) potently induce effector T
cell responses via increased expression levels of co-stimulatory
molecules and pro-inflammatory cytokines such as interleukin (IL)-
12, IL-6 and tumor necrosis factor a (TNFa). In contrast,
tolerogenic DCs are able to reduce pro-inflammatory T cell
responses and induce regulatory T cells or T cell anergy. Several
phenotypic characteristics have been described for tolerogenic
DCs. For example low or intermediate expression levels of
maturation markers, expression of IDO or secretion of IL-10 are
all features of tolerogenic DCs [1]. Due to their ability to dampen
immune responses by the induction of antigen specific regulatory
T cells, tolerogenic DCs have gained interest as a therapeutic
strategy in autoimmune diseases such as rheumatoid arthritis (RA)
[2–7] Recently, we reported that intragastric administration of
carvacrol, a component of the essential oil of Oreganum species,
suppressed proteoglycan induced arthritis (PGIA) a chronic,
progressive and self-maintaining T cell dependent, antibody-
mediated murine model for RA. [8]. Carvacrol was shown to be a
potent co-inducer of heat shock protein 70 (HSP70) in various
antigen presenting cells. In mammalian cells HSP70 expression
can be induced e.g. by thermal stress (TS) and TS has been
described to inhibit as well as to induce the production of several
pro-inflammatory cytokines depending on culture conditions and
cell type [9–12]. Oral carvacrol treatment in mice induced a
regulatory T cell response that could transfer protection in the
PGIA model [8]. Moreover, carvacrol has been described to
exhibit anti-bacterial properties [13], anti-oxidant activity [14]
and anti-inflammatory qualities. Carvacrol was able to suppress
lipopolysaccharide (LPS) induced cyclooxygenase (COX)-2 ex-
pression in human macrophages, possibly by the induction of the
anti-inflammatory transcription factors peroxisome proliferator-
activated receptor (PPAR)-a and PPAR-c [15]. In addition, RA is a chronic autoimmune disease characterized by joint
inflammation resulting in progressive cartilage and bone erosion. Inflammatory cytokines such as TNFa and IL-1b and increased
levels of the Th1 cytokines are involved in its pathogenesis. Administration of biologics such as anti-TNFa, has proven to be
an efficient therapy for RA, but this systemic suppression of the
immune response has unwanted consequences for host defense September 2012 | Volume 7 | Issue 9 | e46336 1 PLOS ONE | www.plosone.org Carvacrol Treated DCs Inhibit Autoimmune Arthritis Flow cytometric analysis of surface markers, HSP70 and Flow cytometric analysis of surface markers, HSP70 and
FoxP3 In vivo treatment and arthritis induction g
p
Increased
expression
of
CCR2
binding
chemokines. Chemokines CCL2, CCL7 and CCL12 were
upregulated after TS treatment but not with carvacrol-TS
treatment measured 0.5 hour after TS (table 2). These b-
chemokines all bind to the chemokine receptor CCR2 which is
expressed on several different immune cells like monocytes, T cell
subsets and granulocytes. To study expression profiles of CCL2,
CCL7 and CCL12 in more detail mRNA levels were measured by
quantitative RT-PCR in a kinetics experiment. As shown in
figure 2b, mRNA levels in TS treated BMDC were upregulated
after TS. Interestingly, in carvacrol-TS treated BMDC upregula-
tion of chemokine mRNA was delayed, but eventually much
higher expression levels were reached. As a control, thymol and p-
cymene, two compounds structurally similar to carvacrol (figure
S1a), were also tested for the induction of CCL2. As shown in
figure S1c p-cymene-TS treated BMDC did not alter CCL2
mRNA expression as compared to TS treated BMDC. In contrast,
thymol-TS treatment induced CCL2 expression comparable to Arthritis was induced in female mice, 16 to 26 weeks old, using a
standard immunization protocol by intraperitoneal injection of
human PG (250 mg) and 2 mg dimethyldioctadecylammonium
bromide (DDA) (Sigma) emulsified in 200 ml PBS on days 0 and 21
[24]. One day prior to the second PG/DDA immunization
BMDC treated as described above were able to recover for four
hours and then pulsed with 250 mg/ml PG. Two hours later
16106 BMDC were resuspended in 200 ml PBS and intraperito-
neally injected into acceptor mice. As a control 200 ml PBS only
was used. Onset and severity of arthritis was determined using a
visual scoring system based on swelling and redness of paws [24]. Analysis of antigen-specific T cell responses Single cell suspensions of spleen and mLN were cultured in
complete medium in flat-bottom plates (Corning) at 26105 cells
per well, in the presence/absence of pOVA (20 mg/ml). After
72 hours supernatants were collected for cytokine assays. Fluor-
esceinated microbeads coated with capture antibodies for simul-
taneous detection of IL-6 (MP5-20F3), IL-2 (JES6-1A12), TNFa
(G281-2626) (BD Biosciences) and IFNc (AN-18) (homemade)
were added to 50 ml of culture supernatant. Cytokines were PLOS ONE | www.plosone.org September 2012 | Volume 7 | Issue 9 | e46336 2 Carvacrol Treated DCs Inhibit Autoimmune Arthritis detected by biotinylated antibodies IL-6 (MP5-32C11), IL-2
(JES6-5H4), TNFa (MP6-XT3), IFNc (XMG1.2) and PE-labeled
streptavidin (BD Biosciences) and analyzed on a Luminex model
100 XYP (Luminex). In parallel cells were used for RT-PCR
quantification of mRNA of IFNc, IL-1b, IL-10 and TGFb. Analysis of mRNA expression by quantitative real time
PCR BMDC were treated as described above. BMDC, mLN or
spleen cells were harvested at indicated time points and stored in
350 ml RLT with 1% b-mercaptoethanol. Total mRNA extraction
with the RNeasy kit (Qiagen), on column DNAse treatment
(Qiagen), and transcription into cDNA using the iScriptTM cDNA
Synthesis kit (Bio-Rad) were carried out according to the
manufacturer’s protocol. Quantitative RT-PCR (3 minutes at
95uC and 40 cycles of 10 seconds 95uC and 45 seconds at 59.5uC)
was performed in a Bio-Rad MyiQ iCycler (Bio-Rad). Amplifica-
tion was done in a total volume of 25 ml using IQTM SYBR
GreenH Supermix (Bio-Rad Laboratories B.V.) with 0.25 mM final
concentrations of primers (table 1). For each sample mRNA
expression was normalized to the detected Ct value of HPRT and,
for BMDC expressed relative to the average of the control group. Gene expression profiling of thermal stress (TS) and
carvacrol-thermal stress (CTS) treated BMDC Previously we have shown that carvacrol in a concentration of
0.1 mM was a strong co-inducer of HSP70 in various antigen
presenting cells [8,18]. To study whether carvacrol pretreatment
besides affecting the heat shock response, also altered the
immunological phenotype of DCs we performed a microarray
analysis on BMDC. Similar to the HSP70 co-inducing effect,
carvacrol treatment alone, without additional stress did not induce
any differentially expressed genes compared to untreated BMDC
(data not shown). Conversely, BMDC treated with carvacrol in
combination with TS or TS alone led to differential expression of
approximately 870 genes with at least a two-fold difference when
measured 0.5 hour following TS (figure 1a). Since, the differential
expression of many of these genes most likely reflected the impact
of the TS treatment we focused on the immunologically relevant
genes as indicated by DAVID analysis (figure 1b). Of the fifty-eight
immunologically relevant genes, genes with a high fold change or
genes with particular interest for DCs were selected for further
research (table 2). Analysis of mRNA expression by quantitative real time
PCR doi:10.1371/journal.pone.0046336.t001 Statistical analysis Statistical analysis was carried out using Prism software (version
4.00, Graphpad software Inc.). Significance level was set at
p#0.05 and one-tailed Student’s t test was applied. Table 1. Primer sequences used for quantitative real time PCR. Table 1. Primer sequences used for quantitative real time PCR. Table 1. Primer sequences used for quantitative real time PCR. Forward primer 59–39
Reverse primer 59–39
HPRT
CTG GTG AAA AGG ACC TCT CG
TGA AGT ACT CAT TAT AGT CAA GGG CA
CCL2
TTA AAA ACC TGG ATC GGA ACC AA
GCA TTA GCT TCA GAT TTA CGG GT
CCL7
GCT GCT TTC AGC ATC CAA GTG
CCA GGG ACA CCG ACT ACT G
CCL12
ATT TCC ACA CTT CTA TGC CTC CT
ATC CAG TAT GGT CCT GAA GAT CA
LIF
GCT ATG TGC GCC TAA CAT GAC
CGC TCA GGT ATG CGA CCA T
AREG
GGG GAC TAC GAC TAC TCA GAG
TCT TGG GCT TAA TCA CCT GTT C
IL-10
TGC CAA GCC TTA TCG GA
ACC TGC TCC ACT GCC TTG CT
MT1
AAG AGT GAG TTG GGA CAC CTT
GA GAC AAT ACA ATG GCC TCC
HSP1A1
AAG AAC GCG CTC GAA TCC TA
GAG ATG ACC TCC TGG CAC TTG T
IFNc
TCA AGT GGC ATA GAT GTG GAA GAA
TGG CTC TGC AGG ATT TTC ATG
IL-1b
CAA CCA ACA AGT GAT ATT CTC CAT G
GAT CCA CAC TCT CCA GCT GCA
TGFb
GCC CTG TAT TCC GTC TCC TCC TTG
CGT AAC CGG CTG CTG ACC
doi:10.1371/journal.pone.0046336.t001 September 2012 | Volume 7 | Issue 9 | e46336 September 2012 | Volume 7 | Issue 9 | e46336 PLOS ONE | www.plosone.org 3 Carvacrol Treated DCs Inhibit Autoimmune Arthritis Figure 1. Gene expression profile of TS and carvacrol-TS treated BMDC as compared to untreated BMDC. Mouse BMDC were incubated
with 0.1 mM carvacrol or were left untreated. After two hours a one hour TS at 42.5uC followed. Samples for microarray analysis were taken 0.5 hour
after TS. A. Venn diagram showing genes differentially expressed (p#0.05) with at least a two-fold change for TS treated BMDC (left circle) and
carvacrol-TS treated BMDC (right circle) compared to untreated BMDC. The numbers in the circles indicate the modulated genes for each condition. The grey area represents the overlap between both conditions. B. Carvacrol Treated DCs Inhibit Autoimmune Arthritis Statistical analysis Heat map of immunological relevant genes that were significantly (p#0.05)
differentially expressed with at least a two-fold change in carvacrol-TS and/or TS treated BMDC compared to untreated BMDC. The heat map shows
the log-ratio of the mean of three independent experiments. doi:10.1371/journal.pone.0046336.g001 Figure 1. Gene expression profile of TS and carvacrol-TS treated BMDC as compared to untreated BMDC. Mouse BMDC were incubated
with 0.1 mM carvacrol or were left untreated. After two hours a one hour TS at 42.5uC followed. Samples for microarray analysis were taken 0.5 hour
after TS. A. Venn diagram showing genes differentially expressed (p#0.05) with at least a two-fold change for TS treated BMDC (left circle) and
carvacrol-TS treated BMDC (right circle) compared to untreated BMDC. The numbers in the circles indicate the modulated genes for each condition. The grey area represents the overlap between both conditions. B. Heat map of immunological relevant genes that were significantly (p#0.05)
differentially expressed with at least a two-fold change in carvacrol-TS and/or TS treated BMDC compared to untreated BMDC. The heat map shows
the log-ratio of the mean of three independent experiments. d i 10 1371/j
l
0046336 001 carvacrol-TS treatment. However, the duration of the elevated
expression levels was shorter. (LIF) and amphiregulin may also contribute to joint inflammation
[25,26]. The mRNA expression levels of IL-1b, LIF and
amphiregulin were upregulated in TS as well as carvacrol-TS
treated BMDC, with a delay but eventually higher expression
levels in BMDC pretreated with carvacrol (table 2, figure 2c and
data not shown). mRNA expression levels of the anti-inflammatory carvacrol-TS treatment. However, the duration of the elevated
expression levels was shorter. Differentially expressed gene expression of RA relevant
cytokines. Important mediators in the pathogenesis of RA are
the pro-inflammatory cytokines IL-6, IL-1b and TNFa. Addition-
ally, other less well known cytokines like leukemia inhibitory factor September 2012 | Volume 7 | Issue 9 | e46336 September 2012 | Volume 7 | Issue 9 | e46336 PLOS ONE | www.plosone.org 4 Carvacrol Treated DCs Inhibit Autoimmune Arthritis Table 2. Genes with a high fold change or particularly
interesting for DCs. Chemokines
F.C. TS
F.C. CTS
CCL2
chemokine (C-C motif) ligand 2
2.97
1.09
CCL7
chemokine (C-C motif) ligand 7
5.24
0.99
CCL12
chemokine (C-C motif) ligand 12
4.91
1.18
Cytokines
IL-1b
interleukin 1 beta
2.36
1.44
LIF
leukemia inhibitory factor
3.77
5.02
AREG
amphiregulin
13.25
5.67
Stress induced immunologically relevant molecules
MT1
metallothionein 1
3837.54
3795.52
HSP1A1
heat shock protein 1A
3.65
50.28
Maturation markers
IL-2Ra (CD25)
interleukin 2 receptor, alpha chain
0.33
0.21
CD40
CD40 antigen
0.59
0.47
CD80
CD80 antigen
0.68
0.48
BMDC were treated as in figure 1. Genes that are particularly interesting for DC
were selected and fold changes (F.C.) of gene expression in TS BMDC treated
with or without carvacrol compared to that of fully untreated BMDC are shown. doi:10.1371/journal.pone.0046336.t002 BMDC. In addition, carvacrol-TS treatment could even further
decrease expression of CD86 and CD25 in BMDC. In contrast,
protein expression of CD40 and CD80 (figure 2e and data not
shown) were not altered. No differences could be detected either in
annexin V/7AAD staining or Dextran-FITC uptake respectively
(data not shown), showing that reduced maturation is not the result
of reduced viability or function of the cells. Table 2. Genes with a high fold change or particularly
interesting for DCs. Suppressed proliferation and activation of antigen
specific T cells As shown in table 2 and figure 2d, expression levels of
MT1 were upregulated after TS treatment but especially after
carvacrol-TS treatment. Interestingly although not detectable in
the microarray, in non-stressed carvacrol treated BMDC mRNA
levels of MT1 were also increased (figure 2d). Expression levels of
HSP70 mRNA and protein increased shortly after both TS and
carvacrol-TS
treatment. However,
carvacrol
enhanced
and
prolonged expression levels (figure 2d and e). In sum, expression
of the immunoregulatory stress related genes MT1 and HSP70
was elevated in TS and especially in carvacrol-TS treated BMDC,
suggesting the induction of DCs with a relatively tolerogenic
phenotype. The structurally similar compound thymol was also
able to act as a co-inducer and elevated expression levels of HSP70
and MT1. Nevertheless, expression levels were lower and duration
of expression was shorter as compared to carvacrol-TS treatment. Pretreatment of DCs with p-cymene did not alter mRNA
expression levels as compared to TS alone (figure S1c and d). Suppressed proliferation and activation of antigen
specific T cells Genes that are particularly interesting for DC
were selected and fold changes (F.C.) of gene expression in TS BMDC treated
with or without carvacrol compared to that of fully untreated BMDC are shown. doi:10.1371/journal.pone.0046336.t002 BMDC were treated as in figure 1. Genes that are particularly interesting for DC
were selected and fold changes (F.C.) of gene expression in TS BMDC treated
with or without carvacrol compared to that of fully untreated BMDC are shown. doi:10.1371/journal.pone.0046336.t002 cytokine IL-10 were also upregulated after both treatments and
followed the same expression profile as IL-1b, amphiregulin and
LIF (figure 2c). In conclusion, mRNA expression levels of pro-
inflammatory RA relevant cytokines as well as the anti-inflamma-
tory cytokine IL-10 were increased after treatment. In the controls
where BMDC were treated with thymol-TS or p-cymene-TS,
amphiregulin mRNA expression was not altered as compared to
TS. IL-10 expression levels were only increased after thymol-TS
treatment as compared to TS treatment (figure S1c). Induction
of
immunoregulatory
stress
related
molecules. TS induces the expression of many stress related
genes and some of these, like metallothionein 1 (MT1) and HSP70
[27] are also considered to be immunomodulatory molecules
[28,29]. As shown in table 2 and figure 2d, expression levels of
MT1 were upregulated after TS treatment but especially after
carvacrol-TS treatment. Interestingly although not detectable in
the microarray, in non-stressed carvacrol treated BMDC mRNA
levels of MT1 were also increased (figure 2d). Expression levels of
HSP70 mRNA and protein increased shortly after both TS and
carvacrol-TS
treatment. However,
carvacrol
enhanced
and
prolonged expression levels (figure 2d and e). In sum, expression
of the immunoregulatory stress related genes MT1 and HSP70
was elevated in TS and especially in carvacrol-TS treated BMDC,
suggesting the induction of DCs with a relatively tolerogenic
phenotype. The structurally similar compound thymol was also
able to act as a co-inducer and elevated expression levels of HSP70
and MT1. Nevertheless, expression levels were lower and duration
of expression was shorter as compared to carvacrol-TS treatment. Pretreatment of DCs with p-cymene did not alter mRNA
expression levels as compared to TS alone (figure S1c and d). Induction
of
immunoregulatory
stress
related
molecules. TS induces the expression of many stress related
genes and some of these, like metallothionein 1 (MT1) and HSP70
[27] are also considered to be immunomodulatory molecules
[28,29]. Suppressed proliferation and activation of antigen
specific T cells TS and carvacrol-TS treated BMDC have increased mRNA
expression levels of certain anti-inflammatory mediators and
display a decreased maturation profile. To investigate whether
the assorted phenotype we observed in mRNA expression of the
BMDC resulted in an immunomodulatory function of treated
BMDC we studied their antigen presenting capacity in vivo. pOVA-specific CFSE-labeled CD4+ T cells were intravenously
transferred into recipient mice one day prior to intraperitoneal
injection of treated and untreated BMDC pulsed with pOVA. Three days later proliferation and phenotype of the pOVA-specific
T cells from spleen and mLN were analyzed. Transfer of TS
treated BMDC induced less T cell proliferation as compared to
untreated BMDC in vivo. Notably, carvacrol-TS treated BMDC
induced even lower proliferation in vivo as shown by the increase in
non-divided cells (figure 3a). In addition, although carvacrol-TS
treated BMDC did not increase FoxP3 expression in the total
CD4+DO11.10+ population, mice treated with carvacrol-TS
treated BMDC had increased FoxP3 expression levels in the
proliferating CD4+DO11.10+ population in the mLN as compared
to untreated BMDC (figure 3b). Moreover, carvacrol-TS treated
BMDC preferentially induced the proliferation of FoxP3+ T cells
over proliferation of FoxP32 T cells. In contrast, with untreated
and TS treated BMDC the percentage of replicated FoxP3+ T
cells of the total FoxP3+ T cells was equal to the replicated FoxP32
T cells of the total FoxP32T cells (figure 3b). Results did not reflect
a difference in survival of the treated BMDC as equal amounts of
transferred viable BMDC could be recovered in spleens of
recipient mice that received untreated, TS treated or carvacrol-
TS treated BMDC (data not shown). To further investigate the
phenotype of the DC primed T cells, splenic cell suspensions were
ex vivo restimulated with pOVA. Secretion of the pro-inflammatory
cytokines IL-2, IL-6, IFNc and TNFa was reduced when mice
were injected with TS or carvacrol-TS treated BMDC as
compared to untreated BMDC. However, reduction was only
significantly lower with carvacrol-TS treated BMDC (figure 3c). Furthermore, mRNA expression levels of the pro-inflammatory
cytokines IFNc and IL-1b were downregulated, whereas expres-
sion levels of IL-10 and TGFb were slightly upregulated (figure 3d). Thus, these data indicated that treated BMDC induced less
activated and less pro-inflammatory T cells and that carvacrol-TS
treated BMDC as compared to untreated BMDC were able to
enhance the proportion of proliferating FoxP3+ T cells. BMDC were treated as in figure 1. Pretreatment of BMDC with carvacrol is essential for
suppression of arthritis Both TS and carvacrol-TS treatment protocols induce a BMDC
with an assorted gene expression profile that is able to suppress
proliferation and activation of antigen-specific T cells. To
determine whether these treated BMDC can suppress PGIA,
mice were intraperitoneally injected with carvacrol-TS treated
BMDC, TS treated BMDC, untreated BMDC or PBS one day
prior to the second PG immunization. PG pulsed and unpulsed
BMDC were compared to distinguish if loading of DCs with an Reduced maturation. As maturation status is an important
hallmark for tolerogenic DC and the activation markers CD40,
CD80 and CD25 (IL2ra) [30] were downregulated in the
microarray (table 2), we assessed protein expression of activation
markers on treated and untreated BMDC. TS treated BMDC
displayed an immature phenotype with lower expression levels of
CD86 and CD25 and MHC class II as compared to untreated September 2012 | Volume 7 | Issue 9 | e46336 PLOS ONE | www.plosone.org 5 Carvacrol Treated DCs Inhibit Autoimmune Arthritis Figure 2. DC fingerprint after TS and carvacrol-TS treatment. Mouse BMDC were incubated with 0.1 mM carvacrol or were left untreated. After two hours a one hour TS at 42.5uC followed. A. Experimental setup. B-D: Cells were collected at indicated time points and mRNA was isolated. With cDNA quantitative RT-PCRs were performed in untreated, TS treated (grey) and carvacrol-TS treated (black) BMDC. Results were depicted relative
to untreated BMDC at the indicated time points. Data are representative of three independent experiments. B. Quantitative RT-PCRs for CCL2, CCL7
and CCL12. C. Quantitative RT-PCRs for LIF, AREG and IL-10. D. Quantitative RT-PCRs for HSP70 and MT1. E. After ON recovery at 37uC, intracellular
HSP70 levels and extracellular CD40, CD86 and MHCII were analyzed. Grey solid: untreated BMDC; black line: TS treated BMDC; grey line: carvacrol-TS
treated BMDC. Results are representative of at least 5 independent experiments. doi:10.1371/journal.pone.0046336.g002 Figure 2. DC fingerprint after TS and carvacrol-TS treatment. Mouse BMDC were incubated with 0.1 mM carvacrol or were left untreated. After two hours a one hour TS at 42.5uC followed. A. Experimental setup. B-D: Cells were collected at indicated time points and mRNA was isolated. With cDNA quantitative RT-PCRs were performed in untreated, TS treated (grey) and carvacrol-TS treated (black) BMDC. Results were depicted relative
to untreated BMDC at the indicated time points. Data are representative of three independent experiments. B. Quantitative RT-PCRs for CCL2, CCL7
and CCL12. C. Pretreatment of BMDC with carvacrol is essential for
suppression of arthritis Quantitative RT-PCRs for LIF, AREG and IL-10. D. Quantitative RT-PCRs for HSP70 and MT1. E. After ON recovery at 37uC, intracellular
HSP70 levels and extracellular CD40, CD86 and MHCII were analyzed. Grey solid: untreated BMDC; black line: TS treated BMDC; grey line: carvacrol-TS
treated BMDC. Results are representative of at least 5 independent experiments. doi:10.1371/journal.pone.0046336.g002 September 2012 | Volume 7 | Issue 9 | e46336 PLOS ONE | www.plosone.org 6 Carvacrol Treated DCs Inhibit Autoimmune Arthritis Figure 3. Carvacrol-TS treated BMDC induce a less pro-inflammatory T cell. Mice were intravenously injected with 16107 CFSE labeled
pOVA-specific T cells one day prior to intraperitoneally injection of 16106 pOVA pulsed untreated, TS treated or carvacrol-TS treated BMDC. Four days
after BMDC injection spleen and mLN were harvested. A. Representative examples of CFSE dilution of pOVA-specific T cells in mLN (left) and
percentage of undivided pOVA-specific T cells in spleen and mLN (right). B. Percentage of FoxP3 expression of pOVA-specific proliferating T cells (left)
and percentage of replicated FoxP3+ cells of total FoxP3+ cells divided by the percentage of replicated FoxP32 cells of total FoxP32 cell population
(right). Data are analyzed by flow cytometry. C-D. Splenic cell suspensions were restimulated with pOVA for 72 hours. Cytokine secretion for IL-2, IL-6,
IFNc and TNFa relative to non-stimulated cells are shown (C). Cytokine expression of IFNc, IL-1b, IL-10 and TGFb was measured by quantitative RT-
PCR. Data are expressed as relative expression to the calibrator HPRT (D). White: untreated BMDC, grey: TS treated BMDC, black: carvacrol-TS treated
BMDC. Values are the mean and SEM of three mice per group. * (p,0.05) and ** (p,0.01). doi:10.1371/journal.pone.0046336.g003 Figure 3. Carvacrol-TS treated BMDC induce a less pro-inflammatory T cell. Mice were intravenously injected with 16107 CFSE labeled
pOVA-specific T cells one day prior to intraperitoneally injection of 16106 pOVA pulsed untreated, TS treated or carvacrol-TS treated BMDC. Four days
after BMDC injection spleen and mLN were harvested. A. Representative examples of CFSE dilution of pOVA-specific T cells in mLN (left) and
percentage of undivided pOVA-specific T cells in spleen and mLN (right). B. Percentage of FoxP3 expression of pOVA-specific proliferating T cells (left)
and percentage of replicated FoxP3+ cells of total FoxP3+ cells divided by the percentage of replicated FoxP32 cells of total FoxP32 cell population
(right). Data are analyzed by flow cytometry. C-D. Splenic cell suspensions were restimulated with pOVA for 72 hours. Pretreatment of BMDC with carvacrol is essential for
suppression of arthritis Cytokine secretion for IL-2, IL-6,
IFNc and TNFa relative to non-stimulated cells are shown (C). Cytokine expression of IFNc, IL-1b, IL-10 and TGFb was measured by quantitative RT-
PCR. Data are expressed as relative expression to the calibrator HPRT (D). White: untreated BMDC, grey: TS treated BMDC, black: carvacrol-TS treated
BMDC. Values are the mean and SEM of three mice per group. * (p,0.05) and ** (p,0.01). doi:10.1371/journal.pone.0046336.g003 auto-antigen was required. As shown in figure 4 and table 3, only
carvacrol-TS treated BMDC that were pulsed with PG signif-
icantly reduced arthritis scores and delayed disease onset in
recipient mice. In summary, although TS treated BMDC show
characteristics of a tolerogenic DC, they are not anti-inflammatory
in PGIA, whereas carvacrol-TS treated BMDC pulsed with
disease associated antigens were able to suppress autoimmune
arthritis. Figure 4. Carvacrol-TS treated BMDC suppress PGIA. Arthritis
was induced by two intraperitoneally immunizations with human PG on
days 0 and 21. One day prior to the second PG immunization mice were
intraperitoneally injected with 16106 PG pulsed untreated, TS treated
or carvacrol-TS treated BMDC. As a control, mice received only PBS. Arthritis severity expressed as the mean and SEM of at least six mice per
group. * (p,0.05) for area under the curve compared to PBS group. doi:10.1371/journal.pone.0046336.g004 Discussion These chemokines can have diverse effects on the
immune response as they attract both pro-inflammatory cells
and also CCR2+FoxP3+CD4+ regulatory T cells. CCR2+ regula-
tory T cells are important regulators in the progression phase of
collagen induced arthritis (CIA) [33]. Furthermore, CCL2 and
CCL7 promote a Th2 response as CCR2+ DCs produce less IL-12
and activated CCR2+ T cells secrete more IL-4 after binding to
CCL2. [34,35]. Enhanced expression of these chemokines might
promote Th2 or regulatory T cell pathways and thereby suppress
arthritis, depending on the micro-environmental context in which
the chemokines are released. It is clear that our microarray analysis of TS and carvacrol-TS
treated BMDC revealed a diverse gene expression profile with
potential tolerogenic features. In addition, the diverse nature and
kinetics of the 58 immunologically relevant genes could at least
partly help explain why there exists such a controversy in literature
about thermal stress as a pro- or anti-inflammatory trigger. Subsequently, maturation status of treated BMDC was inves-
tigated as a possible feature of tolerogenic DCs. Maturation was
decreased after TS treatment and even stronger after pretreatment
with carvacrol. Both TS and carvacrol-TS treated BMDC induced
proliferation of CD4+ T cells, although less compared to untreated
BMDC. This reduced proliferation coincided with reduced
secretion of pro-inflammatory cytokines following 72 hours of
restimulation indicating that the antigen specific T cells had
changed in vivo due to BMDC treatment. In addition, differences
were more pronounced in carvacrol-TS treated BMDC and only
carvacrol-TS treated BMDC preferentially induced the prolifer-
ation of FoxP3+ T cells over the proliferation of FoxP32 T cells. These data suggested proliferation of existing FoxP3+ natural
regulatory T cells or the induction of a regulatory phenotype in
naı¨ve T cells. These combined results are an indication that an
anergic or regulatory T cell response was induced. A second group of genes that showed differential expression
were certain RA relevant cytokines. The IL-1 family member IL-
1b and the IL-6 family member LIF were two genes with elevated
expression levels after both TS and carvacrol-TS treatment in
BMDC. These pro-inflammatory cytokines contribute to joint
inflammation in RA and are not directly indicative of a tolerogenic
phenotype. In contrast, the expression levels of anti-inflammatory
cytokine IL-10 and amphiregulin were increased. Amphiregulin is
increased in the synovial tissue of RA patients [26], but is also
described as an immunomodulatory molecule that is secreted by
ATP treated semi-mature tolerogenic DCs. Discussion Values
indicate mean 6 SEM of n = 6 (PBS, untreated BMDC-pulsed, carvacrol-TS treated BMDC-pulsed and untreated BMDC-unpulsed) or n = 7 (TS treated BMDC-pulsed, TS
treated BMDC-unpulsed and carvacrol-TS treated BMDC-unpulsed). * (p#0.05) compared to PBS group. doi:10.1371/journal.pone.0046336.t003 To test the tolerogenic capacity of carvacrol-TS treated BMDC mice were treated as in figure 4, although here, PG-unpulsed BMDC recipients are also shown. Values
indicate mean 6 SEM of n = 6 (PBS, untreated BMDC-pulsed, carvacrol-TS treated BMDC-pulsed and untreated BMDC-unpulsed) or n = 7 (TS treated BMDC-pulsed, TS
treated BMDC-unpulsed and carvacrol-TS treated BMDC-unpulsed). * (p#0.05) compared to PBS group. doi:10.1371/journal.pone.0046336.t003 macrophages and T and B cells [38–40]. Moreover, repeated
administration of MT1 and 2 during the course of CIA
dramatically reduced incidence and disease severity via induction
of TGFb and down-regulation of pro-inflammatory mediators
[29] and it was shown to induce IL-10 and TGFb producing
regulatory T cells in CIA [41]. Elevated expression levels of both
molecules point toward a tolerogenic DC that functions via the
induction of anti-inflammatory cytokines like IL-10 and TGFb. however the effects of severe TS comparable to our TS protocol
on the immune response are less well known. Fifty-eight
immunologically relevant genes were differentially expressed with
at least a two-fold change and a p-value #0.05, 0.5 hour after TS
and/or carvacrol-TS treatment compared to untreated BMDC
(figure 1b). Although most of these genes are altered after both
treatment protocols, differences were found in the timing of
expression and expression levels as shown by table 1 and figure 2. Especially the lower expression levels of certain maturation
markers and the excessively high levels of potentially regulatory
stress molecules could steer towards a tolerogenic functional
phenotype in carvacrol-TS treated BMDC as compared to TS
treatment alone. The capacity of carvacrol to enhance the stress response and the
expression of certain immunoregulatory mediators induced by TS
is not a unique feature of carvacrol as the structurally similar
compound thymol could also affect some of these same genes. However, carvacrol was the most potent co-inducer of the immune
response and had the largest effect on the expression of
immunological relevant genes making carvacrol the most suitable
compound. One group of genes with elevated expression levels in treated
BMDC were the CCR2 binding chemokines CCL2, CCL7 and
CCL12. Discussion Approaches to induce tolerogenic phenotypes in DCs have been
developed and used successfully in autoimmune models [1,31,32]. Earlier we found that oral administration of carvacrol suppressed
PGIA in a T cell dependent manner [8]. Furthermore, carvacrol
had an anti-inflammatory effect on activated macrophages [15]. In
this study, we demonstrated that treatment with carvacrol and TS
induced a functional tolerogenic DC that, upon transfer, was able
to suppress autoimmune arthritis. The immunological effect of carvacrol on DCs was analyzed by
microarrays. We found that carvacrol acted as co-inducer as no
effects in the absence of TS were seen 0.5 hour after carvacrol
administration. We used severe TS as stimulus as it is known that
carvacrol functions as co-inducer for the heat shock response
[8,18]. Controversy exists as both immune activating and
inhibitory effects have been described for mild TS [10,12], Figure 4. Carvacrol-TS treated BMDC suppress PGIA. Arthritis
was induced by two intraperitoneally immunizations with human PG on
days 0 and 21. One day prior to the second PG immunization mice were
intraperitoneally injected with 16106 PG pulsed untreated, TS treated
or carvacrol-TS treated BMDC. As a control, mice received only PBS. Arthritis severity expressed as the mean and SEM of at least six mice per
group. * (p,0.05) for area under the curve compared to PBS group. doi:10.1371/journal.pone.0046336.g004 PLOS ONE | www.plosone.org September 2012 | Volume 7 | Issue 9 | e46336 7 Carvacrol Treated DCs Inhibit Autoimmune Arthritis Table 3. Mean day of onset and arthritis score. PG pulsed
Day of onset
Maximum arthritis score
PBS
3061.3
8.361.25
Untreated BMDC
+
3662.8
6.961.16
TS treated BMDC
+
3361.9
6.961.39
Carvacrol TS treated BMDC
+
3863.1*
3.861.26*
Untreated BMDC
2
2961.0
8.361.16
TS treated BMDC
2
3062.0
5.561.48
Carvacrol TS treated BMDC
2
3462.9
5.561.37
To test the tolerogenic capacity of carvacrol-TS treated BMDC mice were treated as in figure 4, although here, PG-unpulsed BMDC recipients are also shown. Values
indicate mean 6 SEM of n = 6 (PBS, untreated BMDC-pulsed, carvacrol-TS treated BMDC-pulsed and untreated BMDC-unpulsed) or n = 7 (TS treated BMDC-pulsed, TS
treated BMDC-unpulsed and carvacrol-TS treated BMDC-unpulsed). * (p#0.05) compared to PBS group. doi:10.1371/journal.pone.0046336.t003 To test the tolerogenic capacity of carvacrol-TS treated BMDC mice were treated as in figure 4, although here, PG-unpulsed BMDC recipients are also shown. September 2012 | Volume 7 | Issue 9 | e46336 References 1. Thomson AW, Robbins PD (2008) Tolerogenic dendritic cells for autoimmune
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support. Figure S1
Mouse BMDC were incubated with 0.1 mM
carvacrol, 0.1 mM thymol, 0.1 mM p-cymene or were
left untreated. After two hours a one hour TS at 42.5uC
followed. A. Chemical structures of cavacrol, thymol and p-
cymene. B. Experimental setup. C. Cells were collected at
indicated time points and mRNA was isolated. With cDNA
quantitative RT-PCRs were performed in untreated, TS treated
(grey), p-cymene-TS treated (white), thymol-TS treated (white Figure S1
Mouse BMDC were incubated with 0.1 mM
carvacrol, 0.1 mM thymol, 0.1 mM p-cymene or were
left untreated. After two hours a one hour TS at 42.5uC
followed. A. Chemical structures of cavacrol, thymol and p-
cymene. B. Experimental setup. C. Cells were collected at
indicated time points and mRNA was isolated. With cDNA
quantitative RT-PCRs were performed in untreated, TS treated
(grey), p-cymene-TS treated (white), thymol-TS treated (white Discussion [36]. Expression levels of the immunomodulatory stress related
molecules HSP70 and MT1 were elevated after both treatments,
although up-regulation was more prominent after carvacrol-TS
treatment. This result is in concordance with the co-inducing
capacity of carvacrol on the stress response. HSP70 functions as an
intracellular chaperone, but has also been described as an anti-
inflammatory molecule that inhibits maturation and induces the
secretion of IL-10 in DCs [28,37]. MT1 is a protein that binds
heavy metals and functions as an immunosuppressive agent on In conclusion, this study shows that although TS and carvacrol-
TS treated BMDC share several anti-inflammatory characteristics,
only carvacrol pretreated BMDC can function as tolerogenic DC
in suppressing arthritis. This is probably caused by differences in
the balance between pro- and anti-inflammatory molecules. For
example, both TS and carvacrol-TS treated BMDC express the
pro-inflammatory
cytokines
LIF and
IL-1b, two
mediators
involved in the pathogenesis of RA. In contrast, carvacrol-TS September 2012 | Volume 7 | Issue 9 | e46336 PLOS ONE | www.plosone.org 8 Carvacrol Treated DCs Inhibit Autoimmune Arthritis striped) and carvacrol-TS treated (black) BMDC. Results were
depicted relative to untreated BMDC at the indicated time points. D. After over night recovery at 37uC, intracellular HSP70 levels
were analyzed. Grey solid: untreated BMDC; black line: TS
treated BMDC; grey line: p-cymene/thymol/carvacrol-TS treated
BMDC. treated BMDC also express very high levels of the immunoreg-
ulatory molecules MT1 and HSP70, whereas TS treated BMDC
express lower levels of both molecules. These differences probably
cause the shift toward a tolerogenic function after carvacrol-TS
treatment, but not after TS treatment alone. Our data show that
the combined action of a phytonutrient carvacrol, in combination
with a physiological form of stress, can lead to the induction of a
tolerogenic state in DCs. Author Contributions Conceived and designed the experiments: RS FZ FB. Performed the
experiments: RS JW DK. Analyzed the data: RS DK. Wrote the paper: RS
RvdZ WvE FB. Conceived and designed the experiments: RS FZ FB. Performed the
experiments: RS JW DK. Analyzed the data: RS DK. Wrote the paper: RS
RvdZ WvE FB. experiments: RS JW DK. Analyzed the data: RS DK. Wrote the paper: RS
RvdZ WvE FB. 37. Detanico T, Rodrigues L, Sabritto AC, Keisermann M, Bauer ME, et al. (2004)
Mycobacterial heat shock protein 70 induces interleukin-10 production:
Immunomodulation of synovial cell cytokine profile and dendritic cell
maturation. Clin Exp Immunol 135(2): 336–342. 33. Bruhl H, Cihak J, Schneider MA, Plachy J, Rupp T, et al. (2004) Dual role of
CCR2 during initiation and progression of collagen-induced arthritis: Evidence
for regulatory activity of CCR2+ T cells. J Immunol 172(2): 890–898. 38. Lynes MA, Borghesi LA, Youn J, Olson EA (1993) Immunomodulatory activities
of extracellular metallothionein. I. metallothionein effects on antibody
production. Toxicology 85(2–3): 161–177. References Ostberg JR, Repasky EA (2006) Emerging evidence indicates that physiolog-
ically relevant thermal stress regulates dendritic cell function. Cancer Immunol
Immunother 55(3): 292–298. 10.1007/s00262-005-0689-y. 13. Xu J, Zhou F, Ji BP, Pei RS, Xu N (2008) The antibacterial mechanism of
carvacrol and thymol against escherichia coli. Lett Appl Microbiol. 47(3): 174-
179. 10.1111/j.1472-765X.2008.02407.x. 29. Youn J, Hwang SH, Ryoo ZY, Lynes MA, Paik DJ, et al. (2002) Metallothionein
suppresses collagen-induced arthritis via induction of TGF-beta and down-
regulation of proinflammatory mediators. Clin Exp Immunol 129(2): 232–239. j
14. Yanishlieva NV, Marinova EM, Gordon MH, Raneva VG (1999) Antioxidant
activity and mechanism of action of thymol and carvacrol in two lipid systems. Food Chem 64(1): 59–66. DOI: 10.1016/S0308-8146(98)00086-7. 30. Driesen J, Popov A, Schultze JL (2008) CD25 as an immune regulatory molecule
expressed on myeloid dendritic cells. Immunobiology 213(9–10): 849–858. 10.1016/j.imbio.2008.07.026. 15. Hotta M, Nakata R, Katsukawa M, Hori K, Takahashi S, et al. (2010)
Carvacrol, a component of thyme oil, activates PPARalpha and gamma and
suppresses COX-2 expression. J Lipid Res 51(1): 132–139. 10.1194/
jlr.M900255-JLR200. 31. Torres-Aguilar H, Blank M, Jara LJ, Shoenfeld Y (2010) Tolerogenic dendritic
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autoimmunity. Autoimmun Rev 10(1): 8–17. 10.1016/j.autrev.2010.07.015. 16. Landa P, Kokoska L, Pribylova M, Vanek T, Marsik P (2009) In vitro anti-
inflammatory activity of carvacrol: Inhibitory effect on COX-2 catalyzed
prostaglandin E(2) biosynthesis. Arch Pharm Res 32(1): 75–78. 10.1007/s12272-
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of extracellular metallothionein. II. effects on macrophage functions. J Toxicol
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to expel trichuris muris infection. J Immunol 170(9): 4693–4700. 36. Bles N, Di Pietrantonio L, Boeynaems JM, Communi D (2010) ATP confers
tumorigenic properties to dendritic cells by inducing amphiregulin secretion. Blood 116(17): 3219–3226. 10.1182/blood-2010-01-265611. 41. Huh S, Lee K, Yun HS, Paik DJ, Kim JM, et al. (2007) Functions of
metallothionein generating interleukin-10-producing regulatory CD4+ T cells
potentiate suppression of collagen-induced arthritis. J Microbiol Biotechnol
17(2): 348–358. 37. Detanico T, Rodrigues L, Sabritto AC, Keisermann M, Bauer ME, et al. (2004)
Mycobacterial heat shock protein 70 induces interleukin-10 production:
Immunomodulation of synovial cell cytokine profile and dendritic cell
maturation. Clin Exp Immunol 135(2): 336–342. September 2012 | Volume 7 | Issue 9 | e46336 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10
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The Role of Medicinal Cannabis in Clinical Therapy: Pharmacists' Perspectives
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RESEARCH ARTICLE a11111 OPEN ACCESS Citation: Isaac S, Saini B, Chaar BB (2016) The
Role of Medicinal Cannabis in Clinical Therapy:
Pharmacists' Perspectives. PLoS ONE 11(5):
e0155113. doi:10.1371/journal.pone.0155113 Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Methods Semi-structured interviews with 34 registered pharmacists in Australia were conducted. All
interviews were audio-recorded, transcribed ad verbatim and thematically analysed using
the NVivo software. Editor: Kent E. Vrana, Penn State College of
Medicine, UNITED STATES Editor: Kent E. Vrana, Penn State College of
Medicine, UNITED STATES Received: January 18, 2016
Accepted: April 25, 2016
Published: May 12, 2016 The Role of Medicinal Cannabis in Clinical
Therapy: Pharmacists' Perspectives Sami Isaac☯, Bandana Saini☯, Betty B. Chaar*☯
Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, Australia ☯These authors contributed equally to this work. * betty.chaar@sydney.edu.au ☯These authors contributed equally to this work. * betty.chaar@sydney.edu.au Background Medicinal cannabis has recently attracted much media attention in Australia and across the
world. With the exception of a few countries, cannabinoids remain illegal–known for their
adverse effects rather than their medicinal application and therapeutic benefit. However,
there is mounting evidence demonstrating the therapeutic benefits of cannabis in alleviating
neuropathic pain, improving multiple sclerosis spasticity, reducing chemotherapy induced
nausea and vomiting, and many other chronic conditions. Many are calling for the legalisa-
tion of medicinal cannabis including consumers, physicians and politicians. Pharmacists
are the gatekeepers of medicines and future administrators/dispensers of cannabis to the
public, however very little has been heard about pharmacists’ perspectives. Therefore the
aim of this study was to explore pharmacists’ views about medicinal cannabis; its legalisa-
tion and supply in pharmacy. a1111 Results Emergent themes included stigma, legislation, safety and collaboration. Overall the majority
of pharmacists felt national legalisation of a standardised form of cannabis would be suit-
able, and indicated various factors and strategies to manage its supply. The majority of par-
ticipants felt that the most suitable setting would be via a community pharmacy setting due
to the importance of accessibility for patients. Copyright: © 2016 Isaac 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. Introduction For over 4000 years cannabis more commonly known as marijuana, has been used medicinally,
recreationally and in religious ceremonies in cultures across the globe [1]. Well known in the
public arena, recreational cannabis is the leisurely use of cannabis for its euphoriant effect. Medicinal cannabis, on the other hand is regarded as the use of cannabis ‘to achieve a curative
or remedial effect’ on the symptoms of a medical condition [2]. From a healthcare perspective,
medicinal use of cannabis would refer to use based on a prescription or recommendation by a
registered physician, for a known medical condition, that has evidence demonstrating its indi-
cation and efficacy [3]. For many years, cannabis has been viewed as an illicit substance and this negative repute
has hindered efforts to conduct research into its therapeutic benefits. A plethora of literature
exists investigating the abuse, misuse and side effect profile of cannabis, in the realms of addic-
tion, mental cognition and schizophrenia. More recently however, there has been a gradual increase of research into the therapeutic
benefits associated with the medicinal use of cannabis. With a greater number of well designed
trials providing evidence to support its use for spasticity, chronic neuropathic pain and chemo-
therapy-induced nausea and vomiting, cachexia, as well as appetite stimulation in HIV/AIDS
infection [4]. The expanding body of research into the medicinal application of cannabis has
initiated the development of marketable forms of cannabis internationally, as well as rapid pol-
icy making by governing bodies worldwide. A systematic and meta-analysis into the therapeutic applications of cannabis provided mod-
erate-quality evidence to support the use of cannabinoids for the treatment of chronic pain and
spasticity. For conditions other than pain and spasticity (such as nausea and vomiting, weight
loss in HIV infection, sleep disorders and Tourette syndrome), minor improvements were
noted [4] and more evidence is emerging as more stringent investigations are being
undertaken. Globally, the type and intent of legislation governing cannabis use is complex and varied
with focus on both general and medicinal use. In the case of medicinal cannabis, countries
including the UK, Denmark, Czech Republic, Austria, Sweden, Germany, and Spain [3] have
all formally approved use of cannabis based products of one form or another (Table 1), [5]
thus decriminalising its therapeutic use. Discussion This study explored views of practicing pharmacists, revealing a number of previously
undocumented views and barriers about medicinal cannabis from a supply perspective. There were several ethical and professional issues raised for consideration. These findings
highlight the important role that pharmacists hold in the supply of medicinal cannabis. Funding: The authors have no support or funding to
report. Competing Interests: The authors have declared
that no competing interests exist. 1 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy Additionally, this study identified important factors, which will help shape future policies for
the successful implementation of medicinal cannabis in healthcare. We recommend that
these views and strategies be incorporated in the development of policies and legislations. Additionally, this study identified important factors, which will help shape future policies for
the successful implementation of medicinal cannabis in healthcare. We recommend that
these views and strategies be incorporated in the development of policies and legislations. PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The opioid substitution therapy (OST) is another program aimed at reducing
i k b h
i
d illi it d
i i di id
l [20] Th
i i
f fl
i
ti
b
Table 1. Forms of Cannabinoids, their effects and any registered products (5). Cannabinoids
Source
Pharmacological Effects
Registered Products
Anandamide (Δ8-THC)
Endogenous ligand
Memory/Cognition, Motivation and Pleasure
N/A
Δ9-tetrahydrocanabinol
(THC)
Natural/ Botanical
Psychoactive, # Memory/Cognition, " Pleasure and
Appetite stimulation
See Nabiximols
Cannabidiol (CBD)
Natural/ Botanical
Non-psychoactive, Antiemetic and Antispasmodic
See Nabiximols
Cannabinol (CBN)
Natural/ Botanical
Non-Psychoactive, Anti-inflammatory and
Immunosuppressive
N/A
Nabiximols
Combination of THC
and CBD
See THC & CBD
Sativex = 2.7mg THC + 2.5mg CBD
(oromucosal spray)
Dronabinol
Synthetic THC
Appetite stimulating
Marinol = 2.5mg, 5mg, 10mg (tablet/
capsule)
Nabilone
Synthetic analogue of
THC
Antiemetic and Antispasmodic
Cesamet = 1mg (capsule)
doi:10.1371/journal.pone.0155113.t001 Table 1. Forms of Cannabinoids, their effects and any registered products (5). Cannabinoids
Source
Pharmacological Effects
Registered Products
Anandamide (Δ8-THC)
Endogenous ligand
Memory/Cognition, Motivation and Pleasure
N/A
Δ9-tetrahydrocanabinol
(THC)
Natural/ Botanical
Psychoactive, # Memory/Cognition, " Pleasure and
Appetite stimulation
See Nabiximols
Cannabidiol (CBD)
Natural/ Botanical
Non-psychoactive, Antiemetic and Antispasmodic
See Nabiximols
Cannabinol (CBN)
Natural/ Botanical
Non-Psychoactive, Anti-inflammatory and
Immunosuppressive
N/A
Nabiximols
Combination of THC
and CBD
See THC & CBD
Sativex = 2.7mg THC + 2.5mg CBD
(oromucosal spray)
Dronabinol
Synthetic THC
Appetite stimulating
Marinol = 2.5mg, 5mg, 10mg (tablet/
capsule)
Nabilone
Synthetic analogue of
THC
Antiemetic and Antispasmodic
Cesamet = 1mg (capsule)
doi:10.1371/journal.pone.0155113.t001 Table 1. Forms of Cannabinoids, their effects and any registered products (5). In the Australasian sector, the Australian register of Therapeutic Goods (TGA) has regis-
tered Sativex for future use, but the distributor Novartis™has made the commercial decision
not to make it available [6]. However as of 24th February 2016, The Australian parliament
passed new national laws and amendments to the Narcotics Drug Act 1967 to allow for the
controlled cultivation of cannabis for medical and scientific purposes in Australia [11]. This
followed the Victorian state government’s introduction of legislation into parliament to legalise
medicinal cannabis based on the advice of the Victorian Law Reform Commission’s Report on
Medicinal Cannabis [12]. These legislations are designed to allow access for those in need
while limiting abuse and diversion potential. Introduction In the United State (US) to date, 18 states and Washington DC have legalised recreational
cannabis use, and 23 states in total have legalised the medicinal use of cannabis [6]. The US
Foods and Drugs Administration (FDA) has yet to approve the marketing of products contain-
ing or derived from botanical marijuana extract–despite its legislative status. There are, how-
ever, approved synthetic cannabis derivatives formulated such as dronabinol, the marketed
delta-9-tetrahydrocannabinol (THC) analogue registered as Marinol, for the therapeutic treat-
ment of anorexia in AIDS patients. Also, Cesamet containing the synthetic THC analogue
nabilone, which is approved as a last line antiemetic. [7] The European Medicine Agency has
granted registration of dronabinol [8] for the treatment of central and peripheral neuropathic
pain. It has also registered the nabiximol (Sativex) [9], a combination of cannabidiol and THC,
for the indicated treatment of spasticity in multiple sclerosis and nabilone [10] for the treat-
ment of amyotrophic lateral sclerosis. PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 2 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy In the Australasian sector, the Australian register of Therapeutic Goods (TGA) has regis-
tered Sativex for future use, but the distributor Novartis™has made the commercial decision
not to make it available [6]. However as of 24th February 2016, The Australian parliament
passed new national laws and amendments to the Narcotics Drug Act 1967 to allow for the
controlled cultivation of cannabis for medical and scientific purposes in Australia [11]. This
followed the Victorian state government’s introduction of legislation into parliament to legalise
medicinal cannabis based on the advice of the Victorian Law Reform Commission’s Report on
Medicinal Cannabis [12]. These legislations are designed to allow access for those in need
while limiting abuse and diversion potential. In New Zealand, medicinal cannabis remains ille-
gal, though the recent approval for the use of Elixinol (a cannabidiol usually marked as a die-
tary supplement based on anti-oxidant properties of CBD) in a coma patient suffering status
epilepticus actioned by the health minister on “compassionate grounds” has sparked a spate of
discussion in the medical community. [13–15]
Worldwide, the growing development of cannabis-based medicines has led to greater dis-
cussion among patients, prescribers and policy makers. As prescribers of cannabis, physicians’
perspectives have been documented in studies conducted in the US [16], Canada [17] and
Israel [18]. These results illustrate how medical practitioners’ views have been explored in dif-
ferent national settings especially in Organisation for Economic Cooperation and Development
(OECD) countries. However, in such countries ethical principles in medicine mandate a degree
of separation between prescribing a drug and its supply; thus necessitating the need for inde-
pendent channels of distribution. In most instances this is the role of the pharmacy profession. Thus–should medicinal cannabis be legalised–pharmacists would be responsible for the stock-
ing, handling, ethical supply, counselling and overseeing the safe use of medicinal cannabis. This makes their professional support, opinion and perspective a fundamental aspect to be
explored in order to ensure medicinal cannabis is implemented successfully. The pharmacy profession itself has championed many public health services across several
OECD nations in order to better the community. A key example in the UK and Australia is the
implementation of fundamental harm-minimisation programmes such as the needle exchange
program, which aims to reduce the transmission of blood borne viruses among intravenous
drug users [19]. Aim To explore and investigate Australian pharmacists’ views on medicinal cannabis and their role
in its supply. Objective To explore pharmacists’ perspectives on the facilitators and barriers with respect to: To explore pharmacists’ perspectives on the facilitators and barriers with respect to: • The legalisation of medicinal cannabis in Australia • Dispensing medicinal cannabis in community pharmacies • The support needs in relation to provision of medicinal cannabis in community pharmacies • The support needs in relation to provision of medicinal cannabis in community pharmacies Ethics Statement Prior to commencement of the study (July–November 2015), approval was obtained from the
University of Sydney Human Research Ethics Committee (Ref No. 2015/591). This included
approval for the method of obtaining participant’s consent, utilizing an approved Participant
Information Statement [PIS]. All participants were required to sign a formal standardized Consent Form before starting
the interview and audio-recording, as per University of Sydney HREC requirements. Signed
Consent forms, constituting evidence of informed consent, from every participant were col-
lected and retained. These were stored in hard copies in a dedicated locked cabinet in the
supervising researcher's office. The Role of Medicinal Cannabis in Clinical Therapy contraception, aiming to reduce the risk of unwanted pregnancies, especially in young adults
[21]. In the implementation and supply of each of these public health measures pharmacists’ per-
spectives and pragmatic recommendations have been important. Given the ongoing debate
and controversy surrounding medicinal cannabis, the viewpoint of the pharmacy profession is
just as important for the successful implementation and delivery of medicinal cannabis to
patients worldwide. This is the first study to investigate pharmacists’ perspectives on the supply
and legalisation of medicinal cannabis. PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 In New Zealand, medicinal cannabis remains ille-
gal, though the recent approval for the use of Elixinol (a cannabidiol usually marked as a die-
tary supplement based on anti-oxidant properties of CBD) in a coma patient suffering status
epilepticus actioned by the health minister on “compassionate grounds” has sparked a spate of
discussion in the medical community. [13–15] Worldwide, the growing development of cannabis-based medicines has led to greater dis-
cussion among patients, prescribers and policy makers. As prescribers of cannabis, physicians’
perspectives have been documented in studies conducted in the US [16], Canada [17] and
Israel [18]. These results illustrate how medical practitioners’ views have been explored in dif-
ferent national settings especially in Organisation for Economic Cooperation and Development
(OECD) countries. However, in such countries ethical principles in medicine mandate a degree
of separation between prescribing a drug and its supply; thus necessitating the need for inde-
pendent channels of distribution. In most instances this is the role of the pharmacy profession. Thus–should medicinal cannabis be legalised–pharmacists would be responsible for the stock-
ing, handling, ethical supply, counselling and overseeing the safe use of medicinal cannabis. This makes their professional support, opinion and perspective a fundamental aspect to be
explored in order to ensure medicinal cannabis is implemented successfully. The pharmacy profession itself has championed many public health services across several
OECD nations in order to better the community. A key example in the UK and Australia is the
implementation of fundamental harm-minimisation programmes such as the needle exchange
program, which aims to reduce the transmission of blood borne viruses among intravenous
drug users [19]. The opioid substitution therapy (OST) is another program aimed at reducing
risk behaviours and illicit drug use in individuals [20]. The provision of flu vaccinations by
pharmacists across Europe, Canada, the UK and Australia is an expanded public health pre-
vention service and helps to reduce the burden of disease costs on healthcare systems. In addi-
tion to these services pharmacists have been central in the supply of the emergency 3 / 17 Design of interview An interview protocol (Table 2) was developed based on research literature on medicinal can-
nabis (4, 16–18) and practice experience of the researching team. The semi-structured inter-
views incorporated open-ended questions to enable the exploration of new ideas with prompts
to allow deeper probing and expansion of key issues relating to medicinal cannabis. For partici-
pants who required greater knowledge or awareness about medicinal cannabis, clinical research
papers including a meta-analysis was provided to them for greater familiarity with the topic,
before re-commencing the interview at a later scheduled time. For uniformity, the interviews were conducted by one interviewer (SI) and were between
10–30 minutes in length each. They were audio-recorded following participant consent, tran-
scribed ad verbatim and de-identified. Data were then entered into NVivo (QSR Version 10.0.3
Mac) software for coding. For quality control and to ensure reasonably objective analyses, the
research team independently read and coded transcripts into themes to create a coding scheme
for thematic analysis [22]. Constant comparison of interviews helped extract key perspectives
and ensured a level of consistency and reliability of analysis. These key themes were then used
to generate a driver diagram as seen in Fig 1. The Role of Medicinal Cannabis in Clinical Therapy were targeted in order to capture a variety of perspectives based on location, practice environ-
ment and experience. We sought the perspective of community pharmacists and key stake-
holders of the profession. Table 2. Interview Protocol. Table 2. Interview Protocol. Table 2. Interview Protocol. QUESTIONS
PROMPTS
What are your general thoughts about medicinally used
cannabis? Understanding of legal status? Various uses? Evidence about its potential for medicinal use? Do you believe it might be beneficial to make medicinal
cannabis legal or should it remain illicit? Effect on pharmacy practice? Would you be happy to dispense medicinal
cannabis if made available? Potential problems with medicinal cannabis
from a pharmacist’s point of view? Do you think the substance “cannabis” attracts a
stigma? Links with its side effects when used illicitly? Based on evidence? Is medicinal cannabis in capsule form any
different from other medicines? What do you believe pharmacists should be saying in
this debate? Role of Pharmacy profession? Other healthcare professionals’ roles? doi:10.1371/journal.pone.0155113.t002 Participant demographics A total of 34 respondents met inclusion criteria and participated in the study. The gender dis-
tribution was equivalent, and their level of practice experience varied (Table 3). Registered
pharmacists from across Australia were interviewed, but a greater proportion were pharmacists
practicing in the state of New South Wales. In regards to primary roles between practicing
pharmacists, academics and LRPOs, the majority of participants were solely practicing Recruitment The sampling strategy involved a convenience sampling of Australian pharmacists. To enable
the sampling of a wide variety of views, the inclusion criteria were that the interviewees were
currently registered with the Australian Health Practitioner Regulation Agency as pharmacists,
and willing to express their views on the legalisation of medicinal cannabis. It also employed a
purposive sampling of a subset of Leading Representatives of Professional Organisations
(LRPO) and was followed by passive snowballing as a result of individual requests to partici-
pate. LRPO’s were identified based on their credentials, the prominence of their role in the
Australian pharmacy profession, and their ability to influence the industry as professional lead-
ers and representatives. Their contact details were found in the public domain from various
Internet websites and professional journals. Recruitment was initiated via email, phone and
face-to-face invitation circulation, along with advertisements placed on professional society
newsletters and social media sites of professional organisations. A broad range of locations PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 4 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 QUESTIONS giving them a better quality of life is something that basi-
cally we work for every day.” (Interview #3) is the upmost of our priority . . . giving them a better quality of life is something that basi-
cally we work for every day.” (Interview #3) is the upmost of our priority . . . giving them a better quality of life is something that basi-
cally we work for every day.” (Interview #3) The results are presented under the five following emergent themes: role of the pharmacist,
legislation, safety, stigma and collaboration (Table 4). Each theme was deduced from the data of
quotes extracted from the interview transcripts. Table 4. Themes and sub-themes describing pharmacists’ views on the legalisation and implementa-
tion of medicinal cannabis. Themes
Sub-themes
Role of Pharmacists
Significance
Legislation
Scheduling
Nationalisation
Quality Assurance
Access
Safety
Patient safety
Risk of Abuse (SE)
Safety of the Pharmacy
Stigma
Public
Media
Collaboration
Professional Training & Public Awareness
Unified Communication
doi:10.1371/journal.pone.0155113.t004 QUESTIONS doi:10.1371/journal.pone.0155113.t002 5 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy Fig 1. Driver diagram of emergent themes from interviews (n = 34)
doi:10.1371/journal.pone.0155113.g001 Fig 1. Driver diagram of emergent themes from interviews (n = 34) doi:10.1371/journal.pone.0155113.g001 doi:10.1371/journal.pone.0155113.g001 pharmacists. Although concordant with other representative data, the views, strength of con-
viction and proactive ideas of LRPO participants emanated more resoundingly when compared
to other participants. This was determined via linguistic analysis of the recorded interviews
and interpretation of verbal cues. Interestingly, there were varying levels of knowledge across the differing demographics. Dif-
ferent patterns of awareness were observed, as pharmacists in academia had greater pharmaco-
logical and pharmacotherapeutic knowledge while LRPOs appeared to have a greater sense of
clinical and regulatory awareness than those in other roles. It was also acknowledged by LRPO participants that professional organisations in phar-
macy–like theirs–should take responsibility for the ongoing training and support of members
of the profession when it comes to the legalisation of medicinal cannabis. Overall, the majority of participants expressed support and encouragement for the legalisa-
tion of medicinal cannabis with a sense of duty of care to their patients. “It’s quite exciting. There is finally going to be a treatment option for those that up until
now had no hope and no treatment. And as a pharmacist. . . patients’ health and well-being 6 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy Table 3. Demographic of registered pharmacists’ interviewed (n = 34). Characteristics
n
%
Gender
Male
17
50
Female
17
50
Age
20 to 29
17
50
30 to 39
8
23
40 to 49
4
12
50 to 59
3
9
60+
2
6
Number of years in practice
1
4
12
1 to 5
11
32
6 to 10
6
18
11 to 15
4
12
16 to 20
1
3
21
8
23
Practice Location
New South Wales
27
79
Victoria
5
15
South Australia
1
3
Western Australia
1
3
Primary Roles
Practicing Pharmacists
25
73
Academia
3
9
Leading representatives of professional organisations (LRPO)
6
18
doi:10.1371/journal.pone.0155113.t003 Table 3. Demographic of registered pharmacists’ interviewed (n = 34). is the upmost of our priority . . . Themes PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 7 / 17 The Role of Medicinal Cannabis in Clinical Therapy Role of the Pharmacist The majority of participants expressed agreement that pharmacists would play an essential role
in providing legitimate access to medicinal cannabis. As drug specialists, participating pharma-
cists identified their role as central to the drugs supply, use and safekeeping. “We need to have our input into the matter, I think that it is very important. You know we
are the ones to most likely dispense and supply it.“(Interview #18) They also acknowledged, successful implementation of medicinal cannabis programs
require input from the profession in this contemporary debate and discussions amongst all
involved. “We are all part of the healthcare professional team and in order for us to help the patient
we need to actually work hand-in-hand together and have all different types of opinions
amalgamated into one.” (Interview #22) Legislation Scheduling. The majority of the participants expressed the view that medicinal cannabis
would be best introduced as a controlled substance, which under Australian regulation is cate-
gorised as a dangerous drug or schedule 8 (S8) as opposed to prescription only medicines or
schedule 4 (S4) drugs [23]. Participants felt that this controlled scheduling would be best suited for medicinal cannabis
due to its perceived propensity to be misused. Also based on past experiences with the OST
and misuse of other medicines such as pseudoephedrine for illicit purposes, pharmacists
expressed a desire for stringent guidelines as a means of legislative support. “Initially it would be like a S8 and I think that's the appropriate place for it. S8 gives the
pharmacist some comfort about the level of legislation behind it; checking the medication
and making sure doctors write the prescriptions properly.” (Interview #6) On the other hand some participants who had a greater knowledge on the cannabinoid con-
stituents and their pharmacological effects conveyed a lesser focus on controlled scheduling,
rather a greater focus on its accessibility as a prescription drug. “I don’t see it as a S8 medicine, based on its use. I don’t really see it as the same thing as
methadone. I see it more as a schedule 4.” (Interview #17) In addition to the legislation, some participants suggested the development of comprehen-
sive recording systems (e.g. where medication supply is recorded in pharmacy registers) to be
incorporated. Parallels were drawn with current systems for recording of supplied medications
including: staged supply, methadone subsidiaries, and the clozapine portal. These pharmacists
felt that in order to fulfil their duty of care to “do no harm” [24] as healthcare professionals a
stringent protocol with recording systems in place would be needed. Nationalisation. Some participants stated that the success of implementation of legal
medicinal cannabis supply would depend on a nationalised framework. Pharmacists’ support
for a nationalised framework was to ensure a level of consistency, uniformity and standardisa-
tion across the country (i.e. to avoid inter-state variations). PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 8 / 17 The Role of Medicinal Cannabis in Clinical Therapy “Establishing a nationalised system and accompanying that with the current E-Health
scripts . . . that would help manage this well.” (Interview #8) Quality Assurance. Participants with a broad knowledge of all the multiple constituents of
cannabis stressed a need to have stringent quality assurance protocols. Legislation They expressed the
need for a standard homogenous stable formulation of a high pharmaceutical grade, regardless
of manufacturer. “A committee should recommend a standard formulation containing specific moieties (sic.)
that can be prepared and then that’s the one standard formulation that’s made available
nationally.” (Interview #1) Conversely, a portion of those interviewed revealed a confidence in existing regulatory bod-
ies to govern the standardisation of medicinal cannabis and its key components. “As long as the active ingredient are produced or packaged by a TGA licensed facility, then I
don’t see why there’d be a problem.” (Interview #11) Majority of participants (even those who worked in pharmacies that identified as com-
pounding pharmacies) felt strongly about the initiation of medicinal cannabis as a standardised
pharmaceutical product in order to preserve the medications quality control and minimise any
risk of harm to the patient associated with compounded forms of cannabis. “You can’t just grow a plant because there are variations in the plant itself and in the grow-
ing conditions. I see it as being a medicinally used product. . . I guess much along the lines
that digitalis was standardised and used. I mean over the years, pharmacology has developed
from plants and it’s a lot better when we have standardised and know what the ingredients
are.” (Interview #31) Access. Several avenues for access to medicinal cannabis were proposed. The majority of
participants felt that the most suitable setting would be via a community pharmacy setting due
to the importance of accessibility for chronic and palliative patients. “It should be within a community setting. I think that all palliative care should be. . . in
terms of accessibility, within the community is best.” (Interview #6) This was followed by the suggestion of staged implementation, with supply initiating at clin-
ics or hospitals before being introduced to a community setting. “Initially in a clinic setting and then following good feedback and positive outcomes in a
community setting. . . because it is more readily available.” (Interview #7) Some participants preferred cannabis to be supplied in a hospital environment with the key
reason cited being a more specialised team monitoring its use. A few participants making this
suggestion, also proposed a clinic setting like that used for methadone initiation would mini-
mise the potential for cannabis abuse. PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 Legislation A number of participants were indifferent to the location of supply, suggesting that it could
be successfully supplied in a multiple number of settings in order to make it accessible to all PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 9 / 17 The Role of Medicinal Cannabis in Clinical Therapy patients in various locations and with various needs. A few participants suggested a specialised
cannabis supplier model similar to those existing overseas as means of cannabis supply. patients in various locations and with various needs. A few participants suggested a specialised
cannabis supplier model similar to those existing overseas as means of cannabis supply. Safety Patient Safety (SE. A number of participants were concerned about potential long-term
effects of medicinal cannabis with risks associated with cognitive impairment and psychosis. However, most participants mentioned that all medicines have risks involved and it is a matter
of weighing up those risks with the benefits for each individual undergoing treatment. “There are potential harms associated with its long-term use, like with any medicine. There
is no doubt about that. But throwing the blanket over the whole thing and saying no we
can’t use it because of that, is kind of a way out and unethical.” (Interview #29) Risk of Abuse. A few participants perceived the gaps in current regulations and recording
systems as an opportunity for abuse and misuse of controlled substances. They emphasised the
need to either have more stringent safe keeping protocols or even have special formulations to
avoid these perceived risks. “If it becomes legalised than it may be easier than before to be abused.” (Interview #20) PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 “If it becomes legalised than it may be easier than before to be abused.” (Interview #20) “I’m thinking direct contact with the doctor every time something has been prescribed, no
repeats at all, just constant follow ups with each healthcare professional to make sure it’s not
being abused.” (Interview #27) On the other hand, the majority of participants didn’t deem this perceived risk as a barrier
to the introduction of medicinal cannabis. The mainstream outlook on medicinal cannabis is
that such a formulation for medicinal use makes the risk of abuse or diversion potential almost
insignificant. “From my understanding these medicinal products are not that divertible or desirable...due to
their formulation, so the hype of not using it (in fear of abuse) is unjustified” (Interview #30) Many held the firm view that the profession was one of primary care and prevention and
was well equipped to manage and assist those who need it most. “We are healthcare professionals and if we identify abuse then we refer people to the correct
avenues. That’s our role as pharmacists. We are not here to make people who have a prob-
lem with medicines feel like they’re criminals and we refer them to appropriate health facili-
ties.” (Interview #11) Safety of the Pharmacy. A level of concern was raised by LRPOs, in regards to safety and
security issues for pharmacies. There may be a “higher prevalence of break-ins and people coming in demanding it. That
would be a security issue that would need to be looked into.” (Interview #18) 10 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy As a result, despite the advocacy and encouragement, there was an acknowledgment of the
ethical principle of professional autonomy and of the right of the pharmacist to conscientious
objection, as long as a level of professional duty of care is preserved. “I don’t think we can dictate that all pharmacists dispense it, because individuals may have
their preferences and that is something we may have to accept. I just think we should
encourage all pharmacists to be part of it and participate in it as they have a responsibility
here to dispense these particular products.” (Interview #23) Stigma Public. All participants identified the presence of public stigma associated with medicinal
cannabis. Further to that, many proposed that the current illicit status of medicinal cannabis
has led to this. “Stigma will stay whilst ever it (cannabis) is illegal.” (Interview #19) “Stigma will stay whilst ever it (cannabis) is illegal.” (Interview #19) Many participants identified the lack of public awareness, influence of cultural upbringing,
age and inability to distinguish between medicinal and recreational cannabis as key factors con-
tributing to public stigma. It was apparent that most participants drew upon their previous
experiences with patients on OST and opioid medicines, and would only be resolved with
ongoing public health campaigns and further discussion. “When you say the word cannabis people often just think about. . .the negative aspects and
side effects.” (Interview #16) “Imagining a place like Nimbin or you know a hippie crowd using it.” (Interview #1) “Imagining a place like Nimbin or you know a hippie crowd using it.” (Interview #1) “Imagining a place like Nimbin or you know a hippie crowd using it.” (Interview #1) “Just from my observations, dealing with methadone patients. . .a lot of people look down
on them and treat them badly.” (Interview #18) For some, this public stigmatisation of medicinal cannabis was feared to affect the consumer
and pharmacy/pharmacist rapport that they have established, with concerns that the pharmacy
itself would be perceived in a negative light. “In saying that, I don’t want my patients to think that we are a ‘cannabis pharmacy’ it might
give us (franchise) a bad look in the community.” (Interview #27) Media. Participants discussed how the media had played a predominant role in creating
the awareness about medicinal cannabis whether negative or positive. Some suggested that the
power of the media be harnessed for creating informed awareness on the therapeutic evidence
behind medicinal cannabis and to dissolve negative stigma and rebuild the image of cannabis
in a healthcare setting. “You hear stories in the media. . . with some pushing for it to be legalised and others in the
past highlighting it negative effects and this influences a lot of people.” (Interview #20) Participants cited recent media attention advocating the medical use of cannabis, as a shift
away from conventional views. 11 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy “I don't feel there is a lot of stigma to be honest. I think there's more like a push for it to be
available. I've seen a lot of stories where it's been beneficial.” (Interview #12) Collaboration Professional Training & Public Awareness. While pharmacists acknowledged a lack of
extensive understanding about medicinal cannabis, it was deemed no different to any other
new drug that enters the market. The majority of participants suggested the need for develop-
ment of new training courses and learning opportunities, in order to ensure a greater under-
standing of the effects of medicinal cannabis. “There will need to be education campaigns for pharmacists, consumers and probably all
healthcare professionals around this issue when cannabis is legalised.” (Interview #23) “Pharmacists have a great capacity. . . to learn and then disseminate information. . .to edu-
cate the public.” (Interview #31) Unified Communication. Many participants suggested the development of a collaborative
team of healthcare professionals to discuss the implications of legalising medicinal cannabis in
order to ensure multidisciplinary care. “There needs to be a forum where all key stakeholders who are involved in this issue. . .have
a discussion. They need to raise issues that are going to affect them or their profession and
considerations need to be made so I think that is very key.” (Interview #18) PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy expressed opinions of prescribers worldwide have been relatively more sceptical with negative
attitudes towards the use of cannabis medicinally. In 2005, a nationwide study exploring Amer-
ican physicians’ opinions about medicinal cannabis revealed that their level of positivity about
legalised cannabis was lower than that of the American general public at that time [16]. In 2015
an Israeli study reported that 79% of Israeli physicians expressed cautionary views of support
that medicinal cannabis “could be helpful for chronic and for terminally ill patients” [18]. As
some researchers note, perhaps US physicians are not convinced of cannabis’s health benefits
and believe its use carries risks [25]. Our participants did share these common concerns for
patient safety but expressed that these are universal for all medicines, and for medicinal canna-
bis the benefits outweigh the risks primarily when needed to optimise quality of life in condi-
tions recalcitrant to other treatments. As illustrated in our study results, participants acknowledged the importance of continued
training and learning in regards to information on medicinal cannabis. Similar needs for train-
ing preceding new program implementation have been expressed by pharmacists for other pro-
grams, such as the methadone program offered by pharmacies in many countries. In a 2001
survey conducted by Fleming et al. with North Ireland pharmacists, results demonstrated that
pharmacists were willing to participate in methadone dispensing (OST) with a proviso that
they be offered comprehensive training beforehand [26]. Pharmacists may not be unique in
desiring training prior to supply program implementation. A recent Canadian study conducted
with physicians highlighted their expressed need for “greater knowledge about. . .risks and dos-
ing” before they could implement medicinal cannabis programs successfully [17]. Training is
an essential element in implementing novel treatment. In a trial comprising pharmacists and
opioid dependent consumers in Victoria, Australia, health policy researchers used seven key
pillars to bridge the know-do gap in using buprenorphine for OST [27]. These pillars included
skilled and experienced practitioners, government and policy support, incentives to prescribe
the new treatment, specialist support services, clinical guidelines, training programs and
patient involvement and information. Authors propose that this multi-faceted approach pro-
pelled the uptake of buprenorphine as maintenance therapy for opioid dependent patients in
Victoria [27]. Our study also identified the need for greater collaboration to enhance transparency and
involvement of all stakeholders, including pharmacists. PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 Discussion This is the first study to investigate pharmacists’ views and concerns in relation to the prospec-
tive legalisation of medicinal cannabis in Australia, and one of the few in the world reporting
pharmacists’ viewpoint on this issue. Given the global discussion about medicinal cannabis
currently, there is a clear need to ensure that the views of all stakeholders involved are explored;
particularly that of pharmacists, who have the role of medicine supply. Our study gathered the
opinions of a reasonably heterogeneous sample of pharmacists, including professional leaders,
and results indicated that a majority supported the legislation and decriminalisation of medici-
nal cannabis in order to provide a suitable treatment option to those with refractory and
chronic medical conditions. Participating pharmacists described the need for suitable legisla-
tive and forensic frameworks that would allow legitimate supply under their scrutiny and rec-
ommended several models of supply. Pharmacists the world over, have demonstrated
willingness and capacity for delivering harm minimisation services such as needle exchange
programs and OST. Medicinal cannabis may be viewed in the same vein, where legal formula-
tions dispensed with the purview of a trained and knowledgeable professional may be far safer
than other means of procurement frustrated patients resort to. Pragmatic models offered by
the participants and previous experience with pharmacy-delivered harm minimisation pro-
grams should drive future implementation programs for therapeutic cannabis provision. The
perspective of pharmacists should also be probed in health systems considering legalising
medicinal cannabis. With no other study examining pharmacists’ perspectives to compare with, a comparison
may be drawn with prescriber-based international studies. In contrast to our participants, the PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 12 / 17 The Role of Medicinal Cannabis in Clinical Therapy lacking legislative mandates on medicinal cannabis supply [34]. In order to prevent cases such
as these and to increase pharmacists’ confidence, our study highlighted nationalised legislation
as a key driver to successfully introducing medicinal cannabis into Australian healthcare. Participants in our study also identified safety as a major area of concern. For reasons of safety and reproducibility, introducing standardized forms of synthetic or
extracted cannabinoids supply, although more expensive, rather than extractions of phytocan-
nabinoids in the cannabis sativa plant, would be ideal. However, there are no globally recog-
nised standardized forms or formulations of medicinal cannabis available yet, due to vast
variability in constituents from plant to plant. This could be a future development once medici-
nal cannabis is legalised and research/development is allowed to evolve. Suggestions also
included pharmacovigilance to monitor patient safety, addressing risk of abuse and creating
support to reduce prescribing under duress as well as establishing robust support for pharmacy
safety. Such suggestions were drawn from past experiences with OST and the supply of danger-
ous drugs of addiction. A 2010 survey study by Winstock et al. reported pharmacists’ had expe-
rienced problems with OST clients showcasing “aggression, intoxication and remaining in debt
with the pharmacy” [35] Indeed, increasing reports of pharmacies being robbed or held-up are
noticed globally [36, 37]. And for pharmacies to supply medicinal cannabis this needs to be
specifically addressed. Stigma was identified as a major barrier that needed to be mitigated in order for medicinal
cannabis to be successfully rolled out. The influence of negative stigma on the actual adoption
of such medicines and health programs is revealed in various studies. Comparably our partici-
pants described the stigma around medicinal cannabis paralleled that enveloping methadone
clients. Another recent qualitative Australian study exploring pharmacists’ opinions about
methadone supply as an OST, reported that stigma appeared to be an underlying cause in the
case where pharmacies were still unwilling to provide OST despite increasing demand. It
reported these participants showcasing negative attitudes of “prejudice, cynicism and fear.”
[38] In our study, it was recognised that stigma may be a result of the blurred lines between
medicinal and recreational use of cannabis. To shift public opinion, greater awareness and edu-
cation needs to be implemented, in which clear distinctions between the two uses of cannabis
are defined. All of these themes were triangulated to generate a conceptual framework (Fig 1), which
captures the results of the study and showcases socio-political drivers for the legalisation and
supply of medicinal cannabis through community pharmacies. It is a conceptual map of
themes emerging from ideas expressed by participants in this study. The conceptual map iden-
tifies secondary drivers, such as “safety of patient and pharmacy” for example, which together
formulate primary drivers of change. The four major drivers of change in this particular case
were identified as: 1. Addressing Safety, 2. Drawing up inclusive legislation reflecting all stake-
holders’ concerns, 3. Addressing Stigma and 4. Establishing suitable collaborations. This conceptual framework can be utilised by policy-makers to develop policies of change
based on primary sourced data of pharmacists’ perspectives. This issue also appears to be identified
in other studies; as collated data from surveys with Swiss pharmacists involved in methadone
and needle exchange programs highlighted that “pharmacists do not feel integrated enough in
the network of care of drug misusers and ask for better recognition of their role.” [28] There-
fore from the perspective of practicing pharmacists, it would be important to establish an
inter-professional forum to promote multidisciplinary discussion, collaboration and allow for
consultation to take place across various disciplines. This, along with maintaining open lines of
communication may help mitigate errors and ensure improved patient outcomes following the
facilitation of medicinal cannabis [29, 30]. Successful specialised treatment programs, for
example buprenorphine provision, have often utilised inter-professional training for pharma-
cists and specialist physicians [31, 32]. Similar methods need to be applied in the case of medic-
inal cannabis as well. A major issue emergent from our study was the need for nationalised legislation to maintain
uniform regulatory policies. This view is reflective of international literature that documents
the long-running battle between federal and state law. A US article contained a Californian
Supreme Court ruling in 2005 indicating that “patients who take cannabis in states where its
medicinal use is legal are not shielded from federal prosecution.”[33] In 2008 a survey study by
Reiman et al. reported medicinal cannabis facilities followed a “social model of care” with
patients creating a system of self-dispensing without professional intervention as a result of 13 / 17 S4 File. Safety—Product Standardisation—Formulation theme Quotes.
(DOCX) S5 File. Stigma theme Quotes. (DOCX) S5 File. Stigma theme Quotes. (DOCX) Acknowledgments The researchers wish to thank the Australian pharmacists who participated in the study. The researchers wish to thank the Australian pharmacists who participated in the study. Author Contributions Conceived and designed the experiments: BC BS SI. Performed the experiments: BC BS SI. Analyzed the data: BC BS SI. Contributed reagents/materials/analysis tools: BC. Wrote the
paper: BC BS SI. Limitations Some limitations of this study include the diversity of participants interviewed, which were pri-
marily from a community pharmacy setting, and this is not generalizable to the entire phar-
macy profession. Despite interviewing participants from various locations across Australian a
broader location sampling would provide more widespread and rigorous results. Video record-
ing of the interviews may have also strengthened the study through paralinguistic analysis of
captured non-verbal cues. 14 / 17 PLOS ONE | DOI:10.1371/journal.pone.0155113
May 12, 2016 The Role of Medicinal Cannabis in Clinical Therapy Conclusion Findings of this study (S1–S5 Files) highlighted the perspective of pharmacists who hold the
important role of suppling medicinal cannabis, thereby identifying important factors which
can help shape future policies for the successful implementation of medicinal cannabis in
healthcare. We recommend that these views and strategies be incorporated in the development
of new policies and legislations. Supporting Information pp
g
S1 File. Barrier and Facilitator Quotes. (DOCX)
S2 File. Legislation-Regulation theme Quotes. (DOCX)
S3 File. Role of Pharmacist—Collaboration Quotes. (DOCX)
S4 File. Safety—Product Standardisation—Formulation theme Quotes. (DOCX)
S5 File. Stigma theme Quotes. (DOCX) S2 File. Legislation-Regulation theme Quotes.
(DOCX) S2 File. Legislation-Regulation theme Quotes. (DOCX) S2 File. Legislation-Regulation theme Quotes. (DOCX) S3 File. Role of Pharmacist—Collaboration Quotes. (DOCX) S4 File. Safety—Product Standardisation—Formulation theme Quotes. (DOCX) PLOS ONE | DOI:10.1371/journal.pone.0155113
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SquiggleNet: real-time, direct classification of nanopore signals
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© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give 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
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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. METHOD METHOD *Correspondence:
welchjd@umich.edu
1Department of Computer Science
and Engineering, University of
Michigan, 48109 Ann Arbor, MI, USA
4Department of Computational
Medicine and Bioinformatics,
University of Michigan, 48109 Ann
Arbor, MI, USA SquiggleNet: real-time, direct
classification of nanopore signals Yuwei Bao1, Jack Wadden1,2, John R. Erb-Downward3, Piyush Ranjan3, Weichen Zhou4,
Torrin L. McDonald5, Ryan E. Mills4,5, Alan P. Boyle4,5, Robert P. Dickson3,6,7, David Blaauw3
and Joshua D. Welch1,4* *Correspondence:
welchjd@umich.edu
1Department of Computer Science
and Engineering, University of
Michigan, 48109 Ann Arbor, MI, USA
4Department of Computational
Medicine and Bioinformatics,
University of Michigan, 48109 Ann
Arbor, MI, USA Abstract We present SquiggleNet, the first deep-learning model that can classify nanopore reads
directly from their electrical signals. SquiggleNet operates faster than DNA passes
through the pore, allowing real-time classification and read ejection. Using 1 s of
sequencing data, the classifier achieves significantly higher accuracy than base calling
followed by sequence alignment. Our approach is also faster and requires an order of
magnitude less memory than alignment-based approaches. SquiggleNet distinguished
human from bacterial DNA with over 90% accuracy, generalized to unseen bacterial
species in a human respiratory meta genome sample, and accurately classified
sequences containing human long interspersed repeat elements. Full list of author information is
available at the end of the article Keywords: Deep learning, Read-until, Oxford Nanopore, Raw signal, Real-time Bao et al. Genome Biology (2021) 22:298
https://doi.org/10.1186/s13059-021-02511-y Bao et al. Genome Biology (2021) 22:298
https://doi.org/10.1186/s13059-021-02511-y Background Oxford Nanopore sequencers, such as MinION or PromethION, determine the nucleo-
tide sequence of a DNA or RNA molecule by measuring changes in electrical cur-
rent (called “squiggles”) as the molecule translocates through a protein nanopore. This
approach is fundamentally different from the widely-used Illumina platform and provides
several benefits: the MinION is small, fast, and portable, making it ideal for rapid diagnos-
tics and field work. Because it does not rely upon synchronized nucleotide addition (the
heart of the Illumina sequencing-by-synthesis technology), MinION also produces much
longer reads. To our knowledge, the longest published MinION read is around 2 Mbp
[1], though even longer reads have been reported anecdotally. The changes in electrical
current induced by a DNA or RNA molecule depend on the specific chemical properties
of the nucleotides, including secondary structure interactions and epigenomic modifica-
tions such as methylation. Additionally, the nanopore sequencer can stream the squiggle
data to a computer in real time. The nanopore sequencer can also eject a partially sequenced molecule, a capability
referred to as “Read-Until”. In principle, this enables targeted sequencing without the Bao et al. Genome Biology (2021) 22:298 Page 2 of 16 need for biochemical enrichment. The Read-Until capability allows selective sequencing
of molecules by reversing the voltage across individually selected nanopores, ejecting the
unwanted molecules. The unoccupied nanopores can then sequence different molecules
of interest. Such computational enrichment of target sequences holds great promise for clinical
diagnostics and field research, but realizing this potential requires fast and accurate
approaches for identifying molecules of interest. For example, identifying pathogenic
DNA in a patient lung fluid sample requires bypassing human DNA—which often rep-
resents > 99% of the sequences—to find the pathogen sequences. Biochemical methods
for target sequence enrichment, such as PCR [2–5], hybrid capture [6], or CRISPR/Cas9
enrichment [7, 8] require much more time, expertise, and equipment. In contrast, a com-
putational approach to enriching target sequences provides clear savings of time, labor,
and cost. Previous computational approaches for this problem include (a) perform standard
base calling followed by sequence alignment as in [9] and (b) perform rough base call-
ing to identify and align k-mers [10]. The first approach requires significant computing
resources—such as a graphics processing unit (GPU) and a large genome index database
for the sequence aligner. Background The second approach also requires a large genome index and
multiple CPU cores and can map only non-repetitive references smaller than ∼100 Mbp. Both approaches are based on sequence alignment and thus are limited by sequencing
errors, their reliance on genome indexes, and their inability to capture non-sequence
information such as DNA methylation. To address these limitations, we developed SquiggleNet, the first deep-learning-based
approach for classifying DNA sequences directly from electrical signals. SquiggleNet is
fast, accurate, memory-efficient, and robust to unknown species. It requires only 3000
signals—less than the amount of data generated in one second of sequencing—to clas-
sify the species of a DNA molecule with over 90% accuracy, significantly higher than the
best alignment-based methods. The model requires only 304 KB of RAM and no exter-
nal reference database. SquiggleNet is faster than or on par with the competitors and can
run in real time on a single core of a standard laptop. When tested on a human respira-
tory metagenome sample with a majority of unseen species, our approach achieves >90%
overall accuracy. SquiggleNet: a convolutional neural network for classifying nanopore signals SquiggleNet: a convolutional neural network for classifying nanopore signals
SquiggleNet is a deep neural network that classifies molecules of interest based on
statistical patterns in nanopore conductivity, which are often hard for humans to iden-
tify by eye, automatically extracted from the input data. The overall workflow for
using SquiggleNet to enrich sequences of interest is shown in Fig. 1a. The network
is first trained to recognize certain classes of sequences, such as human vs. bacte-
rial DNA, using labeled examples. Then, as the nanopore sequencer generates raw
electrical signals from a new and unseen sample, SquiggleNet rapidly classifies each
molecule to determine whether it is a sequence of interest. Molecules not of inter-
est are ejected from the nanopore, freeing the pore to sequence a different molecule. In contrast, targeted molecules are sequenced to full length and used for downstream
analysis. Bao et al. Genome Biology (2021) 22:298 Page 3 of 16 (2021) 22:298 Bao et al. Genome Biology Fig. 1 Read-until pipeline overview. a A DNA molecule translocates through a nanopore, generating electric
signals (squiggles). SquiggleNet rapidly classifies the molecule to determine whether it is a sequence of
interest. If the molecule is accepted by the classifier, it is sequenced to full length. Otherwise, the molecule is
ejected from the pore, freeing the pore to sequence another molecule. b SquiggleNet employs
1D-ResNet-styled bottleneck blocks with increasing numbers of filters. Average pooling and a final fully
connected layer are performed after the last convolutional block Fig. 1 Read-until pipeline overview. a A DNA molecule translocates through a nanopore, generating electric
signals (squiggles). SquiggleNet rapidly classifies the molecule to determine whether it is a sequence of
interest. If the molecule is accepted by the classifier, it is sequenced to full length. Otherwise, the molecule is
ejected from the pore, freeing the pore to sequence another molecule. b SquiggleNet employs
1D-ResNet-styled bottleneck blocks with increasing numbers of filters. Average pooling and a final fully
connected layer are performed after the last convolutional block SquiggleNet (Fig. 2b) employs a convolutional architecture, using residual blocks mod-
ified from ResNet [11] to perform one-dimensional (time-domain) convolution over
squiggles. The architecture consists of four blocks with increasing numbers of chan-
nels; each block includes two 1D-ResNet Bottleneck units. Mean pooling followed by a
fully-connected layer with softmax activation allows SquiggleNet to classify sequences
based on the convolutional filters in the last ResNet block. SquiggleNet: a convolutional neural network for classifying nanopore signals The final output is a con-
ditional probability on the sequence labels, which is then used to make the final class
prediction. We experimented with several other approaches, including a recurrent neu-
ral network (RNN) with long short-term memory (LSTM) blocks; gated recurrent units
(GRUs); other types of convolutional blocks; a combination of RNN and convolution;
different convolutional window sizes; and differing model hyperparameters. However,
we found that approaches based on convolution outperformed models using LSTM
blocks, suggesting that local features are sufficient for this problem, and long-range
time-dependent relationships do not add much information. Convolutional architec-
tures without LSTM blocks are also faster to train. Our final architecture gave the
best classification accuracy of any approach we tried and could not be made signif-
icantly smaller without sacrificing performance. Additional details about the model
architecture and hyperparameter choices can be found in the Method section and
Additional file 1. SquiggleNet accurately classifies species directly from squiggles To test the performance of SquiggleNet, we generated four experimental datasets con-
taining a mixture of human and bacterial DNA. The first dataset, HeLa&Zymo, con-
tains 8 bacterial species from the Zymo mixture [12] and HeLa cells. The species Page 4 of 16 Bao et al. Genome Biology (2021) 22:298 Bao et al. Genome Biology Fig. 2 Overall performance across five test datasets: accuracy, true positive rate (TPR, RECALL), true negative
rate (TNR), precision, and the AUROC score of the model trained on the HeLa&Zymo training set, and tested
on five test sets with bacterial sequences as the target Fig. 2 Overall performance across five test datasets: accuracy, true positive rate (TPR, RECALL), true negative
rate (TNR), precision, and the AUROC score of the model trained on the HeLa&Zymo training set, and tested
on five test sets with bacterial sequences as the target Fig. 2 Overall performance across five test datasets: accuracy, true positive rate (TPR, RECALL), true negative
rate (TNR), precision, and the AUROC score of the model trained on the HeLa&Zymo training set, and tested
on five test sets with bacterial sequences as the target Fig. 2 Overall performance across five test datasets: accuracy, true positive rate (TPR, RECALL), true negative
rate (TNR), precision, and the AUROC score of the model trained on the HeLa&Zymo training set, and tested
on five test sets with bacterial sequences as the target labels were obtained through Minimap2 [13] alignment. The other three datasets
(Human&Zymo_b12, Human&Zymo_b34, and Human&Zymo_b56) contain a mixture of
human GM12878 DNA and DNA from Zymo High Molecular Weight mixture with 7 bac-
terial species [14]. To avoid systematic error from the alignment algorithms, we obtained
reliable ground-truth species labels for these three datasets by attaching a nucleotide
sequence barcode to each DNA molecule indicating whether the molecule is from human
or bacteria. Note that our direct biochemical labeling strategy allows us to independently
assess the accuracy of species determination from base calling followed by read align-
ment; this is important for our application, since we expect that SquiggleNet may be able
to outperform purely sequence-based approaches by leveraging other information from
the electrical signals. Further dataset details can be found in the Method section and
Additional file 1. We trained SquiggleNet using more than two million reads from the first dataset
(HeLa&Zymo), which contains equal proportions of HeLa and bacterial sequences. SquiggleNet accurately classifies species directly from squiggles We
used 3000 signals from each read, the equivalent of about 300 nucleotides. We discarded
the first 1500 signals of each read (an overestimation of the adapter length), to remove
potential pore noise and adapter sequences, which could confound training. Thus Squig-
gleNet requires a total of 4500 signals, which is equivalent to about 1 second of sequencing
time. (The exact time and number of nucleotides depends on the translocation speed, Bao et al. Genome Biology (2021) 22:298 Page 5 of 16 Bao et al. Genome Biology which varies per pore and molecule over the course of the sequencing experiment.) How-
ever, using this exact amount of signal is not crucial; we verified that using fewer signals
did not significantly change the results (see below). Our best-performing model was
trained on the HeLa&Zymo dataset, which contains the largest number of sequenced
reads. This dataset also lacks species-specific barcodes, and we were careful to remove
the sequencing adapters and species barcodes before extracting the 3000 signals used
for classification (Methods). Thus, there is no way that the classifier could “cheat” by
using the barcodes to classify the species. When we instead trained the model on the
Human&Zymo datasets and tested on HeLa&Zymo, the model accuracy was nearly iden-
tical but slightly lower, possibly due to the smaller number of training samples (see
Additional file 1: Figure S1). Overall, the model classifies each molecule as bacterial or human with over 90% accu-
racy across different test datasets using only 3000 signals per read (see Fig. 2). The clas-
sifier generalizes well to different lab preparations, flow cells and proportions of species. For the first three datasets (HeLa&Zymo, Human&Zymo_b12, Human&Zymo_b34), the
true positive rates (TPR, also Recall) and the true negative rates (TNR) are all above or
around 90%. The precision and AUROC scores are all about 90% as well. Even for samples
with significantly more human than bacterial DNA (Human&Zymo_b56 and Respiratory
Metagenome), the accuracy and recall both remain high. We used the method of integrated gradients (IG) [15] to investigate the features influ-
encing SquiggleNet’s classification decisions. The IG method computes the amount of
gradient change for each corresponding input, and by doing so, offers interpretation
on which part of the input contributes the most to the model’s decision. SquiggleNet accurately classifies species directly from squiggles Inspecting
these IG results (Additional file 1: Figure S2) shows that SquiggleNet predictions are
most strongly influenced by positions where the signal changes direction, changes by
a large amount, and/or changes from one nucleotide to another. This suggests that
SquiggleNet has learned filters related to the nucleotide composition of the signal and
uses the results to make classification decisions. Further details are contained in the
Additional file 1. To demonstrate that the results are robust to the amount of signal removed from
the beginning of the read at test time, we also tested our pre-trained model on the
Human&Zymo_b34 dataset with only the first 1000 signals per read removed. We chose
the number 1000 because this is a closer estimation of the adapter length [16], and at
test time, we would like to make the decision as soon as possible to enable real-time
read selection. When testing the model on sequences with the first 1000 signals removed,
the results were nearly identical to those obtained from conservatively removing 1500
signals: 89.35% accuracy, 90% true positive rate, and 86.9% true negative rate. Thus it
appears that, as long as the initial pore noise, adaptors, and barcodes are removed from
the training sequences, the model is able to make an accurate and fast decision at test
time. This robustness also allows flexibility if, for example, different sequencing datasets
use sequencing adapters of different lengths. Remarkably, we find that SquiggleNet achieves significantly higher accuracy from 3000
signals than base calling followed by sequence alignment using the same amount of sig-
nal. This result gives crucial context for interpreting the accuracy of our model and
suggests that the convolutional filters may detect some non-sequence features that help
with species classification, such as chemical modification of nucleotides by methylation. Bao et al. Genome Biology (2021) 22:298 Page 6 of 16 Bao et al. Genome Biology Indeed, we found that the bacterial and human DNA sequences in our dataset show sig-
nificant methylation differences, with significantly more methylated cytosines in human
sequences and significantly more methylated adenines in bacterial sequences (see Addi-
tional file 1: Table S2 for details). For the Human&Zymob_56 dataset, the target to non-target sequence ratio is 1 to
99. The overall accuracy, TNR, and AUROC score are around 90%. The TPR (Recall) is
closely following, above 87%. The precision, however, is about 1/10 of the other cases. SquiggleNet accurately classifies species directly from squiggles This is due to the extremely low concentration of the target sequences (Zymo bacterial
species), and the precision calculation is diluted by the overwhelming number of false
positive reads. Nevertheless, this is acceptable, since we want to preserve as many tar-
geted reads as possible (high recall) due to the low target read concentration. All true
positives and false positives will be sequenced to full length, and thus can be further pro-
cessed in the downstream analysis. Considering that the Human&Zymo_b56 dataset has
99× more non-targeted reads than targeted, whereas only ∼10× more reads were falsely
identified as positive compared to the HeLa&Zymo dataset, this model demonstrated
strong ability to filter out non-targeted reads, and has high potential to improve through-
put (see below). Overall, the model that was trained on only the HeLa&Zymo dataset
yields high performance across different testing datasets, highlighting the robustness of
the model. Interestingly, SquiggleNet performance varies systematically across bacterial taxa. The
network classifies human vs. bacterial DNA with 90% accuracy, but some bacterial species
are easier to distinguish from human sequences than others. The eight bacterial species
in the Zymo mixture are related according to the taxonomy tree shown in Fig. 3. The top
three species—Pseudomonas aeruginosa (Pse), Salmonella enterica (Sal), and Escherichia
coli (Esc)—are gram-negative bacteria and are most easy to identify, while the bottom five
species are gram-positive bacteria and are harder to distinguish from human DNA. It is
not clear what specific features of the gram-negative bacteria make them easier to identify,
but this behavior may be related to species differences in GC-content or the amount of
methylation. SquiggleNet identifies species not seen during training In real-world applications, samples may contain species whose genomes are not in the
training samples. We thus investigated whether the model can identify unseen species. To do this, we performed a leave-one-out analysis, removing each of the bacterial species Fig. 3 Taxonomy tree and accuracy per species. Taxonomy tree for the eight species in our dataset grouped
in color and their corresponding accuracy breakdown per species. The accuracy for distinguishing bacterial
sequences from human was highest for the red branch, intermediate for the blue group, and lowest for the
brown group Fig. 3 Taxonomy tree and accuracy per species. Taxonomy tree for the eight species in our dataset grouped
in color and their corresponding accuracy breakdown per species. The accuracy for distinguishing bacterial
sequences from human was highest for the red branch, intermediate for the blue group, and lowest for the
brown group Bao et al. Genome Biology (2021) 22:298 Page 7 of 16 separately during training, then putting it back during testing to challenge SquiggleNet’s
generalization ability. For the held-out species comparisons, we used 400k and 20k reads
from the HeLa&Zymo dataset for training and testing, respectively. During each training run, we removed one of the eight Zymo bacterial species from
the training dataset. We then compared the test accuracy from the classifier trained
on seven bacterial species plus human with the performance of the same model on
two different testing sets containing the eighth held-out species. The dataset we call
Test-Uniform/HeLa includes all eight species (including the one held out during train-
ing), evenly distributed, and balanced to contain equal numbers of HeLa and bacterial
molecules. The dataset we refer to as Test-One/HeLa includes only the single held-out
species and HeLa, in equal proportions. The unknown species identification results can be found in Fig. 4. The red bars are
the test accuracy results without held-out species. The left-most column is the perfor-
mance of a training run with all 8 bacterial species as a reference for cross-testing run
performance comparison. Across different runs, the test accuracies, not including held-out species, are around
84–86%. For each Test-Uniform/HeLa experiment, accuracy of classifying the held-out
species was ∼83–85%, only about 1% lower compared to when the species was seen
during training. This shows that the model was able to accurately identify sequences
from bacterial species that were not seen during training. SquiggleNet identifies species not seen during training Genome Biology (2021) 22:298 Bao et al. Genome Biology Escherichia coli (Esc) actually increased by ∼1–4% compared to their validation accu-
racies. The remaining four gram-positive species had a minor performance increase or
drop within 4%. Staphylococcus aureus had the largest performance drop among all, but
the accuracy was still above 75%. In summary, these two sets of experiments show that even when one species was not
seen during training time, SquiggleNet was still able to identify it with high confidence. SquiggleNet identifies species not seen during training For the Test-One/HeLa
experiment, the test performance is more influenced by the taxonomic position of the
held-out species. Since the testing datasets only include human DNA and the one species
that was held out, we expected performance to drop even more than the previous
Test-Uniform/HeLa experiment. However, the test accuracies of the first three gram-
negative bacterial species, Pseudomonas aeruginosa (Pse), Salmonella enterica (Sal), and Fig. 4 Performance of SquiggleNet on unseen species. Each column (except “All”) is a model trained on a
Zymo/HeLa 1:1 mix without the held-out species. For each species, the red bar shows the test accuracy on all
species minus the held-out species; this number provides a baseline against which to compare performance
on the held-out species. Blue bars show the accuracy of each trained model on Test-Uniform/HeLa, a test set
with all eight Zymo bacterial species included and HeLa in a 1:1 ratio. Brown bars show the accuracy of each
model on Test-One/HeLa, a test set with only the single unseen species and HeLa in a 1:1 ratio Fig. 4 Performance of SquiggleNet on unseen species. Each column (except “All”) is a model trained on a
Zymo/HeLa 1:1 mix without the held-out species. For each species, the red bar shows the test accuracy on all
species minus the held-out species; this number provides a baseline against which to compare performance
on the held-out species. Blue bars show the accuracy of each trained model on Test-Uniform/HeLa, a test set
with all eight Zymo bacterial species included and HeLa in a 1:1 ratio. Brown bars show the accuracy of each
model on Test-One/HeLa, a test set with only the single unseen species and HeLa in a 1:1 ratio Fig. 4 Performance of SquiggleNet on unseen species. Each column (except “All”) is a model trained on a
Zymo/HeLa 1:1 mix without the held-out species. For each species, the red bar shows the test accuracy on all
species minus the held-out species; this number provides a baseline against which to compare performance
on the held-out species. Blue bars show the accuracy of each trained model on Test-Uniform/HeLa, a test set
with all eight Zymo bacterial species included and HeLa in a 1:1 ratio. Brown bars show the accuracy of each
model on Test-One/HeLa, a test set with only the single unseen species and HeLa in a 1:1 ratio Page 8 of 16 Page 8 of 16 Bao et al. SquiggleNet identifies bacterial DNA in a human respiratory metagenome sample To further test the generalizability and practicality of SquiggleNet, we tested the best
performing model (trained on the Hela&Zymo dataset) on a dataset collected from sev-
eral clinical human samples. We collected the data following the procedures in [17]. The ground-truth labels were obtained using our previously published read alignment
pipeline[18]. The dataset includes 324,526 human reads and 341 bacteria and other (less
than 0.6%), a human:bacterial ratio of 951:1. Some of the dominant bacteria groups
include Prevotella (29%), Neisseria (20%), and Rothia (11%). However, less than 3% of the
bacterial species overlap with the training dataset. The full taxonomic composition can
be found in Fig. 5 and [17]. Fig. 5 SquiggleNet accuracy by species in human respiratory metagenome sample[19]. Some of the
dominant bacterial groups include Neisseria (23%), Bacteriodales (21%), and Firmicutes (20%). Less than 3% of
the bacterial species overlap with the training dataset Fig. 5 SquiggleNet accuracy by species in human respiratory metagenome sample[19]. Some of the
dominant bacterial groups include Neisseria (23%), Bacteriodales (21%), and Firmicutes (20%). Less than 3% of
the bacterial species overlap with the training dataset Bao et al. Genome Biology (2021) 22:298 Bao et al. Genome Biology (2021) 22:298 Page 9 of 16 Bao et al. Genome Biology (202 Even though the model was trained on dataset Hela&Zymo, it achieved 90.8% overall
accuracy in the Respiratory Metagenome dataset, 72.5% true positive rate, and 90.9% true
negative rate (Fig. 2). The AUROC score is 0.817. The precision is about 1/5 of that in
dataset Human&Zymo_b56. As with the unbalanced Human&Zymo dataset, the preci-
sion is diluted by the extremely low concentration of bacteria, but the model still achieves
high recall—which is critical to retrieve all the bacterial reads for downstream analysis. The Zymo community of the dataset on which the model was trained has very little
overlap (< 3%) with the bacterial species found in the Respiratory Metagenome dataset. The genome information in this testing dataset was mostly unseen and unknown for the
trained model. However, it still achieved a true positive rate of 72.5%. This shows that
SquiggleNet is able to extract common bacterial genome features and distinguish them
from the human genome sequencing raw signals. The generalizability of SquiggleNet sig-
nificantly increases the potential applications of our method. As shown in Fig. 5, different
species were classified with different accuracy. SquiggleNet identifies bacterial DNA in a human respiratory metagenome sample The model is therefore, expected to be
even more accurate if it can be fine-tuned in a dataset with closer range of species. SquiggleNet is more accurate and efficient than previous approaches We next compared the performance and efficiency of SquiggleNet against the current
state-of-the-art methods: Guppy+Minimap2 and UNCALLED. This experiment was con-
ducted on dataset Human&Zymo_b34 with 1:4 Human and Zymo mix. All the analysis
was done on a single-usage Intel(R) Xeon(R) CPU E5-2697 v3 @ 2.60GHz machine with
a single TITAN Xp GPU. We benchmarked the running time required to classify 712,000 reads (178 fast5 files
with 4000 sequences each and 3000 signals per read, adapters and barcodes removed). SquiggleNet took 806.74 s (13 min 27 s) to finish processing all on GPU (Fig. 6). When
tested on a 3.5-GHz Dual-Core Intel Core i7 Macbook Pro, SquiggleNet finished pro-
cessing all the files in 2630.58 s (43 min 51 s). With the Guppy+Minimap2 method [9],
sequences were base called using Guppy [20] with 4 callers and 4 runners/GPU, and we
used 32 threads for sequence alignment with Minimap2 [13]. It took 742.602 s (12 min
23 s) for Guppy to finish base calling 3000 signals and another 25.673 s for Minimap2
to finish the alignment, about the same amount of time as SquiggleNet. The accuracy Fig. 6 Processing time and accuracy comparison. The processing time of SquiggleNet with 300 bp of input is
among the lowest, and yet the accuracy is the highest among the three methods. For the other two
alignment-based methods, with longer input length, the processing time grows drastically, whereas the
accuracy gain is limited Fig. 6 Processing time and accuracy comparison. The processing time of SquiggleNet with 300 bp of input is
among the lowest, and yet the accuracy is the highest among the three methods. For the other two
alignment-based methods, with longer input length, the processing time grows drastically, whereas the
accuracy gain is limited Bao et al. Genome Biology (2021) 22:298 Page 10 of 16 Bao et al. Genome Biology of Guppy+Minimap2, however, was 79%, more than 10% lower than SquiggleNet. Using
the full length of the input sequence increased the accuracy of Guppy+Minimap2 to 91%,
but the processing time increased dramatically. With UNCALLED, 32 threads were used
to process 3000 signals, 6000 signals, and full-length reads respectively. It took at least
1277.28 seconds (21 min 17 s) to finish the 3000 signals, but the accuracy was below 50%. SquiggleNet is more accurate and efficient than previous approaches With longer input length, the accuracy increased to 60% (82% for full-length), but the full
length accuracy was still lower than SquiggleNet with 3000 signals. Meanwhile, the pro-
cessing time grows drastically as well. SquiggleNet is, therefore, faster and more accurate
than either Guppy+Minimap2 or UNCALLED. Note that the accuracy values that we report take into account all reads, including
those that Minimap2 could not align. In contrast, the UNCALLED paper reported that
the method was able to recover 94% of the alignments identified by Minimap2, but
this number does not take into account the reads that Minimap2 failed to align. Cru-
cially, our datasets have molecular barcodes, allowing us to determine the true species
even for reads that Minimap2 failed to align. Furthermore, the 94% accuracy reported
by the UNCALLED paper is based on using the entire sequence, whereas we only used
significantly less information (3000 signals) to classify the reads. We also observed that over 90% of the SquiggleNet processing time on GPU and over
40% of the processing time on CPU is spent on loading data from the disk. The actual
classification time for a batch of 500 reads is about 0.06 seconds on GPU and about 0.8
seconds on CPU (one thread). When streaming directly from the nanopore sequencer in
real time, this loading time can be significantly reduced. SquiggleNet also offers significant advantages in terms of space requirements (Table 1),
requiring only 304 KB to store the model parameters. The run-time space usage is dom-
inated by the storage required for each mini-batch of sequences, rather than the model
parameters. Guppy, however, is a much larger deep-learning model, and the smallest
pre-trained option available through Oxford Nanopore Community [20] is 5.5MB. On
top of that, however, the Guppy+Minimap2 method also requires a customized database
for Minimap2 reference. In this experiment, the human and Zymo reference database
takes 3.2GB. UNCALLED is currently operational only on CPU. Similarly, it also takes
a reference database to build a Burrows-Wheeler index, which is an extra 3.2 GB in
this experiment. Therefore, SquiggleNet requires much less space than the other two
methods. SquiggleNet identifies reads containing human long interspersed repeat elements Classifying species is useful for many applications, but distinguishing among different
loci from the same species would significantly expand the settings in which Squig-
gleNet can be applied. To investigate whether SquiggleNet can be used to enrich loci
of interest within a single species, we analyzed data from a recently published protocol Table 1 Method requirement comparison
SquiggleNet
SquiggleNet
Guppy+Minimap2
UNCALLED
Equipment (t = thread)
GPU
CPU (t = 1)
GPU+CPU (t = 32)
CPU (t = 32)
Space requirement↓
≤GPU Max
≤Mem Max
≤GPU Max + 3.2GB
≤Mem Max + 3.2GB
Model size↓
304 KB
304 KB
5.5/40/116 MB Table 1 Method requirement comparison
SquiggleNet
SquiggleNet
Guppy+Minimap2
UNCALLED
Equipment (t = thread)
GPU
CPU (t = 1)
GPU+CPU (t = 32)
CPU (t = 32)
Space requirement↓
≤GPU Max
≤Mem Max
≤GPU Max + 3.2GB
≤Mem Max + 3.2GB
Model size↓
304 KB
304 KB
5.5/40/116 MB Table 1 Method requirement comparison Bao et al. Genome Biology (2021) 22:298 Page 11 of 16 Bao et al. Genome Biology [21] that enriches specific families of interspersed repeat elements using Cas9-directed
adapter ligation (Fig. 7a). This experimental enrichment is effective, but still fewer
than 50% of reads contain the repeats of interest [21]. As previously described [22],
we used BLASTn [23] on the base-called sequence of each read to label the reads as
target (repeat-containing) or non-target (no repeat). We focused specifically on a sin-
gle class of interspersed repeats, human-specific long interspersed elements (L1Hs),
which was the family most effectively enriched by the Cas9-directed ligation protocol. Using these labels, we trained SquiggleNet on a balanced dataset containing approxi-
mately 170,000 reads from each class. Note that this is about tenfold less training data
than we used in the human vs. bacterial classification experiments above. As with the
species classification experiments, we discarded the first 1500 signals from each read,
then used the next 3000 for training or testing. Despite the smaller training dataset, we
found that SquiggleNet was extremely effective at identifying reads containing L1Hs ele-
ments (Fig. 7b, c). Our model achieved more than 92% accuracy with a true positive
rate above 93%. These results indicate that using SquiggleNet in a read-until setting to
enrich long interspersed repeats would provide a significant benefit compared to Cas9
enrichment only. SquiggleNet improves throughput by enabling computationally targeted sequencing To assess the potential improvement in sequencing throughput that SquiggleNet could
provide, we developed a mathematical model to compare the total number of base pairs
and total sequencing time needed to obtain a certain number of targeted sequences with
and without SquiggleNet. Fig. 7 Identifying reads containing human long interspersed repeat elements. a Diagram of experimental
strategy for enriching human mobile elements, including interspersed repeats. A guide RNA specific to each
repeat class directs Cas9 to cut the DNA and ligate a sequencing adapter. However, adapters are also ligated
to some sequences without repeat elements. Subsequent nanopore sequencing produces both target and
non-target reads. b Pie charts of the proportion of L1Hs repeat elements from Cas9 enrichment only vs. SquiggleNet classification. c Classification metrics demonstrating SquiggleNet’s ability to distinguish reads
with or without L1Hs repeats Fig. 7 Identifying reads containing human long interspersed repeat elements. a Diagram of experimental
strategy for enriching human mobile elements, including interspersed repeats. A guide RNA specific to each
repeat class directs Cas9 to cut the DNA and ligate a sequencing adapter. However, adapters are also ligated
to some sequences without repeat elements. Subsequent nanopore sequencing produces both target and
non-target reads. b Pie charts of the proportion of L1Hs repeat elements from Cas9 enrichment only vs. SquiggleNet classification. c Classification metrics demonstrating SquiggleNet’s ability to distinguish reads
with or without L1Hs repeats Fig. 7 Identifying reads containing human long interspersed repeat elements. a Diagram of experimental
strategy for enriching human mobile elements, including interspersed repeats. A guide RNA specific to each
repeat class directs Cas9 to cut the DNA and ligate a sequencing adapter. However, adapters are also ligated
to some sequences without repeat elements. Subsequent nanopore sequencing produces both target and
non-target reads. b Pie charts of the proportion of L1Hs repeat elements from Cas9 enrichment only vs. SquiggleNet classification. c Classification metrics demonstrating SquiggleNet’s ability to distinguish reads
with or without L1Hs repeats Bao et al. Genome Biology (2021) 22:298 Bao et al. Genome Biology (2021) 22:298 Page 12 of 16 Bao et al. Genome Biology The detailed derivation of the model can be found in Additional file 1. The most influ-
ential hyper-parameters include average target sequence length ¯z, average non-target
sequence length ¯h, and target sequence concentration c. SquiggleNet improves throughput by enabling computationally targeted sequencing Several other tunable hyper-
parameters, including the waiting time to eject one molecule and begin sequencing
another; the total number of active pores in a flow cell; the sequencing speed; and the
total number of targeted sequences, did not significantly influence the predicted increase
in throughput (see Additional file 1). We chose the values for these less influential param-
eters based on the empirical time requirements and accuracy of SquiggleNet and the
sample means from the real sequencing data. In Fig. 8, we picked the average non-target/target sequence length ratio as one axis,
and the target sequence concentration as the other axis to demonstrate the total number
of base pairs (left) and total sequencing time (right) that a regular nanopore sequenc-
ing pipeline would require, compared to those of a pipeline with SquiggleNet, in order
to obtain a fixed number of targeted reads. Based on the properties of our sequencing
datasets, we set average sequencing speed to be 450 base pairs per second and the total
number of active pores in a flow cell to be 500. We also used the following parameters
based on SquiggleNet’s empirical performance: TPR = 0.9, TNR = 0.9, sequencing time =
1s, and classification decision time = 0.8 s (time required for SquiggleNet on CPU). We show the predicted gains in throughput and sequencing time for a range of the most
important hyperparameters (Fig. 8). When the average non-target read length is about
20 times longer than the target read length, and the sample contains over 90% non-target
sequences, it would take a nanopore sequencing pipeline ∼10 times longer than Read-
Until pipeline with SquiggleNet to achieve a fixed number of targeted reads. The regular
nanopore sequencing pipeline would also have to sequence ∼10 times more base pairs
than the Read-Until pipeline. Even if we set these parameters much more pessimistically,
the model still predicts about a 5-fold gain in throughput and time. These numbers are
also in the same ballpark as the 4.5× enrichment reported in the UNCALLED paper [10],
supporting the plausibility of our mathematical model. We therefore conclude that Read-
Until with SquiggleNet holds great promise to improve target read throughput, saving
sequencing time and resources. Fig. 8 Throughput and sequencing time comparison without/with read-until. SquiggleNet improves throughput by enabling computationally targeted sequencing When the average non-target
read length is about 20 times longer than the target read length, and sample contains over 90% non-target
reads, a normal sequencing pipeline would have to sequence ∼10 times more base pairs (left) than
Read-Until pipeline with SquiggleNet to achieve a fixed number of targeted reads. The ratio is about 10 for
the required sequencing time as well (right) Fig. 8 Throughput and sequencing time comparison without/with read-until. When the average non-target
read length is about 20 times longer than the target read length, and sample contains over 90% non-target
reads, a normal sequencing pipeline would have to sequence ∼10 times more base pairs (left) than
Read-Until pipeline with SquiggleNet to achieve a fixed number of targeted reads. The ratio is about 10 for
the required sequencing time as well (right) Bao et al. Genome Biology (2021) 22:298 Bao et al. Genome Biology (2021) 22:298 Page 13 of 16 Bao et al. Genome Biology Discussion The success of our approach suggests that the raw sequencing signals generated by
nanopore sequencing contain rich information for identifying target sequences from
background sequences. Such features could include different DNA modification patterns,
codon frequencies, GC content, or even DNA shape or RNA secondary structure. Fur-
thermore, because these features are primarily local in nature, only a small amount of
sequencing signal is required. In contrast, approaches that rely on sequence information
alone require much more signal (more sequencing time), are susceptible to base-calling
errors, and do not leverage non-sequence information. We also note that different reads go through the pores at different speeds. Future
work could also include an event detector and a scaler into the classifier, which may fur-
ther improve performance. Additionally, the squiggles from different MinION flow cells
show systematic run-to-run differences. Thus, the data preprocessing and normalization
procedures that we employed are crucial for generalizing across datasets. We tested the capability of our model to enrich bacterial DNA in the presence of
more abundant human DNA. Human and bacterial DNA are significantly different, which
makes this classification task feasible. We also demonstrated that SquiggleNet can iden-
tify target sequences in other contexts, such as interspersed repeat elements within
the human genome. This suggests that SquiggleNet could be used for targeted enrich-
ment of other genomic loci, such as commonly mutated cancer genes or regions that
are highly polymorphic across the human population. We also anticipate that Squig-
gleNet will be useful for distinguishing viral DNA or RNA sequences from host molecules
within infected cells. Rapidly identifying targeted sequences could be helpful in numer-
ous clinical settings, including cancer diagnosis, respiratory pathogen identification, and
coronavirus testing. Model architecture SquiggleNet is a 1D-ResNet-based binary classifier (Fig. 1). The first layer of 1D-CNN
is comparable to the first layer of Guppy[20], but with significantly fewer channels (20
instead of 512). After that, there are four layers of 1D-ResNet, and each layer includes two
BottleNeck blocks. The number of channels for each layer increases by a factor of 1.5, and
each BottleNeck block decreases the string size with a stride of 2. We perform average
pooling after the final convolutional layer, followed by a fully connected layer. We also
experimented with other architectures (see Additional file 1). Data collection We generated five datasets using a MinION sequencer (Table 2) for this paper. The first
four datasets used the standard Rapid Sequencing Kit (SQK-RAD004) protocol on a FLO-
106D MinION Flow Cell. The HeLa&Zymo dataset used the ZymoBIOMICS Microbial
Community DNA Standard, and datasets 2–4 used the ZymoBIOMICS HMW DNA Stan-
dard with different barcodes specified in Table 2. Details about the dataset composition
can be found in Additional file 1. The Respiratory Metagenome data collection method
can be found in [17]. Because base calling and sequence alignment of noisy nanopore reads can result in sys-
tematic errors and is not a completely reliable source of ground truth, we used barcodes
to label the sequences in the three Human&Zymo datasets as either bacteria or human Table 2 Datasets description
Name
Ratio
Train
Validation
Test
Note
HeLa&Zymo
1:1
2.4M
40k
54k
Human&Zymo_b12
1:1
1.72M
40k
44k
Barcode 1 and 2
Human&Zymo_b34
1:4
224k
Barcode 3 and 4
Human&Zymo_b56
99:1
100k
Barcode 5 and 6
Respiratory Metagenome
951:1
324,867
Real patient samples Bao et al. Genome Biology (2021) 22:298 Page 14 of 16 Bao et al. Genome Biology before mixing them together. The labels obtained in this way thus represent reliable
ground truth. Each extracted signal read was normalized with fast5 scaling and offset. All reads were
also normalized using Z-scored median absolute deviation. The extreme signal values
with a modified z-score larger than 3.5 were replaced by the average of closest neighbors. Training and evaluation Additional file 2: Review history. Acknowledgements
We thank members of the CELab, including Satish Narayanasamy, Reetu Das, and Prof. Jenna Wiens for their helpful
suggestions and advice during this project. Supplementary Information
The online version contains supplementary m y
The online version contains supplementary material available at https://doi.org/10.1186/s13059-021-02511-y. Additional file 1: Supplement. Details on data collection, model parameters, hyperparameter tuning, details on the
efficiency comparison experiment method, details on the theoretical throughput estimation model are included in
the Supplement. Additional experiment results, including DNA methylation, model interpretation, model
performance trained on a different dataset, model performance on different test dataset composition ratio and on
odd/even chromosomes, are also presented in the Supplement [24–28]. Additional file 2: Review history. Training and evaluation Our best-performing model was trained on the HeLa&Zymo dataset with binary
cross-entropy loss. The dataset was split into training, validation, and testing sets. The Human&Zymo_b12, Human&Zymo_b34, Human&Zymo_b56, and Respiratory
Metagenome datasets were used to assemble testing sets for the best-performing model. As a separate analysis, we also trained on the Human&Zymo datasets and tested on the
HeLa&Zymo datasets. The performance of this model was nearly identical but slightly
worse than the model trained on HeLa&Zymo (see Additional file 1 for details). Our best-performing model was trained on the HeLa&Zymo dataset with binary
cross-entropy loss. The dataset was split into training, validation, and testing sets. The Human&Zymo_b12, Human&Zymo_b34, Human&Zymo_b56, and Respiratory
Metagenome datasets were used to assemble testing sets for the best-performing model. As a separate analysis, we also trained on the Human&Zymo datasets and tested on the
HeLa&Zymo datasets. The performance of this model was nearly identical but slightly
worse than the model trained on HeLa&Zymo (see Additional file 1 for details). The Adam optimizer was used for over 6 epochs on each dataset, with a learning rate
of 1e-3 and batch size of 1000. The model was initialized using Kaiming initialization in
fan-out mode. Batch normalization was conducted within each Bottleneck block. For the human interspersed repeat analysis, we used a balanced training dataset with
equal numbers of reads that contained and did not contain an L1Hs element (about
170,000 reads each). We identified L1Hs elements using BLAST on the base-called reads
with an e-value cutoff of 1 × 10−5, as previously described [21]. We trained for 3 epochs
using the Adam optimizer. Evaluation metrics include overall accuracy, true positive rate
(TPR, Recall), true negative rate (TNR), Precision, and area under receiver operating char-
acteristic curve (AUROC) when running the model on different test datasets. Speed and
memory comparisons were performed on the same Intel(R) Xeon(R) CPU E5-2697 v3 @
2.60 GHz machine with a single TITAN Xp GPU. Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s13059-021-02511-y. Additional file 1: Supplement. Details on data collection, model parameters, hyperparameter tuning, details on the
efficiency comparison experiment method, details on the theoretical throughput estimation model are included in
the Supplement. Additional experiment results, including DNA methylation, model interpretation, model
performance trained on a different dataset, model performance on different test dataset composition ratio and on
odd/even chromosomes, are also presented in the Supplement [24–28]. Funding g
The authors wish to acknowledge support from NIH grants R01HG010883 (JW), R21HG011493 (AB and RM), and
R01HL144599 and R21AI137669 (RD). Availability of data and materials We have uploaded our data to SRA (SRP296988) [29, 30]. The human metagenome dataset has been previously
published in [17]. The Cas9 enrichment dataset is previously published and available on the SRA (BioProject accession
PRJNA699027)[21]. The code is available at a DOI-assigning repository Zenodo (https://doi.org/10.5281/zenodo.4728278)
and under the MIT license in our Github repository [31]: https://github.com/welch-lab/SquiggleNet. Peer review information Anahita Bishop was the primary editor of this article and managed its editorial process and peer review in collaboration
with the rest of the editorial team. Ethics approval and consent to participate
No ethical approval was necessary for this study. Ethics approval and consent to participate
No ethical approval was necessary for this study. No ethical approval was necessary for this study. Review history
The review history is available as Additional file 2. The review history is available as Additional file 2. Authors’ contributions YB and JDW conceived the idea of SquiggleNet. YB designed and implemented the model and analyzed the data. JW
generated datasets 1 to 4. JRE, PR, and RPD provided the human-pathogen dataset and generated Fig. 5. YB and JDW
wrote the paper. JRE and DB offered suggestions and advice. TM, WZ, RM, and AB generated and annotated the Cas9
enrichment dataset. All authors read and approved the final manuscript. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors’ information Twitter: @baobaoyaobaobao (Yuwei Bao); @LabWelch (Joshua D. Welch). Author details
1 1Department of Computer Science and Engineering, University of Michigan, 48109 Ann Arbor, MI, USA. 2Department of
Electrical and Computer Engineering, University of Michigan, 48109 Ann Arbor, MI, USA. 3Division of Pulmonary and
Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, 48109 Ann Arbor, MI,
USA. 4Department of Computational Medicine and Bioinformatics, University of Michigan, 48109 Ann Arbor, MI, USA. 5Department of Human Genetics, University of Michigan Medical, 48109 Ann Arbor, MI, USA. 6Department of
Microbiology and Immunology, University of Michigan Medical School, 48109 Ann Arbor, MI, USA. 7Michigan Center for
Integrative Research in Critical Care, 48109 Ann Arbor, MI, USA. p
,
y
g
,
,
,
p
Microbiology and Immunology, University of Michigan Medical School, 48109 Ann Arbor, MI, USA. 7Michigan Center for
Integrative Research in Critical Care, 48109 Ann Arbor, MI, USA. Received: 5 March 2021 Accepted: 4 October 2021 Received: 5 March 2021 Accepted: 4 October 2021 Received: 5 March 2021 Accepted: 4 October 2021 Declarations Ethics approval and consent to participate
No ethical approval was necessary for this study Ethics approval and consent to participate
No ethical approval was necessary for this study. Acknowledgements
h
k
b
f We thank members of the CELab, including Satish Narayanasamy, Reetu Das, and Prof. Jenna Wiens for their helpful
suggestions and advice during this project. Page 15 of 16 (2021) 22:298 Bao et al. Genome Biology Review history Review history
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sequences by hybridization (dash): using cas9 to remove unwanted high-abundance species in sequencing libraries
and molecular counting applications. Genome Biol. 2016;17(1):41. https://doi.org/10.1186/s13059-016-0904-5. 8. Gu W, Crawford ED, O Donovan BD, Wilson MR, Chow ED, Retallack H, DeRisi JL. Depletion of abundant
sequences by hybridization (dash): using cas9 to remove unwanted high-abundance species in sequencing libraries
and molecular counting applications. Genome Biol. 2016;17(1):41. https://doi.org/10.1186/s13059-016-0904-5. 9. Payne A, Holmes N, Clarke T, Munro R, Debebe BJ, Loose M. Readfish Enables Targeted Nanopore Sequencing of
Gigabase-sized Genomes. https://doi.org/10.1038/s41587-020-00746-x. and molecular counting applications. Genome Biol. 2016;17(1):41. https://doi.org/10.1186/s13059-016-0904-5. 9. Payne A, Holmes N, Clarke T, Munro R, Debebe BJ, Loose M. Readfish Enables Targeted Nanopore Sequencing of
Gigabase-sized Genomes. https://doi.org/10.1038/s41587-020-00746-x. g pp
p
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9. Payne A, Holmes N, Clarke T, Munro R, Debebe BJ, Loose M. Readfish Enables Targeted Nanopore Sequencing of
Gigabase-sized Genomes. https://doi.org/10.1038/s41587-020-00746-x. 10. Kovaka S, Fan Y, Ni B, Timp W, Schatz MC. Targeted Nanopore Sequencing by Real-time Mapping of Raw Electrical
Signal with UNCALLED. https://doi.org/10.1038/s41587-020-0731-9. Page 16 of 16 Bao et al. Genome Biology (2021) 22:298 Bao et al. Genome Biology 11. He K, Zhang X, Ren S, Sun J. Deep Residual Learning for Image Recognition. Publisher’s Note
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Faktor Faktor Yang Berhubungan Dengan Kedisiplinan Penggunaan Alat Pelindung Diri (APD) Pada Pekerja Di PT Perkebunan Lembah Bhakti Aceh Singkil
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T ,2,3 PENDAHULUAN 41 Journal of Health and Medical Science
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Halaman 41-50 42 Journal of Health and Medical Science
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100 Journal of Health and Medical Science
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100 44 Journal of Health and Medical Science
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Halaman 41-50 KESIMPULAN penyakit akibat kerja terhadap
dengan p value 0,001 pengetahuan terhadap
dengan p value 0,001 sikap terhadap
dengan p value 0,009 masa kerja terhadap
dengan p value 0,037 DAFTAR PUSTAKA 49 Journal of Health and Medical Science
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Rapid Changes in Synaptic Strength After Mild Traumatic Brain Injury
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Rapid Changes in Synaptic Strength
After Mild Traumatic Brain Injury 1 Department of Biology, Boston University, Boston, MA, United States, 2 Department of Neurobiology, Edmond & Lily Safra
Center for Brain Sciences, Hebrew University of Jerusalem, Jerusalem, Israel Traumatic brain injury (TBI) affects millions of Americans annually, but effective
treatments remain inadequate due to our poor understanding of how injury impacts
neural function. Data are particularly limited for mild, closed-skull TBI, which forms
the majority of human cases, and for acute injury phases, when trauma effects and
compensatory responses appear highly dynamic. Here we use a mouse model of mild
TBI to characterize injury-induced synaptic dysfunction, and examine its progression
over the hours to days after trauma. Mild injury consistently caused both locomotor
deficits and localized neuroinflammation in piriform and entorhinal cortices, along with
reduced olfactory discrimination ability. Using whole-cell recordings to characterize
synaptic input onto piriform pyramidal neurons, we found moderate effects on excitatory
or inhibitory synaptic function at 48 h after TBI and robust increase in excitatory inputs in
slices prepared 1 h after injury. Excitatory increases predominated over inhibitory effects,
suggesting that loss of excitatory-inhibitory balance is a common feature of both mild
and severe TBI. Our data indicate that mild injury drives rapidly evolving alterations in
neural function in the hours following injury, highlighting the need to better characterize
the interplay between the primary trauma responses and compensatory effects during
this early time period. Edited by:
Qi Yuan, Edited by:
Qi Yuan,
Memorial University of Newfoundland,
Canada Memorial University of Newfoundland,
Canada Reviewed by:
Anne-Marie M. Oswald,
University of Pittsburgh, United States
Maxime Lévesque,
McGill University, Canada Keywords: traumatic brain injury, piriform cortex, synapse, excitatory-inhibitory balance, neuroinflammat *Correspondence:
Ian G. Davison
idavison@bu.edu ORIGINAL RESEARCH
published: 26 April 2019
doi: 10.3389/fncel.2019.00166 INTRODUCTION An estimated 2.8 million Americans suffer a traumatic brain injury (TBI) every year (Taylor
et al., 2017), leading to debilitating symptoms such as seizures, cognitive impairment, emotional
instability, and sensory deficits (Rimel et al., 1981; Levin et al., 1987, 1990; Lew et al., 2007b; McKee
et al., 2009). Mild injuries are most common, accounting for ∼80% of cases (Cassidy et al., 2004). Although mild TBI causes little to no gross damage to neural tissue, its effects often persist for
months or even years, significantly impacting employment and quality of life (Rimel et al., 1981;
Carroll et al., 2004). Repeated injury significantly worsens both pathology as well as cognitive and
motor impairments (Laurer et al., 2001; DeFord et al., 2002; Mouzon et al., 2012). Despite the high
prevalence of mild TBI, treatment options remain limited due to our poor understanding of how
injury affects neural function, especially in the acute phase after trauma. Specialty section:
This article was submitted to
Cellular Neurophysiology,
a section of the journal
Frontiers in Cellular Neuroscience
Received: 07 December 2018
Accepted: 08 April 2019
Published: 26 April 2019
Citation:
Witkowski ED, Gao Y, Gavsyuk AF,
Maor I, DeWalt GJ, Eldred WD,
Mizrahi A and Davison IG (2019)
Rapid Changes in Synaptic Strength
After Mild Traumatic Brain Injury. Front. Cell. Neurosci. 13:166. doi: 10.3389/fncel.2019.00166 Specialty section:
This article was submitted to
Cellular Neurophysiology,
a section of the journal
Frontiers in Cellular Neuroscience Received: 07 December 2018
Accepted: 08 April 2019
Published: 26 April 2019 Keywords: traumatic brain injury, piriform cortex, synapse, excitatory-inhibitory balance, neuroinflammation Behavioral Testing Our data indicate that
mild injury causes rapid disruptions in a cortical region that
has received little attention, emphasizing the need to consider
dynamic effects during immediate post-TBI periods that will be
vital to identifying interventions matched to different phases of
the injury response. Mice All experiments were performed in adult male and female
C57BL/6J mice 2–5 months of age. Animals were group housed in
Boston University’s animal care facility on a 12 h light/dark cycle
with ad libitum access to food and water. All procedures were Citation: Witkowski ED, Gao Y, Gavsyuk AF,
Maor I, DeWalt GJ, Eldred WD,
Mizrahi A and Davison IG (2019)
Rapid Changes in Synaptic Strength
After Mild Traumatic Brain Injury. Front. Cell. Neurosci. 13:166. doi: 10.3389/fncel.2019.00166 Substantial evidence points to deficits in both structural and functional connectivity after mild
TBI. Diffuse axonal injury is common, particularly in long-range projections, suggesting potential
disruptions in network connectivity (Pandit et al., 2013; Caeyenberghs et al., 2014). TBI is also
strongly linked to loss of excitatory-inhibitory (E-I) balance, acute seizures and post-traumatic
epilepsy in both humans and animal models, and reduced thresholds for pharmacologically induced April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 1 Witkowski et al. Synaptic Changes in Mild TBI seizures in rodents (Lee and Lui, 1992; Nilsson et al., 1994;
Annegers et al., 1998; Chadwick, 2000; Golarai et al., 2001; Zanier
et al., 2003; Kharatishvili et al., 2006). While overt seizures are
less common in mild injury, E-I imbalances have also been
described. The hippocampus has shows increased excitability and
spontaneous firing within 24 h of mild TBI in vitro, and signs of
reduced inhibition at 48 h in vivo (Reeves et al., 1997; Griesemer
and Mautes, 2007). However, other studies found opposing
effects, with early increases in inhibition onto CA1 pyramidal
cells and reduced excitatory efficacy 1 week later (Santhakumar
et al., 2000; Witgen et al., 2005). While cortical effects are less
well characterized, existing data also suggest disparate and time-
varying effects on E-I balance. Activity in somatosensory cortex
was suppressed within 24 h after injury in vivo (Alves et al.,
2005; Johnstone et al., 2013), but in vitro data showed increased
excitatory input and action potential firing from 48 h to 1 week
post-TBI, and epileptiform activity 1–4 weeks later (Yang et al.,
2010; Greer et al., 2012). performed in accordance with the Boston University Institutional
Animal Care and Use Committee. Injury Model Mild TBI was induced with a modified Marmarou model where a
weight is dropped onto a metal helmet attached to the animal’s
skull (Marmarou et al., 1994) and the animal is unrestrained
to allow free movement of the head and body to recapitulate
the acceleration and shear forces characteristic of human injury
(Meaney and Smith, 2011; Kane et al., 2012). Prior to injury,
animals were briefly anesthetized, the scalp over the midline
was removed, and a stainless steel cylinder was cemented onto
the midline at bregma (4 mm diameter; 4 mm tall; Zap Gel,
Pacer; C&B Metabond, Parkell). After 3–7 days, mice were briefly
anesthetized with isoflurane, given buprenorphine analgesia
(0.125 mg/kg, subcutaneous) and placed on top of perforated foil
in a custom-made TBI apparatus. A 150–200 g weight secured to
fishing line (Stren high impact, Pure Fishing, Inc.) was dropped
150–240 cm through a guide tube onto the helmet implant,
propelling the animal through the foil onto foam padding below
(5 cm upholstery foam, Mybecca). The fishing line was set to a
length that would prevent the possibility of double-hit injury. Animals were monitored in a cage on a heating pad until
sternal. Sham groups received identical treatment including brief
anesthesia with isoflurane, analgesia and placement in the injury
apparatus, and the only difference between groups was the lack of
weight drop impact on the helmet implant. While some discrepancies may arise from differences in injury
model or experimental system, the complex temporal dynamics
of injury responses could also be a key factor. Ding et al. (2011)
showed that cortical activity underwent a transition from initial
suppression to dramatically elevated excitability at approximately
2 h following injury, indicating that TBI responses can evolve
rapidly during this early time window. While TBI effects begin
to manifest immediately, few studies have addressed the acute
periods spanning the first few hours after injury. Of these, only a
handful have made direct intracellular measurements of cellular
physiology or synaptic function (Griesemer and Mautes, 2007),
so that the neural mechanisms underlying dynamic changes in
cortical responsiveness remain open. Behavioral Testing We confirmed the efficacy of our injury model by investigating
vestibulomotor deficits using a custom rotarod apparatus. Mice
were placed on a rod that remained stationary for 10 s and then
steadily accelerated from 4 to 40 rpm over 5 min. Performance
was quantified as the latency to fall. Naïve mice were tested
daily (2 trials, 5 min inter-trial interval) for 5 days before
injury, at 1 h following TBI on each injury day, and daily for
1 week afterwards. To assess olfactory acuity, we trained mice
to perform a binary discrimination between pairs of odorants
using a recently described automated home-cage training system
(Maor et al., 2019). Briefly, a nose poke into a sampling port
triggered pseudorandom delivery of one of two odors. Following
a 400 ms delay to enforce a sensory sampling period, mice
indicated their choice either by licking in response to the S+ cue
to receive a water reward during the 1.6 s response window, or
by withholding licking for the S−cue, after which they were free
to initiate another trial. S−licks triggered a 2 s timeout period. While mice were allowed to drink freely, all water intake required
correct performance in the discrimination task. Mice typically
learned to discriminate monomolecular compounds within 4–
6 days. Injury was delivered after animals reached a group average
criterion of >80%. Animals recovered on a heating pad for 1 h,
and were then returned to the odor testing apparatus within
1.5–2 h after injury. p
p
Here we examined how mild TBI impacts both excitatory
and inhibitory synaptic interactions in cortical circuits, including
both a commonly used 48 h time point, but also testing at 1 h
post-injury to probe acute injury stages. Using a modified weight
drop model allowing free head movement typical of human
injury, we consistently found localized neuroinflammatory
responses in piriform cortex and neighboring entorhinal areas. To test for associated functional changes, we used whole-cell
recordings in acute brain slices to examine synaptic input onto
piriform pyramidal neurons at 1 and 48 h after injury. Synaptic
function was largely normal at 48 h, but excitatory inputs were
substantially increased at 1 h post-TBI. Data Analysis Data Analysis
Animals were pseudo-randomly assigned to sham or TBI
groups. Spontaneous synaptic currents were analyzed in Igor
Pro (WaveMetrics, Oregon) using Taro Tools1. Raw traces were
smoothed using a 3rd order Savitzky-Golay with a 25-point
moving window. Event detection thresholds were set between 4
and 6 pA based on noise levels in each recording, and events
were inspected visually in each recording to ensure sensitive
detection and avoid false positives. The mean thresholds used for
experimental comparisons between sham and injury groups were
statistically equivalent and varied by <0.3 pA in all cases. Any
slow drift in baseline holding current during the recording was
removed using a cubic polynomial fit to ensure thresholds were
applied consistently to all events. For evoked EPSCs and IPSCs,
we calculated the time to peak, width at half maximum amplitude,
and exponential decay constants using custom Matlab routines. Decay was best fit with a double exponential of the form I = A ∗
(e−T1∗t + e−T2∗t), where A is maximum amplitude of the current
I, t is time, and T1 and T2 are fast and slow decay constants. y
Values from pooled data are reported as mean ± SEM. Statistical significance for group means was calculated using
2-sample t-tests, or using 2-sample Kolmogorov–Smirnov tests
(K–S) comparing distributions of event amplitude and frequency,
as noted in the text. K–S tests were performed on group-
averaged distributions, calculated individually for each neuron
and averaged over all cells in each experimental group. The
density of activated microglia across brain areas was analyzed
with ANOVA. For t-tests, a p-value of <0.05 was considered
statistically significant. For more sensitive K–S tests, p < 0.01 was
considered significant. Electrophysiology p y
gy
Animals were deeply anesthetized with ketamine/xylazine and
transcardially perfused with ice-cold slicing solution containing,
in mM: 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3,
75 sucrose, 10 glucose, 1.3 ascorbic acid, 0.5 CaCl2, and 7
MgCl2. Acute coronal slices (300 µm) of piriform cortex were
cut with a vibratome (VT1200S, Leica, Buffalo Grove, IL,
United States) using the same solution, and then transferred to
ACSF containing, in mM: 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26
NaHCO3, 20 sucrose, 2 CaCl2, and 1.5 MgCl2. Both slicing and
recordings solutions were continuously oxygenated with 95/5%
O2/CO2. Slices were maintained at 29◦C for 30 min for recovery,
and then slowly returned to room temperature (approximately
22◦C) for incubation and recording. For recording, pyramidal
neurons were visualized with a two-photon microscope (Ultima,
Prairie Technologies, Middleton, WI, United States) using
a 40× 0.8NA objective and Dodt contrast imaging. Whole
cell voltage clamp recordings were made with electrodes (3–
7 M tip resistance) filled with internal solution containing,
in mM: 115 CsMeSO4, 10 HEPES buffer, 10 phosphocreatine
disodium, 5 QX314-Cl, 4 MgATP, 0.3 NaGTP, and 0.2 EGTA. Recorded cells were visualized with Alexa 594 to confirm cell
type. Membrane voltage was not corrected for liquid junction
potential. Spontaneous and evoked excitatory and inhibitory
postsynaptic currents (EPSCs and IPSCs) were recorded from
superficial pyramidal cells with a Multiclamp 700B amplifier
(Molecular Devices, Sunnyvale, CA, United States) and digitized
at 10 kHz (National Instruments PCI-6321) using custom
Matlab routines (Mathworks, Natick, MA, United States). Evoked
synaptic responses were elicited by stimulating the lateral
olfactory tract with glass microelectrodes (1–3 M tip resistance) 1https:sites.google.com/site/tarotoolsmember/ Immunohistochemistry In a separate cohort, we examined microglial activation with
immunostaining for Iba1, a common neuroinflammatory marker April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 2 Witkowski et al. Synaptic Changes in Mild TBI filled with ACSF using a stimulation isolator (World Precision
Instruments, Sarasota, FL, United States). filled with ACSF using a stimulation isolator (World Precision
Instruments, Sarasota, FL, United States). filled with ACSF using a stimulation isolator (World Precision
Instruments, Sarasota, FL, United States). for microglia and macrophages, at time points immediately after
two injuries and 24 h after three injuries. Mice were heavily
anesthetized with isoflurane and transcardially perfused with
ice-cold artificial cerebral spinal fluid (ACSF) followed by 4%
paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were
harvested and post-fixed overnight in 4% paraformaldehyde
in 0.1 M PB at 4◦C before undergoing a sucrose series and
cryosectioning at 40 µm. Sections were blocked with 5% normal
donkey serum in 0.1 M PB with 0.3% Triton X (PBTx) for 1 h
at 25◦C then incubated overnight in anti-Iba1 rabbit polyclonal
antibody 019-19741 (1:1000; Wako Chemicals, United States)
diluted in PBTx at 4◦C. The primary antibody was visualized by
incubating with Alexa Fluor 488 donkey anti-rabbit secondary
(1:500; Jackson ImmunoResearch Laboratories, West Grove, PA,
United States) diluted in PBTx for 2 h at 25◦C. Sections were
imaged on a FluoView 300 confocal microscope (Olympus,
Center Valley, PA, United States) using a 20× 0.5NA objective
and analyzed with ImageJ (NIH, Bethesda, MD, United States). We quantified the total number of Iba1-positive microglia
in four different brain areas (somatosensory, entorhinal, and
piriform cortex, as well as hippocampus). We also counted the
number of visual identified cells with anatomical characteristics
associated with activation, such as swollen somata, and calculated
a normalized density based on the total measured area in
each brain area. for microglia and macrophages, at time points immediately after
two injuries and 24 h after three injuries. Mice were heavily
anesthetized with isoflurane and transcardially perfused with
ice-cold artificial cerebral spinal fluid (ACSF) followed by 4%
paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were
harvested and post-fixed overnight in 4% paraformaldehyde
in 0.1 M PB at 4◦C before undergoing a sucrose series and
cryosectioning at 40 µm. Immunohistochemistry Sections were blocked with 5% normal
donkey serum in 0.1 M PB with 0.3% Triton X (PBTx) for 1 h
at 25◦C then incubated overnight in anti-Iba1 rabbit polyclonal
antibody 019-19741 (1:1000; Wako Chemicals, United States)
diluted in PBTx at 4◦C. The primary antibody was visualized by
incubating with Alexa Fluor 488 donkey anti-rabbit secondary
(1:500; Jackson ImmunoResearch Laboratories, West Grove, PA,
United States) diluted in PBTx for 2 h at 25◦C. Sections were
imaged on a FluoView 300 confocal microscope (Olympus,
Center Valley, PA, United States) using a 20× 0.5NA objective
and analyzed with ImageJ (NIH, Bethesda, MD, United States). We quantified the total number of Iba1-positive microglia
in four different brain areas (somatosensory, entorhinal, and
piriform cortex, as well as hippocampus). We also counted the
number of visual identified cells with anatomical characteristics
associated with activation, such as swollen somata, and calculated
a normalized density based on the total measured area in
each brain area. Mild Injury Induces Behavioral Deficits
and Neuroinflammation 71 ± 9% for sham and TBI; p = 0.012; t(15) = 2.866; 2-sample t-test; n = 9 sham, 8 TBI
mice]. ∗and ∗∗∗indicate p < 0.05 and p < 0.01. FIGURE 1 | Transient Vestibulomotor Deficits after TBI. (A) Timeline of locomotor testing. Latency to fall was measured daily as indicated by numbers. (B) Fraction of
mice remaining on the rotarod over time for testing 1 day before injury, as well as TBI days 1 and 3. Performance was similar for sham and TBI groups before injury,
but was reduced on injury days (day 1: p = 0.019; k = 0.667; day 3: p = 0.034, k = 0.639; 2-sample K–S test; n = 9 sham, 8 TBI mice). (C) Mean latency to fall
averaged over 3-day windows before, during, and after injury. Performance was decreased in TBI mice relative to pre-injury levels [85 ± 11 vs. 55 ± 13 s for baseline
and injury days; p = 0.0002, t(23) = 4.394, paired t-test; n = 8 mice] and relative to the sham group during the injury period [92 ± 11 vs. 55 ± 13 s for sham and TBI;
p = 0.002; t(49) = –3.242; 2-sample t-test; n = 9 sham, 8 TBI mice]. (D) Group data normalized to pre-injury baseline [injury phase: 131 ± 10% vs. 70 ± 16% for
sham and TBI; p = 0.005; t(15) = 3.321; recovery phase: 118 ± 14% vs. 71 ± 9% for sham and TBI; p = 0.012; t(15) = 2.866; 2-sample t-test; n = 9 sham, 8 TBI
mice]. ∗and ∗∗∗indicate p < 0.05 and p < 0.01. following week, it was still reduced relative to sham animals at
days 8–10 (Figure 1D: 118 ± 14% vs. 71 ± 9% of pre-injury
levels for sham and TBI; p = 0.012; n = 9 sham, 8 TBI mice). This partial recovery is consistent with other mild TBI studies
(Koliatsos et al., 2011; Yang et al., 2013). Overall, we found that
injury induces a reliable and relatively long-lasting motor deficit
consistent with mild TBI. sham and TBI; p = 0.002; n = 9 sham, 8 TBI mice). Within
the TBI group, injury also significantly reduced latency to fall
relative to initial baseline levels (85 ± 11 vs. 55 ± 13 s for 3
baseline and injury days; p = 0.0002; n = 8). Mild Injury Induces Behavioral Deficits
and Neuroinflammation We induced mild TBI using an unrestrained weight-drop model
where free head movement recapitulates the acceleration and
shear forces characteristic of human injury (Meaney and Smith,
2011; Kane et al., 2012). We began with a multiple-injury
paradigm, delivering three repeated impacts at 24 h intervals
(150 g, 150–190 cm), mimicking the repetitive TBI that is most
strongly associated with human neuropathology (McKee et al.,
2009). We first confirmed that our model induced behavioral
deficits and neuropathology consistent with mild TBI effects in
previous studies (Hamm et al., 1994; Mouzon et al., 2012; Yang
et al., 2013; Donat et al., 2017). We assessed vestibulomotor
performance using a rotarod assay, starting 5 days before the
injury series and continuing for 1 week afterwards (Figure 1A). While pre-injury performance was similar in both groups, TBI
significantly reduced latency to fall relative to sham mice over
the three injury days (Figures 1B,C: 92 ± 12 vs. 55 ± 13 s for April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 3 Synaptic Changes in Mild TBI Witkowski et al. FIGURE 1 | Transient Vestibulomotor Deficits after TBI. (A) Timeline of locomotor testing. Latency to fall was measured daily as indicated by numbers. (B) Fraction of
mice remaining on the rotarod over time for testing 1 day before injury, as well as TBI days 1 and 3. Performance was similar for sham and TBI groups before injury,
but was reduced on injury days (day 1: p = 0.019; k = 0.667; day 3: p = 0.034, k = 0.639; 2-sample K–S test; n = 9 sham, 8 TBI mice). (C) Mean latency to fall
averaged over 3-day windows before, during, and after injury. Performance was decreased in TBI mice relative to pre-injury levels [85 ± 11 vs. 55 ± 13 s for baseline
and injury days; p = 0.0002, t(23) = 4.394, paired t-test; n = 8 mice] and relative to the sham group during the injury period [92 ± 11 vs. 55 ± 13 s for sham and TBI;
p = 0.002; t(49) = –3.242; 2-sample t-test; n = 9 sham, 8 TBI mice]. (D) Group data normalized to pre-injury baseline [injury phase: 131 ± 10% vs. 70 ± 16% for
sham and TBI; p = 0.005; t(15) = 3.321; recovery phase: 118 ± 14% vs. Mild Injury Induces Behavioral Deficits
and Neuroinflammation Performance in the
sham group improved slightly over the same time period, likely
from a practice effect (75 ± 11 vs. 92 ± 12 s, for 3 baseline and
injury days; p = 0.007; n = 9 mice). Deficits were also apparent
after normalizing each animal’s performance to pre-injury levels
(Figure 1D: 131 ± 10 vs. 70 ± 16% for sham and TBI groups
averaged over 3 injury days; p = 0.005; n = 9 sham, 8 TBI mice). The greatest deficits occurred on the 1st and 3rd days of injury
(Figure 1B) (p = 0.019 and p = 0.034 for the 1st and 3rd day
of injury; n = 9 sham, 8 TBI mice). While performance of the
TBI group recovered steadily to near-baseline levels over the In addition to behavioral disruptions, TBI commonly leads
to neuropathology including pro-inflammatory responses in
astrocytes and microglia, which develop a swollen ‘activated’
morphology, migrate to the injury site, and release inflammatory
cytokines (Kreutzberg, 1996; Streit, 2000; Nimmerjahn et al.,
2005; Loane and Byrnes, 2010). Microglial activation is reliably
seen across diverse TBI models (Kaur et al., 1995; Davalos et al., April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 4 Witkowski et al. Synaptic Changes in Mild TBI Mild injury leads to microglial activation in lateral inferior temporal lobes. (A–H) Iba1 immunostaining reveals microglial morphology in diverse brain
h following repeated mild injury. In most regions, microglia appeared largely similar between sham and TBI mice (A–D), but piriform (E,F) and entorhinal
consistently showed clusters with a swollen appearance characteristic of neuroinflammatory response. Microglia with activated morphology were only
ound in sham mice. Scale bars, 50 and 10 µm. (I) Heat maps comparing microglial activation across brain areas in individual animals. (J) Injury did not
(Continued) FIGURE 2 | Mild injury leads to microglial activation in lateral inferior temporal lobes. (A–H) Iba1 immunostaining reveals microglial morphology in diverse brain
regions at 24 h following repeated mild injury. In most regions, microglia appeared largely similar between sham and TBI mice (A–D), but piriform (E,F) and entorhinal
areas (G,H) consistently showed clusters with a swollen appearance characteristic of neuroinflammatory response. Microglia with activated morphology were only
infrequently found in sham mice. Scale bars, 50 and 10 µm. (I) Heat maps comparing microglial activation across brain areas in individual animals. Mild Injury Induces Behavioral Deficits
and Neuroinflammation (J) Injury did not
(Continued) April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 5 Witkowski et al. Synaptic Changes in Mild TBI FIGURE 2 | Continued
alter total microglial density, including both activated and non-activated cells [4.252 ± 0.279 vs. 4.575 ± 0.394 microglia/100 µm2 for sham and TBI; p = 0.529;
t(6) = –0.668; 2 sample t-test; n = 3 sham, 4 TBI mice]. (K) Piriform and entorhinal areas showed the largest increases in microglial activation (piriform: 0 ± 0 vs. 0.194 ± 0.097 activated microglia/100 µm2 for sham and TBI; entorhinal: 0.010 ± 0.008 vs. 0.173 ± 0.080 activated microglia/100 µm2 for sham and TBI). p = 0.652; n = 9 mice, 14 cells for sham, 9 mice, 17 cells for
TBI). Frequency was similarly unchanged (Figure 3E: 5.4 ± 1.2
vs. 7.2 ± 1.4 Hz for sham and TBI; p = 0.898; n = 9 mice, 14
cells for sham; 9 mice, 17 cells for TBI). While the more sensitive
Kolmogorov–Smirnov (K–S) test did reveal a significant shift
in the distributions of both amplitude and interevent intervals
(Figures 3B,D: p = 0.009 and p = 0.0003 for amplitude and
interval; n = 1750 sham, 1377 TBI events), changes in synaptic
function were less pronounced than in other models of moderate-
to-severe TBI (Witgen et al., 2005; Cantu et al., 2015). Overall,
our data showed significant but comparatively limited effects on
excitatory synaptic inputs. 2005; Shitaka et al., 2011; Goldstein et al., 2012; Xu et al.,
2016; Tagge et al., 2018). To probe neuroinflammatory responses,
we used Iba1 immunohistochemistry to label microglia and
macrophages (Imai et al., 1996; Ito et al., 1998) and examined
their morphology across a wide range of brain areas including
neocortex, hippocampus, striatum, and piriform and entorhinal
cortices. While microglia in sham animals appeared normal
(Figures 2A,C,E,G), TBI led to pronounced morphological
changes including swollen somata and processes indicative of
activation (Figures 2F,H). Neuroinflammation was apparent at
24 h following 3 injuries, and in one case immediately following
the second injury (Figure 2: n = 3 sham, 4 TBI mice). In
each brain region, we quantified the number and density of
cells showing anatomical changes characteristic of an activated
state (Figures 2I–K). Mild Injury Induces Behavioral Deficits
and Neuroinflammation Microglial activation was reliably localized
to the lateral inferior temporal lobes, particularly to piriform
and entorhinal cortices (Figures 2F,H) and olfactory tubercle
(not shown), regions that have received little attention in TBI. Activated cells were often concentrated near the cortical surface
(Figures 2F,H). Interestingly, we rarely found activation directly
under the injury site near somatosensory cortex or in the
hippocampus (Figures 2B,D), which has commonly been seen
after more severe injury (Reeves et al., 1995, 1997; Santhakumar
et al., 2000, 2001; Witgen et al., 2005), perhaps due to differences
in severity, impact site, or head movement in our model. Overall,
however, these reliable and localized inflammatory responses are
consistent with mild injury. Traumatic brain injury has also been linked to changes in
inhibitory function (Reeves et al., 1997; Witgen et al., 2005; Smith
et al., 2015), which plays a prominent role in balancing local
excitatory networks in piriform (Luna and Schoppa, 2008; Franks
et al., 2011; Suzuki and Bekkers, 2011; Sheridan et al., 2014; Large
et al., 2016; Bolding and Franks, 2018). Recording from the same
pyramidal neurons, we examined inhibitory input by holding
at excitatory reversal potential (+5 mV; Figure 3F). Similar to
excitatory input, there was little to no change in either mean
amplitude or frequency of sIPSCs after injury (Figures 3H,J:
amplitude: 16.0 ± 1.2 vs. 15.6 ± 0.8 pA for sham and TBI;
p = 0.750; frequency: 5.8 ± 0.7 vs. 5.1 ± 0.4 Hz for sham and TBI;
p = 0.754; n = 9 mice, 14 cells for sham; 9 mice, 17 cells for TBI). Again, the higher-sensitivity K–S test revealed a shift toward
increased sIPSC intervals after injury (Figure 3I: p = 0.026,
n = 2744 sham, 3213 TBI events), but this effect was moderate in
size and did not reach our statistical criterion. Altogether, while
injury altered the distribution of both excitatory and inhibitory
inputs between sham and TBI animals, there was little effect on
mean amplitude or frequency, suggesting that mild trauma either
had a relatively minor impact on synaptic transmission or that
compensatory effects were already recruited at 48 h after injury. Mild TBI Has Moderate Effects on
Synaptic Input Onto Pyramidal Cells
at 48 h Traumatic brain injury-induced deficits may arise at least in
part from disrupted synaptic communication. While prior work
has shown both increased cellular excitability (Griesemer and
Mautes, 2007) and diffuse axonal injury leading to functional
disconnection (Pandit et al., 2013), there are few direct
measurements of how synaptic input is affected by mild TBI. The consistent presence of neuroinflammation in piriform led
us to use this region to probe the functional effects of mild
injury. We used whole-cell recordings in acute brain slices to
test for TBI-induced changes in synaptic strength, examining
both spontaneous and evoked synaptic input onto superficial
pyramidal cells, the major excitatory cell type in this brain
area (Figure 3). Finally, to further probe how injury acts on piriform circuit
function, we measured electrically evoked responses upon
stimulating afferent axons of the lateral olfactory tract (LOT),
which provides sensory input from olfactory bulb. LOT responses
contain multiple components reflecting local interactions within
piriform, including direct excitatory input from olfactory bulb,
additional intracortical excitation from other local pyramidal
neurons, and local inhibition from both feedforward and
feedback inhibitory neurons (Suzuki and Bekkers, 2007, 2011;
Luna and Schoppa, 2008; Franks et al., 2011; Large et al.,
2016). Given the importance of E-I balance in cortical function
in general (Xue et al., 2014), and in piriform in particular
(Bolding and Franks, 2018), we tested how injury affects the
relative contributions of excitation and inhibition in piriform
pyramidal cells. Similar to spontaneous measurements, we
first isolated excitatory inputs by holding at −70 mV. LOT
stimulation produced rapid onset, presumably monosynaptic Despite finding reliable microglial activation in piriform,
we observed only moderate changes in spontaneous synaptic
currents measured 48 h after our repeated 3-injury mild
TBI series. The mean amplitude of excitatory events was
not statistically different between sham and TBI groups
(Figures 3A,C: 10.8 ± 1.0 vs. 10.1 ± 1.2 pA for sham and TBI; April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 6 Synaptic Changes in Mild TBI Witkowski et al. FIGURE 3 | Mild TBI has comparatively minor effects on spontaneous synaptic input 48 h post-injury. (A) Whole cell recordings of excitatory input onto superficial
pyramidal cells in piriform cortex. (B,C) Amplitudes of excitatory events showed moderate changes after TBI. Left, cumulative histograms (p = 0.009; k = 0.056; K–S
test; n = 1750 sham, 1377 TBI events). Mild TBI Has Moderate Effects on
Synaptic Input Onto Pyramidal Cells
at 48 h (F) Spontaneous inhibitory inputs
onto the same superficial pyramidal cell recorded at excitatory reversal potential. (G,H) Cumulative histograms and mean values for sIPSC amplitude, which were
unchanged by TBI. (I,J) Cumulative histogram of interevent interval and mean frequency values for inhibitory events, which were also unaffected. [p = 0.026;
k = 0.038; K–S test; n = 2744 sham, 3213 TBI events; 5.8 ± 0.7 vs. 5.1 ± 0.4 Hz for sham and TBI; p = 0.754; t(29) = 0.317; 2 sample t-test; n = 9 mice, 14 cells
for sham; 11 mice, 17 cells for TBI]. ∗indicates p < 0.01 in K–S tests. which could also affect the interplay between excitation and
inhibition independently of peak amplitude. We found no effect
on response kinetics as assessed by time to peak, width at half
maximum, or fast or slow decay time constants (Figures 4C–E:
peak time: 11.9 ± 1.0 vs. 11.9 ± 1.2 ms for eEPSCs, 19.3 ± 0.9 vs. 20.0 ± 0.9 ms for eIPSC; half width: 23.4 ± 1.3 vs. 22.0 ± 2.0 ms
for eEPSCs, 45.6 ± 2.8 vs. 49.5 ± 4.9 ms for eIPSCs; fast decay
constant: 10.4 ± 0.7 vs. 10.4 ± 0.8 for eEPSCs, 31.6 ± 3.5
vs. 39.2 ± 6.4 ms for eIPSCs). These data further suggest
that mild injury either has relatively minor impact on synaptic
communication, or that effects have largely recovered at 48 h. EPSCs, as well as a secondary component presumably due to local
recurrent circuits. For consistency, we adjusted stimulus intensity
to elicit similar levels of input in each cell (∼200 pA). We
then measured corresponding GABAergic currents at the same
stimulus intensity while holding at excitatory reversal (+5 mV). Inhibitory currents scaled with excitatory responses but were
typically larger, consistent with previous data in both piriform
and neocortex and reflecting the higher conductance of GABAA
receptors (Franks et al., 2011; Isaacson and Scanziani, 2011;
Haider et al., 2012; Xue et al., 2014; Large et al., 2016, 2018). We quantified an E/I ratio for each cell based on the peak
amplitudes of excitatory and inhibitory currents. Consistent
with the minor effects on spontaneous input, E/I balance was
not significantly altered at 48 h after injury (Figures 4A,B:
0.316 ± 0.033 vs. 0.386 ± 0.040 for sham and TBI; p = 0.185; n = 9
mice, 16 cells for sham; 9 mice, 15 cells for TBI). Mild TBI Has Moderate Effects on
Synaptic Input Onto Pyramidal Cells
at 48 h Right, mean values [10.8 ± 1.0 vs. 10.1 ± 1.2 pA for sham and TBI; p = 0.652, t(29) = –0.456; 2-sample t-test; n = 9 mice,
14 cells for sham; 11 mice, 17 cells for TBI]. (D,E) Interevent interval and mean frequency for excitatory events. The sEPSC interval distribution shifted slightly toward
smaller values after TBI (p = 0.0003; k = 0.074; K–S test; n = 1750 sham, 1377 TBI events) but mean values were not significantly changed [5.4 ± 1.2 vs. 7.2 ± 1.4 Hz for sham and TBI; p = 0.898, 2-sample t-test; t(29) = 0.129; n = 9 mice, 14 cells for sham; 11 mice, 17 cells for TBI]. (F) Spontaneous inhibitory inputs
onto the same superficial pyramidal cell recorded at excitatory reversal potential. (G,H) Cumulative histograms and mean values for sIPSC amplitude, which were
unchanged by TBI. (I,J) Cumulative histogram of interevent interval and mean frequency values for inhibitory events, which were also unaffected. [p = 0.026;
k = 0.038; K–S test; n = 2744 sham, 3213 TBI events; 5.8 ± 0.7 vs. 5.1 ± 0.4 Hz for sham and TBI; p = 0.754; t(29) = 0.317; 2 sample t-test; n = 9 mice, 14 cells
for sham; 11 mice, 17 cells for TBI]. ∗indicates p < 0.01 in K–S tests. FIGURE 3 | Mild TBI has comparatively minor effects on spontaneous synaptic input 48 h post-injury. (A) Whole cell recordings of excitatory input onto superficial
pyramidal cells in piriform cortex. (B,C) Amplitudes of excitatory events showed moderate changes after TBI. Left, cumulative histograms (p = 0.009; k = 0.056; K–S
test; n = 1750 sham, 1377 TBI events). Right, mean values [10.8 ± 1.0 vs. 10.1 ± 1.2 pA for sham and TBI; p = 0.652, t(29) = –0.456; 2-sample t-test; n = 9 mice,
14 cells for sham; 11 mice, 17 cells for TBI]. (D,E) Interevent interval and mean frequency for excitatory events. The sEPSC interval distribution shifted slightly toward
smaller values after TBI (p = 0.0003; k = 0.074; K–S test; n = 1750 sham, 1377 TBI events) but mean values were not significantly changed [5.4 ± 1.2 vs. 7.2 ± 1.4 Hz for sham and TBI; p = 0.898, 2-sample t-test; t(29) = 0.129; n = 9 mice, 14 cells for sham; 11 mice, 17 cells for TBI]. Mild TBI Has Moderate Effects on
Synaptic Input Onto Pyramidal Cells
at 48 h We also tested
for potential changes in the time course of synaptic responses, Frontiers in Cellular Neuroscience | www.frontiersin.org Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury 11.9 ± 1.2 ms for sham and TBI; p = 0.630; t(26) = 0.488; t-test; IPSCs: 19.3 ± 0.9 vs. 20.0 ± 0.9 ms for sham and TBI;
p = 0.580; t(27) = –0.56; t-test]. (D,E) Duration (width at half max) and decay kinetics were also unchanged [half width – EPSCs: 23.4 ± 1.3 vs. 22.0 ± 2.0 ms for
sham and TBI; p = 0.294; t(26) = 1.07; t-test; IPSCs: 45.6 ± 2.8 vs. 49.5 ± 4.9 ms; p = 0.491; t(27) = –0.699; t-test; decay time – EPSCs: 10.4 ± 0.7 vs. 10.4 ± 0.8 ms; p = 0.308; t(26) = 1.040; t-test; IPSCs: 31.6 ± 3.5 vs. 39.2 ± 6.4 ms; p = 0.302; t(27) = –1.051; t-test]. For EPSC kinetics, n = 9 mice, 14 cells for
sham; 9 mice, 15 cells for TBI; for IPSC kinetics, n = 9 mice, 13 cells for sham; 9 mice, 14 cells for TBI. n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI). Mean
frequency showed a slight but non-significant upward trend as
well (Figure 5E: 2.8 ± 0.4 and 4.0 ± 0.4 Hz for sham and TBI;
p = 0.086, n = 8 mice, 18 cells for sham; 12 mice, 30 cells for
TBI). These data indicate that mild injury does in fact cause
robust effects on excitatory synaptic function, but that these are
short-lived compared to more severe models. we observed. TBI responses can evolve rapidly in vivo during
the hours after injury, including successive periods of reduced
and heightened excitability (Kharatishvili et al., 2006; Ding
et al., 2011; Hansen et al., 2018). Compensatory effects can
also contribute to recovery as early as 48 h (Griesemer and
Mautes, 2007; Greer et al., 2012), perhaps accounting for the
apparent stability in synaptic input. To address the direct effects
of TBI at early post-injury phases independently of longer-term
compensatory changes, we instead delivered a single weight drop
and measured synaptic activity in slices prepared 1 h after impact. Data for this early injury phase are particularly limited, especially
for mild closed-skull TBI (Reeves et al., 2000; Griesemer and
Mautes, 2007). Specifically, we probed for changes in synaptic
input and E-I balance linked to seizure and post-traumatic
epilepsy in human injury (Annegers et al., 1998). While loss of E-I
balance has been previously described, the direction and timing
of effects has been inconsistent across studies. Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury The moderate synaptic changes seen at 48 h post-injury were
unexpected given the behavioral and neuroinflammatory effects April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 7 Synaptic Changes in Mild TBI Witkowski et al. FIGURE 4 | Excitatory-inhibitory balance remains stable at 48 h post-injury. (A) Excitatory and inhibitory inputs in the same piriform pyramidal cell upon electrical
stimulation of the LOT. (B) The ratio of excitatory to inhibitory amplitudes was largely unchanged at 48 h after TBI [0.316 ± 0.033 vs. 0.386 ± 0.040 for sham and
TBI; p = 0.185; t(29) = –1.357; t-test; n = 9 mice, 16 cells for sham; 9 mice, 15 cells for TBI]. (C) Response kinetics (time to peak) were unchanged for both EPSCs
and IPSCs [EPSCs: 11.9 ± 1.0 vs. 11.9 ± 1.2 ms for sham and TBI; p = 0.630; t(26) = 0.488; t-test; IPSCs: 19.3 ± 0.9 vs. 20.0 ± 0.9 ms for sham and TBI;
p = 0.580; t(27) = –0.56; t-test]. (D,E) Duration (width at half max) and decay kinetics were also unchanged [half width – EPSCs: 23.4 ± 1.3 vs. 22.0 ± 2.0 ms for
sham and TBI; p = 0.294; t(26) = 1.07; t-test; IPSCs: 45.6 ± 2.8 vs. 49.5 ± 4.9 ms; p = 0.491; t(27) = –0.699; t-test; decay time – EPSCs: 10.4 ± 0.7 vs. 10.4 ± 0.8 ms; p = 0.308; t(26) = 1.040; t-test; IPSCs: 31.6 ± 3.5 vs. 39.2 ± 6.4 ms; p = 0.302; t(27) = –1.051; t-test]. For EPSC kinetics, n = 9 mice, 14 cells for
sham; 9 mice, 15 cells for TBI; for IPSC kinetics, n = 9 mice, 13 cells for sham; 9 mice, 14 cells for TBI. FIGURE 4 | Excitatory-inhibitory balance remains stable at 48 h post-injury. (A) Excitatory and inhibitory inputs in the same piriform pyramidal cell upon electrical
stimulation of the LOT. (B) The ratio of excitatory to inhibitory amplitudes was largely unchanged at 48 h after TBI [0.316 ± 0.033 vs. 0.386 ± 0.040 for sham and
TBI; p = 0.185; t(29) = –1.357; t-test; n = 9 mice, 16 cells for sham; 9 mice, 15 cells for TBI]. (C) Response kinetics (time to peak) were unchanged for both EPSCs
and IPSCs [EPSCs: 11.9 ± 1.0 vs. Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury Enhanced excitatory input was accompanied by a comparable
shift toward shorter sIPSC intervals at 1 h post-injury
(Figures 5F,I: p = 1e−08; n = 2664 sham, 6810 TBI events),
although
mean
frequency
was
not
significantly
affected
(Figure 5J: 2.7 ± 0.3 and 3.8 ± 0.4 Hz for sham and TBI;
p = 0.07, n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI). While mean inhibitory amplitude was also unchanged, there was
a slight but statistically significant shift in the sIPSC amplitude
distribution (Figures 5G,H: p = 0.0002, n = 2664 sham, 6810
TBI events; 12.8 ± 0.6 vs. 13.3 ± 0.5 pA for sham and TBI;
p = 0.512; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for
TBI). Overall, injury drove clear increases in the rate of input
onto pyramidal neurons that were comparable for excitatory
and inhibitory synapses. The amplitude of excitatory inputs also
showed a substantial increase that far outweighed the less robust
changes in inhibitory amplitude. Despite the use of a single rather than a repetitive injury
paradigm, TBI-induced changes in synaptic function were
considerably more pronounced at 1 h post-injury than at 48 h
after three injuries. TBI significantly shifted the distributions
for both sEPSC amplitude and interval toward larger and more
frequent events (Figures 5A,B,D: p = 1e−08 for amplitude;
p = 1e−08 for frequency; n = 2016 sham, 4050 TBI events). Mean
sEPSC amplitudes were also significantly increased (Figure 5C:
8.8 ± 0.4 vs. 11.0 ± 0.5 pA for sham and TBI; p = 0.0025; Elevated excitatory input, if not matched by increases in
inhibition, could lead to a potential imbalance in these circuit
elements during early injury phases. To test this idea, we again April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 8 Synaptic Changes in Mild TBI Witkowski et al. FIGURE 5 | Rapid increases in excitatory input 1 h after mild TBI. (A) Example traces showing spontaneous EPSCs at 1 h after a single injury. (B,C) Cumulative
histograms and mean values for sEPSC amplitudes. Excitatory events were significantly larger in the TBI group [p = 1e-08, k = 0.122; K–S test; n = 2016 sham,
4050 TBI events; 8.8 ± 0.4 vs. Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury 11.0 ± 0.5 pA for sham and TBI; p = 0.0025; t(46) = 3.201; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. (D,E) Cumulative histograms for sEPSC interval and bar plot of mean frequency. TBI also decreased the interval between excitatory inputs (p = 1e-08, k = 0.101;
K–S test; n = 2016 sham, 4050 TBI events), although mean frequency showed only a trend toward increase [2.8 ± 0.4 vs. 4.0 ± 0.4 Hz for sham and TBI;
p = 0.086; t(46) = 1.755; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. (F) Example traces showing spontaneous inhibitory inputs. (G,H)
TBI caused a slight shift toward larger amplitudes (p = 0.0002, k = 0.049; K–S test; n = 2664 sham, 6810 TBI events), although mean sIPSC size was unaffected
[12.8 ± 0.6 vs. 13.3 ± 0.5 pA for sham and TBI; p = 0.512; t(46) = 0.661; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. (I,J) TBI altered
the distribution toward more frequent inhibitory inputs (p = 1e-08; k = 0.102; K–S test; n = 2664 sham, 6810 TBI events), although mean frequency showed only a
trend [2.7 ± 0.3 vs. 3.8 ± 0.4 Hz for sham and TBI; p = 0.070; t(46) = 1.860; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. ∗and ∗∗∗
indicate p < 0.05 and p < 0.01 for t-tests, and p < 0.01 and p < 0.0001 for K–S tests. FIGURE 5 | Rapid increases in excitatory input 1 h after mild TBI. (A) Example traces showing spontaneous EPSCs at 1 h after a single injury. (B,C) Cumulative
histograms and mean values for sEPSC amplitudes. Excitatory events were significantly larger in the TBI group [p = 1e-08, k = 0.122; K–S test; n = 2016 sham,
4050 TBI events; 8.8 ± 0.4 vs. 11.0 ± 0.5 pA for sham and TBI; p = 0.0025; t(46) = 3.201; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. (D,E) Cumulative histograms for sEPSC interval and bar plot of mean frequency. Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury TBI also decreased the interval between excitatory inputs (p = 1e-08, k = 0.101;
K–S test; n = 2016 sham, 4050 TBI events), although mean frequency showed only a trend toward increase [2.8 ± 0.4 vs. 4.0 ± 0.4 Hz for sham and TBI;
p = 0.086; t(46) = 1.755; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. (F) Example traces showing spontaneous inhibitory inputs. (G,H)
TBI caused a slight shift toward larger amplitudes (p = 0.0002, k = 0.049; K–S test; n = 2664 sham, 6810 TBI events), although mean sIPSC size was unaffected
[12.8 ± 0.6 vs. 13.3 ± 0.5 pA for sham and TBI; p = 0.512; t(46) = 0.661; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. (I,J) TBI altered
the distribution toward more frequent inhibitory inputs (p = 1e-08; k = 0.102; K–S test; n = 2664 sham, 6810 TBI events), although mean frequency showed only a
trend [2.7 ± 0.3 vs. 3.8 ± 0.4 Hz for sham and TBI; p = 0.070; t(46) = 1.860; 2 sample t-test; n = 8 mice, 18 cells for sham; 12 mice, 30 cells for TBI]. ∗and ∗∗∗
indicate p < 0.05 and p < 0.01 for t-tests, and p < 0.01 and p < 0.0001 for K–S tests. measured the excitatory and inhibitory components of evoked
LOT responses at 1 h after TBI. E-I ratios showed considerable
variability across cells, and while there was a trend toward
greater excitation, this shift was non-significant (Figures 6A–C:
0.700 ± 0.065 vs. 0.858 ± 0.060 for sham and TBI; p = 0.082;
n = 9 mice, 28 cells for sham; 12 mice, 40 cells for TBI). There
was also no significant change in response kinetics for either
EPSCs or IPSCs, including time to peak, width at half maximum,
and decay constants (Figures 6D–F: peak time: 11.2 ± 0.8 vs. 12.9 ± 0.6 ms for eEPSCs, 17.5 ± 0.8 vs. 19.3 ± 1.1 ms for
eIPSCs; half width: 23.8 ± 2.1 vs. 27.3 ± 2.0 ms for eEPSCs,
43.9 ± 3.5 vs. 50.5 ± 2.8 ms for eIPSCs; fast decay constant:
12.9 ± 1.2 vs. 16.7 ± 1.7 for eEPSCs, 36.2 ± 3.8 vs. 43.4 ± 3.1 ms
for eIPSC), although all of these parameters consistently showed
slight increases. Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury Finally, we tested for potential changes in the
short-term dynamics of synaptic strength that could alter E-I
balance and circuit function, by stimulating the LOT with a measured the excitatory and inhibitory components of evoked
LOT responses at 1 h after TBI. E-I ratios showed considerable
variability across cells, and while there was a trend toward
greater excitation, this shift was non-significant (Figures 6A–C:
0.700 ± 0.065 vs. 0.858 ± 0.060 for sham and TBI; p = 0.082;
n = 9 mice, 28 cells for sham; 12 mice, 40 cells for TBI). There
was also no significant change in response kinetics for either
EPSCs or IPSCs, including time to peak, width at half maximum,
and decay constants (Figures 6D–F: peak time: 11.2 ± 0.8 vs. 12.9 ± 0.6 ms for eEPSCs, 17.5 ± 0.8 vs. 19.3 ± 1.1 ms for
eIPSCs; half width: 23.8 ± 2.1 vs. 27.3 ± 2.0 ms for eEPSCs,
43.9 ± 3.5 vs. 50.5 ± 2.8 ms for eIPSCs; fast decay constant:
12.9 ± 1.2 vs. 16.7 ± 1.7 for eEPSCs, 36.2 ± 3.8 vs. 43.4 ± 3.1 ms
for eIPSC), although all of these parameters consistently showed
slight increases. Finally, we tested for potential changes in the
short-term dynamics of synaptic strength that could alter E-I
balance and circuit function, by stimulating the LOT with a train of 5 pulses at 10 Hz. Again, the relative amplitude of both
excitatory and inhibitory currents was slightly increased relative
to sham for all subsequent pulses in the train, but this effect
was not significant (Figures 6G–J). In general, injury led to
minor effects on LOT-evoked responses that were in a direction
consistent with spontaneous data, but were less robust and did
not reach statistical significance. Despite the lack of significant changes in amplitude and
kinetics of evoked monosynaptic responses, cells from the
TBI group frequently showed unusual extended barrages of
synaptic input that could persist for several seconds after the
stimulus (Figures 7A,B). While a small fraction of neurons
in the sham group displayed a smaller degree of prolonged
activity, its prevalence and intensity were strongly increased
after TBI (Figure 7C: 16.0% vs. 56.4% of cells from sham
and TBI groups, respectively). Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury While the source of this long-
lasting reverberating activity is unclear, it suggests a disruption April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 9 Synaptic Changes in Mild TBI Witkowski et al. | Injury has limited effects on evoked E-I balance 1 h post-injury. (A,B) Excitatory and inhibitory inputs in piriform pyramidal cells elicited by LOT
(C) Excitatory-inhibitory ratio was slightly but non-significantly increased after TBI [0.700 ± 0.065 vs. 0.858 ± 0.060 for sham and TBI; p = 0.082;
6; 2 sample t-test; n = 9 mice, 28 cells for sham; 12 mice, 40 cells for TBI]. (D) Time to peak is not significantly changed by injury [EPSCs: 11.2 ± 0.8 vs. ms for sham and TBI; p = 0.074; t(59) = –1822; 2 sample t-test; IPSCs: 17.5 ± 0.8 vs. 19.3 ± 1.1 ms; p = 0.280; t(61) = –1.090; t-test]. (E,F) Width at
um and decay time constant were not significantly affected [half width – EPSCs: 23.8 ± 2.1 vs. 27.3 ± 2.0 ms; p = 0.266; t(59) = –1.122; IPSCs:
vs. 50.5 ± 2.8 ms; p = 0.151; t(61) = –1.454; decay time constant: EPSCs: 12.9 ± 1.2 vs. 16.7 ± 1.7 ms; p = 0.148; t(59) = –1.466; IPSCs: 36.2 ± 3.8
3.1 ms; p = 0.152; t(61) = –1.450]. 2-sample t-test used for all kinetics comparisons: EPSCs, n = 9 mice, 22 cells and 12 mice, 39 cells for sham and TBI;
9 mice, 23 cells and 9 mice, 40 cells for sham and TBI. (G,H) Example responses to 10-Hz trains of LOT stimulation. (I,J) Short-term synaptic plasticity
ry (left) and inhibitory (right) responses, which was slightly but not significantly enhanced (EPSCs: p > 0.14 for all pulses; IPSCs: p = >0.19 for all pulses). d effects on evoked E-I balance 1 h post-injury. (A,B) Excitatory and inhibitory inputs in piriform pyramidal cells elicited by LOT FIGURE 6 | Injury has limited effects on evoked E-I balance 1 h post-injury. (A,B) Excitatory and inhibitory inputs in piriform pyramidal cells elicited by LOT
stimulation. (C) Excitatory-inhibitory ratio was slightly but non-significantly increased after TBI [0.700 ± 0.065 vs. 0.858 ± 0.060 for sham and TBI; p = 0.082;
t(66) = 1.76; 2 sample t-test; n = 9 mice, 28 cells for sham; 12 mice, 40 cells for TBI]. Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury (D) Time to peak is not significantly changed by injury [EPSCs: 11.2 ± 0.8 vs. 12.9 ± 0.6 ms for sham and TBI; p = 0.074; t(59) = –1822; 2 sample t-test; IPSCs: 17.5 ± 0.8 vs. 19.3 ± 1.1 ms; p = 0.280; t(61) = –1.090; t-test]. (E,F) Width at
half-maximum and decay time constant were not significantly affected [half width – EPSCs: 23.8 ± 2.1 vs. 27.3 ± 2.0 ms; p = 0.266; t(59) = –1.122; IPSCs:
43.9 ± 3.5 vs. 50.5 ± 2.8 ms; p = 0.151; t(61) = –1.454; decay time constant: EPSCs: 12.9 ± 1.2 vs. 16.7 ± 1.7 ms; p = 0.148; t(59) = –1.466; IPSCs: 36.2 ± 3.8
vs. 43.4 ± 3.1 ms; p = 0.152; t(61) = –1.450]. 2-sample t-test used for all kinetics comparisons: EPSCs, n = 9 mice, 22 cells and 12 mice, 39 cells for sham and TBI;
IPSCs, n = 9 mice, 23 cells and 9 mice, 40 cells for sham and TBI. (G,H) Example responses to 10-Hz trains of LOT stimulation. (I,J) Short-term synaptic plasticity
for excitatory (left) and inhibitory (right) responses, which was slightly but not significantly enhanced (EPSCs: p > 0.14 for all pulses; IPSCs: p = >0.19 for all pulses). in the normal circuit interactions that typically limit activity
in piriform to brief time windows (Luna and Schoppa, 2008;
Bolding and Franks, 2018). due to differences in how these data emphasize afferent versus
intracortical inputs. Overall, however, our major finding at
1 h post-injury was a rapid enhancement of excitatory input
that appeared to outweigh increases in inhibition, which was
paralleled by unusual long-lasting bouts of synaptic input In general, relative levels of excitation and inhibition were
less affected in evoked than in spontaneous inputs, perhaps April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 10 Synaptic Changes in Mild TBI Witkowski et al. FIGURE 7 | Prolonged, reverberating synaptic responses after mild TBI. (A) Typical brief responses to LOT stimulation in sham animals, which showed only rare
cases of late synaptic activity (right). Dots indicate individual synaptic currents. (B) Examples of unusually long-lasting barrages of synaptic input after injury. (C) The
prevalence of prolonged activity is greatly increased by TBI (16.0% vs. 56.4% for sham and TBI). FIGURE 7 | Prolonged, reverberating synaptic responses after mild TBI. Mild TBI Drives Rapid Loss of E-I
Balance at 1 h Post-Injury (A) Typical brief responses to LOT stimulation in sham animals, which showed only rare
cases of late synaptic activity (right). Dots indicate individual synaptic currents. (B) Examples of unusually long-lasting barrages of synaptic input after injury. (C) The
prevalence of prolonged activity is greatly increased by TBI (16.0% vs. 56.4% for sham and TBI). immediately after sham treatment, likely due to stress from
removal from the homecage and brief anesthesia, accuracy
rapidly recovered to previous levels within approximately 4 h. In contrast, most TBI animals refrained from drinking for up
to 12–24 h after mild injury, apart from occasional sporadic
trials. Error rates then rose considerably in subsequent trials. Interestingly, initial errors were biased toward ‘miss’ responses,
suggesting that mice may not have recognized the target odor,
or alternatively could have acquired hypersensitivity (Figure 8C). Performance remained degraded for a period of ∼48 h, far
outlasting the effects of sham treatment, before slowly recovering
to pre-injury levels (Figure 8D). To quantify the loss of
discrimination, we calculated the average group performance
for the period from 24 to 48 h after injury, which dropped to
62.8% during this period compared to an average of 83.2% before
TBI (Figure 8E). These results indicate that disrupted synaptic
communication in piriform is associated with a sensory deficit in
odor discrimination that persists for a period of ∼48 h, although
effects on other task-related brain areas may contribute as well. persisting for seconds after stimulation. Together, these data
suggest that loss of E-I balance is a common feature of both mild
and severe injury. Mild Injury Leads to Sensory Deficits in
Odor Discrimination To address the functional correlates of synaptic disruption and
inflammatory responses in piriform, we used a behavioral assay
to test how sensory acuity was affected by mild injury. We trained
mice to discriminate between pairs of odorants in a Go/No-
go paradigm, where licking in response to the S+ odor was
rewarded by delivery of a water drop, and the second S−odor
required animals to withhold licking to avoid receiving a timeout
(Figure 8A). We used a recently described automated training
system where mice are continuously group housed in their home
cage and task performance is monitored throughout using RFID
tags (Maor et al., 2019). Animals were allowed to drink freely at all
times, but all water intake required correct discrimination. This
approach maintained normal physiological status at the time of
injury, avoiding potential confounds due to combining TBI with
water restriction. Frontiers in Cellular Neuroscience | www.frontiersin.org DISCUSSION Mice typically learned the initial task structure and began
discriminating between odors within ∼4–6 days. Sham and
injury treatments were performed after animals reached a mean
performance level of 80%, where they reliably licked in response
to the rewarded S+ cue and withheld licking to the S−odor
(Figure 8B). While there was a brief drop in performance While several studies have characterized changes in neural
activity early after TBI, there is a lack of consensus on the
direction of E-I shifts, and little work has directly addressed
how synaptic interactions are affected acutely after trauma, While several studies have characterized changes in neural
activity early after TBI, there is a lack of consensus on the
direction of E-I shifts, and little work has directly addressed
how synaptic interactions are affected acutely after trauma, April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 11 11 Synaptic Changes in Mild TBI Witkowski et al. E 8 | Olfactory deficits after mild injury. (A) Go/No-go odor discrimination task. Animals sampled from an odor port containing a lick tube, and after a brief
ported their choice of odor A (S+, rewarded) vs. odor B (S–, unrewarded). (B) Block of 24 example trials for one animal preceding injury, grouped by S+ and
rs to illustrate accurate performance. Rasters indicate licks, and green and red indicate correct and incorrect choices. The first 2 licks for each trial are shown. mple trials for the same animal following injury. (D) Group data shows a brief drop in performance due to sham treatment that recovers after ∼4 h (mean
cy prior to injury, 0.793 ± 0.024 and 0.83 ± 0.014 for sham and TBI; shading shows mean ± 2 SD). Mice performed few trials during the 12–24 h after injury,
hich they showed a drop in performance lasting until ∼48 h. (E) Pre- and post-injury accuracy for sham and TBI groups. Sham mice had indistinguishable
ance during the 12 h pre-injury and the 12–24 h post-recovery period [0.790 ± 0.010 vs. 0.822 ± 0.018, respectively; p = 0.145, t(10) = –1.58, paired
t-test; n = 6 time points pre and post-injury]. TBI mice showed a significantly loss of performance over the 24–48 h post-injury period, the point where they
d consistent sampling, compared to the 12 h pre-injury [pre, 0.832 ± 0.007 vs. DISCUSSION post, 0.628 ± 0.040; p = 0.0027; t(16) = 3.547; 2 sample t-test; n = 6 pre
post-injury time points] ∗∗∗indicates p < 0.01. FIGURE 8 | Olfactory deficits after mild injury. (A) Go/No-go odor discrimination task. Animals sampled from an odor port containing a lick tube, and after a brief
delay reported their choice of odor A (S+, rewarded) vs. odor B (S–, unrewarded). (B) Block of 24 example trials for one animal preceding injury, grouped by S+ and
S– odors to illustrate accurate performance. Rasters indicate licks, and green and red indicate correct and incorrect choices. The first 2 licks for each trial are shown. (C) Example trials for the same animal following injury. (D) Group data shows a brief drop in performance due to sham treatment that recovers after ∼4 h (mean
accuracy prior to injury, 0.793 ± 0.024 and 0.83 ± 0.014 for sham and TBI; shading shows mean ± 2 SD). Mice performed few trials during the 12–24 h after injury,
after which they showed a drop in performance lasting until ∼48 h. (E) Pre- and post-injury accuracy for sham and TBI groups. Sham mice had indistinguishable
performance during the 12 h pre-injury and the 12–24 h post-recovery period [0.790 ± 0.010 vs. 0.822 ± 0.018, respectively; p = 0.145, t(10) = –1.58, paired
sample t-test; n = 6 time points pre and post-injury]. TBI mice showed a significantly loss of performance over the 24–48 h post-injury period, the point where they
resumed consistent sampling, compared to the 12 h pre-injury [pre, 0.832 ± 0.007 vs. post, 0.628 ± 0.040; p = 0.0027; t(16) = 3.547; 2 sample t-test; n = 6 pre
and 12 post-injury time points] ∗∗∗indicates p < 0.01. particularly in mild forms of TBI. Here we characterized
synaptic changes in piriform cortex, which showed consistent
neuroinflammatory responses in our mild injury model and
has been linked to generation and propagation of seizures in
other paradigms (Piredda and Gale, 1985; Halonen et al., 1994;
Vismer et al., 2015). Excitatory synaptic function showed only
a relatively moderate increase at an intermediate 48-h time
point, even using a repeated-injury paradigm. This contrasts
with more severe models such as fluid percussion injury and controlled cortical impact where changes last for days to weeks
(Santhakumar et al., 2001; Witgen et al., 2005; Cantu et al.,
2015). Injury and Excitatory-Inhibitory Balance Thus,
while trauma responses appear to vary with brain region and
injury model, they can also occur within minutes and evolve
over time. Our data are consistent with dynamic changes in
neural function, showing rapid changes in synaptic efficacy
appearing within an hour of injury but largely resolving over the
ensuing 48 h. The presence of considerable recovery even with
repeated impacts suggests that early time windows should be a
major focus in characterizing the effects of mild injury. Given
the substantial recovery of synaptic function, we did not make
additional measurements at later time points. The mechanisms that drive initial synaptic changes and
subsequent recovery are unclear. In addition to direct mechanical
damage, TBI triggers numerous secondary cascades, including
mitochondrial dysfunction and elevation of glutamate and
intracellular calcium (Povlishock and Katz, 2005; Blennow
et al., 2012; Giza and Hovda, 2014). Loss of homeostasis
and depolarization can occur within minutes (Blennow et al.,
2012; Giza and Hovda, 2014), and may contribute to the
changes we observe. Axonal injury may also disrupt synaptic
function but is typically thought to occur over hours to
days except in cases of severe TBI (Povlishock and Katz,
2005). Recovery may occur through a variety of compensatory
mechanisms that adjust excitability and synaptic strength to
maintain balanced network activity. A wide range of cellular and
synaptic adaptations occur within 24–48 h after perturbation
of activity (Turrigiano et al., 1998; Ehlers, 2003; Turrigiano
and Nelson, 2004; Keck et al., 2013), and can appear as
quickly 1–2 h (Ibata et al., 2008). Structural adaptations
such as axonal sprouting and changes in dendritic spines
also occur rapidly in response to disruptions such as stroke,
injury, or loss of sensory input (Grutzendler et al., 2002;
Majewska et al., 2006; Brown et al., 2008; Keck et al., 2011). While long-lasting synaptic changes are common in more
severe injury (Santhakumar et al., 2000; Witgen et al., 2005;
Kharatishvili et al., 2006; Yang et al., 2010; Alwis et al., 2012;
Cantu et al., 2015; Smith et al., 2015), our data suggest Multiple mechanisms may underlie early enhancement of
excitatory transmission. Increased sEPSC amplitudes could result
from strengthening of individual postsynaptic sites via increased
receptor content, as with classical activity-dependent plasticity
mechanisms (Turrigiano et al., 1998; Ehlers, 2003; Turrigiano
and Nelson, 2004; Ibata et al., 2008; Keck et al., 2013). Injury and Excitatory-Inhibitory Balance While TBI affected both excitatory and inhibitory inputs, changes
in excitation appeared to be more robust, with clear effects
on both amplitude and frequency of spontaneous inputs. Loss
of E-I balance underlies seizures and epilepsy, which are
common outcomes of severe injuries in both humans and animal
models (Annegers et al., 1998; Pitkanen et al., 2009). The role
of the hippocampus in temporal lobe seizures and memory
loss has made it a strong focus of TBI work (Chang and
Lowenstein, 2003). Hippocampal function depends on its highly
recurrent circuit organization, which is shared by piriform cortex,
where pyramidal cells receive thousands of local intracortical
connections spanning distances of ≥2mm (Johnson et al., 2000;
Franks et al., 2011). Piriform’s highly interconnected excitatory
network requires strong inhibition to prevent runaway activity
(Johnson et al., 2000; Franks et al., 2011; Bolding and Franks,
2018), and is also strongly linked to seizures and epilepsy. Anterior piriform contains “area tempestas,” a region highly
sensitive to chemically induced seizures (Piredda and Gale,
1985; Gale, 1988), which are propagated to other areas by
posterior piriform and neighboring regions (Halonen et al.,
1994; Tortorella et al., 1997). Seizure-induced hyperactivity and
oxidative stress cause localized neuronal loss and atrophy in
piriform cortex in both humans and animal models of temporal
lobe epilepsy (Candelario-Jalil et al., 2001; Chen and Buckmaster,
2005; Pereira et al., 2005). Despite its involvement in seizure
activity in other contexts, piriform has not to our knowledge been
examined after TBI. The effects of TBI are often characterized at 24 h or
later after injury, reflecting the need to understand chronic
pathophysiology. However, a few observations suggest that
trauma drives complex changes on more rapid timescales. EEG
recordings revealed hyperactivation and brief seizure activity in
rat parietal cortex 1–2 min after cortical compression injury,
followed by a post-ictal depression lasting ≥2 h (Nilsson et al.,
1994). In contrast, in vivo Ca2+ imaging showed a decrease
in hippocampal activity within seconds of blast injury in mice
that recovered after ∼60 min (Hansen et al., 2018). Similarly,
multi-unit firing in rat neocortex was initially suppressed at
5–15 min after cortical compression, followed by subsequent
hyper-responsiveness appearing approximately 2 h after injury
(Kharatishvili et al., 2006; Ding et al., 2011). A parallel sequence
of suppression followed by hyperexcitability was seen in cortical
EEG data after fluid percussion, including epileptic activity
emerging hours to weeks later (Kharatishvili et al., 2006). DISCUSSION At 1 h after TBI, however, excitatory synaptic input
was strongly enhanced and predominated over less pronounced
changes in inhibitory input, despite the use of a single-injury
rather than a repetitive TBI protocol. Synaptic changes were
paralleled by reduced performance in an olfactory discrimination
task, where recovery also followed a time course of ∼48 h. Our
data suggest that the synaptic consequences of mild TBI evolve April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 12 Synaptic Changes in Mild TBI Witkowski et al. that adaptive mechanisms may already be engaged by 48 h
after mild injury. rapidly over the hours following injury, highlighting a need to
better understand the initial effects of trauma. Injury and Excitatory-Inhibitory Balance Injury
can also increase membrane excitability, however, and changes
in levels of resting activity in the piriform network may also play
a role. We did not block action potentials in order to measure
spontaneous and evoked responses in the same cell, so we cannot
clearly distinguish between synaptic input from spontaneous
vesicle fusion and release driven by firing in other piriform
neurons. Since local connections are typically composed of
multiple synaptic contacts (Franks and Isaacson, 2006), elevated
spontaneous activity would also generate additional larger
events. Similarly, changes in inhibitory frequency may reflect
changes in either presynaptic release probability, spontaneous
firing, or both. Given the bias toward enhancement of spontaneous excitatory
input, the lack of significant changes in evoked E-I balance was
unexpected. We note that we did find a strong trend toward
increased excitation at 1 h after TBI (p = 0.082), as well as
high variability consistent with heterogeneous injury effects in
both animal models and humans (Carroll et al., 2004; Lingsma
et al., 2010; Xiong et al., 2013). Another possibility is that
TBI acts preferentially on intracortical associational synapses April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 13 Synaptic Changes in Mild TBI Witkowski et al. responses during early phases of TBI. Long-term pathology
and/or neurodegeneration are initiated by processes occurring
at the time of injury (McKee et al., 2009; Goldstein et al.,
2012), and a deeper understanding of early time windows will
be critical for targeting appropriate interventions to different
injury phases. While our in vitro approach allowed detailed
synaptic characterization, it may not reveal all of the factors
contributing to injury in the intact brain, potentially accounting
for why vestibulomotor deficits persisted at 48 h even though
synaptic changes had largely recovered. Slicing removes a
large portion of network interactions and substantially reduces
background activity (Lipton and Whittingham, 1984; Moyer
and Brown, 2002; Hájos et al., 2009), potentially countering
the effects of hyperexcitability. TBI also dramatically reduces
cerebral blood flow (Golding, 2002; DeWitt and Prough, 2003;
Kenney et al., 2016), compounding trauma with metabolic
stress. Slices are perfused with oxygenated recording solutions
that could alleviate these energetic factors. Interestingly, the
increases in excitatory input we see here have parallels with
synaptic responses in hypoxia, stroke, and epilepsy (Fleidervish
et al., 2001; Bolay et al., 2002; Hofmeijer and Putten, 2012;
Naylor et al., 2013). FUNDING This work was supported by Boston University startup funds. Injury and Excitatory-Inhibitory Balance It will be important for future work to
examine how mild injury acts in vivo to better capture the
interactions between synaptic disruptions, altered excitability,
and oxidative stress. rather than ascending afferent inputs. Intracortical inputs will
be emphasized in spontaneous measurements as their more
proximal dendritic location will be less affected by electrotonic
filtering (Bathellier et al., 2009). LOT stimulation primarily
drives afferent inputs onto distal dendrites, followed by a smaller
secondary input from intracortical circuits. Determining the
cause of the E-I imbalance is an important question for future
studies. Pharmacological methods may help test for selective
changes in ascending pathways versus local intracortical circuits
(Tang and Hasselmo, 1994; Franks and Isaacson, 2005). ETHICS STATEMENT This study was performed in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. All
animals were handled according to approved Institutional
Animal Care and Use Committee (IACUC) protocols (#17-017)
of Boston University. Disruptions in piriform are consistent with a range of sensory
deficits described in both human injury and animal models,
including visual and auditory systems as well as olfaction
(Ciuffreda et al., 2007; Lew et al., 2007a; Alwis et al., 2012;
Proskynitopoulos et al., 2016). Reduced odor discrimination
ability is consistent with the relatively localized microglial
activation we found in piriform. Interestingly, changes in
synaptic function and discrimination ability shared broadly
similar time courses, being most severe at earlier time windows
and largely recovering by approximately 48 h. As we used a
diffuse injury model, and did not record from other brain
regions, changes in other areas may also contribute to loss
of discrimination ability. Further work will be needed to test
whether synaptic disruptions are confined to piriform or extend
more widely across brain areas. More generally, sensory assays
have potential as a simple and quantitative way to assess TBI
severity, both during early post-injury phases as well as longer-
term effects (Bressler et al., 2017). AUTHOR CONTRIBUTIONS EW, YG, AG, IM, AM, and ID designed and performed the
experiments. EW, GD, WE, and ID analyzed the data. EW, GD,
WE, and ID wrote the manuscript. Involvement of Piriform in Injury j
y
We consistently found neuroinflammation in piriform and
surrounding areas, which have previously received little attention
in TBI research. While trauma-induced damage has been
described in diverse brain areas, including somatosensory
and prefrontal cortex, the majority of work has focused on
hippocampus (Reeves et al., 1995, 1997; Santhakumar et al., 2000,
2001; Golarai et al., 2001; Zanier et al., 2003; Witgen et al., 2005;
Griesemer and Mautes, 2007; Hansen et al., 2018). While we
did find mild hippocampal neuropathology in one case, it was
inconsistent across animals. The reasons for selective damage
in piriform and entorhinal areas are unclear but may be due
to the free head movement allowed by our model. Activated
microglia were most prominent in ventrolateral areas opposite
the dorsal impact site, consistent with a coup-contrecoup effect
causing damage on the opposite side of the brain (Courville,
1942). Piriform’s extensive axonal pathways may also make it
particularly sensitive to diffuse axonal injury, which tends to
affect long-range projections (Pandit et al., 2013; Caeyenberghs
et al., 2014). Afferent inputs to piriform course ≥5 mm along
the base of the brain, and both intracortical fibers and piriform
projections extend over longer distances than those in neocortex
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Davison. 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. Copyright © 2019 Witkowski, Gao, Gavsyuk, Maor, DeWalt, Eldred, Mizrahi and
Davison. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). Frontiers in Cellular Neuroscience | www.frontiersin.org April 2019 | Volume 13 | Article 166 REFERENCES The use, distribution or reproduction in
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developing nervous system. Nat. Rev. Neurosci. 5, 97–107. April 2019 | Volume 13 | Article 166 Frontiers in Cellular Neuroscience | www.frontiersin.org 17
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O mesmo e o outro: uma leitura ‘dupla’ do conto “92”, de Dalton Trevisan
|
Acta Scientiarum. Human and Social Sciences
| 2,004
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cc-by
| 4,971
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Leonel Lopes* e Evely Vânia Libanori Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900, Maringá, Paraná, Brasil. *Autor para correspondência. e-
mail: lieve@brturbo.com.br RESUMO. Amparados no princípio de que Dalton Trevisan, ao reestruturar seus contos e
sintetizá-los em um procedimento de decomposição e de recomposição, recria um novo conto
que é concomitantemente o mesmo e outro, o presente artigo analisa os contos “92”, da obra
Ah, é? e “Última corrida de touros em Curitiba”, publicado em O Pássaro de cinco asas, com
o objetivo de demonstrar a maneira pela qual se constrói essa concomitância. Ao mesmo
tempo, discorreremos sobre os momentos em que a participação do leitor é solicitada como
condição para que os dois contos acima comuniquem seus efeitos estéticos. Analisando o
corpus, concluímos que a concomitância se deve ao trabalho do autor de eliminar e de deixar
marcas textuais, intencionalmente ou não, as quais permitem ao leitor utilizar-se do texto de
modo a retirar dele leituras novas e também resgatar significações do conto original. ABSTRACT. The same and the other: a double reading of Dalton Trevisan’s short story
“92”. Based on the principle that Dalton Trevisan recreates a new short story that is
concomitantly the same and the other, the present paper analyses the short stories “92” and
"Última corrida de touros em Curitiba". The objective is to demonstrate the way in which this
concomitance is built. At the same time, some considerations about the reader's participation
in the two short stories' aesthetic effects are made. Analysing the corpus, results show that the
concomitance is due to the author's job of eliminating and leaving textual marks, intentionally
or not, which allow the reader to give the text new readings and also to rescue meanings from
the original short story. Key words: Dalton Trevisan, minimization, reader. Acta Scientiarum. Human and Social Sciences Introdução Utilizando-se
desse artifício de repetição, a obra daltoniana
apresenta a síntese de um mundo no qual não há
mudanças significativas no ser humano, que parece
viver em um tempo circular, agarrado a antigas regras
de comportamento. sexual, traições etc.), de personagens (João, Maria,
Pedro, Dinorá etc.) e de situações e frases que
permeiam a vida das personagens residentes na
“fictícia” Curitiba de Dalton Trevisan. Utilizando-se
desse artifício de repetição, a obra daltoniana
apresenta a síntese de um mundo no qual não há
mudanças significativas no ser humano, que parece
viver em um tempo circular, agarrado a antigas regras
de comportamento. seus familiares. A indiferença sofrida por João leva
não apenas à sua degradação, quando é desvalorizado
em relação a um cachorro, mas também ao seu
próprio anulamento, uma vez que a família o esquece
para recolher roupas e fechar as janelas durante a
chuva. A
auto-estima
da
personagem
decai
profundamente em uma escala gradativa que passa de
inseto asqueroso à coisa, chegando à extinção do eu,
como veremos. Uma terceira manifestação do projeto do autor
curitibano, e não necessariamente a última, consiste
em recortar partes de seus contos e apresentá-las, com
algumas alterações, como um novo trabalho. É com
ela que trabalharemos mais detidamente. Escolhemos
como corpus a ministória “92” por ser ela parte do
exemplo máximo do trabalho de síntese de Dalton
Trevisan: o livro de ministórias Ah, é?. Tal obra
consiste em recortes, em partes de vários de seus
contos
que
são
reapresentados
com
algumas
modificações e intitulados por meio de números. Cada recorte se torna um novo trabalho e pode, então,
adquirir outros sentidos, o que não significa perder
algumas possibilidades significativas do conto de
origem, pois se mantém organicamente ligado a ele. A raiz, a procedência do nosso corpus é o conto
“Última corrida de touros em Curitiba”, que servirá
de base comparativa para explicitar a maneira pela
qual a “92” torna-se um novo conto que,
simultaneamente, aproxima-se e se distancia do
anterior. A deterioração da família é aqui apresentada
tendo como conseqüência principal a solidão
existencial de João, que não pode contar consigo nem
com os seus parentes. Restam-lhe apenas seus
pensamentos e questionamentos sobre sua condição
ôntica em um presente degradado, saudoso de um
passado em que era considerado um ser humano
independente. Introdução Seu estado atual corresponde ao de um
velho inválido que, como um bebê, baba, dorme e faz
suas necessidades fisiológicas em qualquer horário e
local. Tal estado transita para um aspecto mais grotesco
e degradante no momento em que João manifesta, por
meio da voz do narrador, que se sente “(...) barata
leprosa com caspa na sobrancelha” (Trevisan, 1994:
67). Pode-se dizer que essa expressão cria um vazio a
ser preenchido pelo leitor e seu preenchimento
interrompe a “assimetria” entre texto e leitor. A
assimetria, de que fala Iser (1979: 88), resulta do fato
de que os códigos de interação estão fragmentados no
texto, existem como potencialidade e necessitam ser
compreendidos pelo leitor de modo que se estabeleça
a comunicação. Essa expressão comparativa e
hiperbólica constitui uma significação no processo
narrativo do conto de Dalton Trevisan, pois esse
acúmulo de traços grotescos tem por efeito a
desumanização da personagem. O fato de João sentir-
se uma “barata leprosa”, um inseto asqueroso, está
diretamente ligado à sua deformação física e à sua
imobilidade, mas não somente a elas. A condição de
“leprosa” reitera o estado morto-vivo de João porque
corresponde a um ser que sofre no corpo as marcas da
morte. Morte não somente física, mas principalmente
afetiva e social, pois, além da acepção de doença
contagiosa, é um coloquialismo usado para designar
pessoas que não servem para nada. Em uma
sociedade em que o homem parece ter valor somente
enquanto força de trabalho ou acúmulo de capital, a
“doença” e a invalidez de João levam-no a viver,
assim como as baratas e os ratos, à margem dessa
sociedade, representada no conto pela família que o
desvaloriza como ser humano, tratando-o com
desprezo e negligência. Acta Scientiarum. Human and Social Sciences Introdução organicamente ligado ao outro. Ao mesmo tempo,
discorreremos sobre os momentos em que a
participação do leitor é solicitada como condição para
que a ministória “92”, como a denomina o próprio
autor, comunique seus efeitos estéticos. A ministória “92”, objeto de nossa análise, é
componente integrante do volume de ministórias Ah,
é? de Dalton Trevisan (1994). Tal volume pode ser
considerado o estágio mais avançado e significativo
de um projeto literário daltoniano de constante
refeitura de seus contos. Um dos objetivos principais
desse projeto é a minimização dos contos, que se
tornam cada vez mais concisos, a ponto de se
tornarem haicais, porém haicais não-tradicionais, pois
do modelo oriental só mantêm o caráter sintético. Amparados no princípio de que Dalton Trevisan, ao
reestruturar seus contos e sintetizá-los em um
procedimento de decomposição e de recomposição,
recria um novo conto que é, concomitantemente, o
mesmo e outro, propomo-nos a analisar o conto “92”,
com o objetivo de demonstrar a maneira pela qual se
constrói essa concomitância. A estratégia narrativa
adotada pelo autor solicita um tipo de leitor que
conheça sua proposta literária e também o conto
“primeiro” que foi resgatado e transformado,
resultando em um texto diferente que se mantém O trabalho de constante refeitura dos contos
daltonianos se manifesta de vários modos, no entanto,
esses modos mantêm ligações entre si e tem como
resultado textos que revelam os traços do produto que
lhes deu origem, mas, ao mesmo tempo, tornam-se
um artefato estético singular. Uma dessas manifestações consiste na revisão que
Dalton Trevisan faz de seus livros a cada nova
edição. Buscando “enxugar” os contos, o autor exclui
ou modifica palavras e formas verbais, de modo a
deixar a narrativa isenta de expressões que podem ser
resgatadas pelo leitor através do seu conhecimento
gramatical e do contexto textual. Uma outra faceta
desse projeto, e talvez a mais importante, manifesta-
se por meio da constante retomada, em diversos
livros, de temas (a guerra conjugal, conflitos entre
interesses mesquinhos, busca incessante do prazer Maringá, v. 26, n. 2, p. 195-200, 2004 196 Lopes e Libanori Lopes e Libanori sexual, traições etc.), de personagens (João, Maria,
Pedro, Dinorá etc.) e de situações e frases que
permeiam a vida das personagens residentes na
“fictícia” Curitiba de Dalton Trevisan. A ministória “92” em duas leituras De novo um homem,
não barata leprosa com caspa na sobrancelha - e, a
sombra das folhas na cabecinha trêmula, adormece.”
(Trevisan, 1994: 67). Geralmente, nos contos de
Dalton Trevisan, as personagens masculinas utilizam-
se de expressões zoomórficas e grotescas para
expressar o estado degradante em que se encontram. São homens que, na maioria das vezes, passaram pela
queda do “rei da terra”, deixaram de ser reconhecidos
como fortes, poderosos e viris. Os tipos são vários:
velhos solitários e saudosos de um tempo em que as
mulheres não os repudiavam, maridos cujas esposas
os obedeciam e os temiam, homens inválidos por uma
doença que pôs fim a um reinado de desmando e
ostentação sexual, cônjuges abandonados pelas
esposas
que
imploram
a
elas
um
retorno,
“arrependidos” de tratá-las como escravas etc. Nessa primeira leitura, é possível vislumbrar o
abuso e o descaso que os doentes e/ou velhos sofrem
pelos parentes. Dalton Trevisan utiliza geralmente
esse tema como forma de crítica social. Porém,
muitas vezes, a crítica não busca um tom
maniqueísta, não apresenta apenas uma faceta das
personagens. Cabe ao leitor uma maior atenção com a
“92” para perceber que a vítima também pode ter seus
momentos de algoz. Partindo, então, para uma segunda leitura, temos,
como já referido, que, no jardim, João sente um
desejo ardente de voltar a ser “homem”, em oposição
à sua atual invalidez. Para Eco, (1979: 56-58) a
recorrência de situações no conjunto de obras de um
autor deve ser um elemento a ser considerado pelo
leitor a fim de ampliar as possibilidades significativas
do texto. No ato da leitura, o leitor deve ativar a sua
habilidade de percepção para compreender que a
mensagem verbal, no caso, o desejo de João de voltar
a ser “homem”, está relacionada a outras obras do
escritor. O leitor deverá, então, buscar, no seu
repertório intertextual, o suporte para o ato de
congenialidade com o autor, evitando, assim, Desse modo, a zoomorfização atribui a eles um
teor trágico, e assim, revestindo-se de vítima, tentam
camuflar um “passado” de algoz. O que João pode ter
feito para a esposa durante seu “reinado” é retribuído
pelo modo como ela o trata durante a invalidez. Destarte, a mulher passa de mártir a carrasco, em uma
inversão de papéis que corresponde à vingança da
esposa pelo sofrimento que o marido lhe causou. A ministória “92” em duas leituras Ânsia essa que é quebrada pelo fato de terem-no
esquecido na chuva, trazendo de volta a sensação de
inumanidade e de anulamento. Ânsia essa que é quebrada pelo fato de terem-no
esquecido na chuva, trazendo de volta a sensação de
inumanidade e de anulamento. Como inferido acima, o que leva a personagem a
sentir-se assim não são somente as marcas deixadas
pelo
derrame
cerebral,
mas
sobretudo
pela
negligência e pelo desinteresse familiar. Destarte, há,
no conto, uma inversão irônica, pois o inumano não
seria João, e sim sua família, que age de forma cruel
para com ele. O conto reflete, desse modo, uma
sociedade que tem valores corrompidos, pois nela o
ser humano é julgado por sua aparência, auto-
suficiência e aptidão ao trabalho, e não por valores
como solidariedade, honestidade, companheirismo
etc. Essa crítica social é apresentada em um contexto
familiar
específico,
atingindo
uma
instância
universal, pois traz à discussão um dos modos como a
violência é exercida dentro do lar, um problema que
se repete constantemente e em toda parte. Atualizando, por essa perspectiva, os códigos de
comunicação empregados pelo autor, tem-se, em
“92”, a figura de quem já foi o “homem”, o “rei da
terra”, e que, devido à atual condição de ente passivo,
dependente e grotesco, deseja reinar novamente,
voltando a ser a mesma pessoa que fora antes do
derrame cerebral. Analisando o artifício daltoniano de repetição dos
mesmos nomes próprios na maioria dos contos,
verificamos que “João”, por se tratar de um nome
bastante comum, inclui a personagem em um vasto
número de anônimos. João passa a ser todos e, ao
mesmo tempo, nenhum. O nome é inexpressivo e está
coerente com o estado da personagem, um “alguém”
que perde sua individualidade e deixa de ter
importância na sociedade. João não tem voz ativa na
narrativa, seus pensamentos são retratados por um
narrador em terceira pessoa, reforçando, assim, sua
subordinação e dependência em relação aos outros. A
inexpressividade do nome, o silêncio e a imobilidade
reafirmam a condição de João, pois, em conjunto, são
marcas de passividade, opressão e morte. Colabora para essa leitura a expressão zoomórfica
que se segue ao anseio de João em retornar à posição
de “rei da casa”: “Primeira vez depois do insulto
cerebral aquela ânsia de viver. Acta Scientiarum. Human and Social Sciences A ministória “92” em duas leituras Em uma primeira leitura da “92”, tem-se a
personagem central João em uma situação de
dependência, de alguém que não governa seus
músculos. Para se locomover, ele necessita ser
carregado em uma cadeira de braços pela família, que
não toma cuidados básicos para amenizar o
sofrimento de João, deixando-o sentado na cadeira
dura e com roupa imprópria à temperatura local. Não
é possível identificá-lo como velho apenas pelo seu
estado atual, pois o derrame cerebral que o deixou
paralisado pode manifestar-se também em pessoas
jovens. A ausência de coordenação muscular da
personagem pode ser percebida, ainda, pelo fato de a
cabeça estar caída no peito, de um fio de baba
escorrer pelo queixo e de ter a boca deformada. Essas
manifestações colaboram para que tenhamos dele
uma visão grotesca que, ao ser relacionada à sua
solidão em um canto do quintal, remete-nos à
situação daquelas pessoas abandonadas em asilos. Cria-se, então, uma outra imagem de João, a de
vítima da indiferença familiar. Tal imagem é
reforçada pelo lirismo da personagem em alegrar-se
com o canto do pássaro e com as flores, atitude de um
ser sensível que contrasta com a insensibilidade de A expressão hiperbólica “barata leprosa com
caspa na sobrancelha” tem também a função de
traduzir o estado de espírito de João, sua falta de
esperança, o que cria um contraste com a ânsia de
vida que sente, por alguns momentos, no jardim. Maringá, v. 26, n. 2, p. 195-200, 2004 197 Uma leitura ‘dupla’ do conto “92” subjetivismos. Na mitologia de Dalton, homem e
mulher têm papéis pré-estabelecidos por uma cultura
baseada em conceitos patriarcais. O “homem” possui
a acepção do varão, do macho, que vê em seu vigor
sexual o poder sem limites. Considerado o “rei da
terra”, é ele quem manda e a quem todos devem
servir e acatar. Já a mulher, aos olhos dessa sociedade
falocêntrica,
é
julgada
um
ser
inferior. Conseqüentemente, deve agir como serva e aceitar as
reinações do seu ‘senhor’, como a traição, a
bebedeira, a vadiagem, a exploração, a agressão física
e sexual, além de sofrer com a eterna desconfiança do
homem em relação à sua fidelidade. Trata-se de uma
concepção arcaica dos papéis matrimoniais, mas que
ainda tem reflexos na atualidade, principalmente em
cidades provincianas, como a que Dalton retrata na
maioria de suas obras. A ministória “92” em duas leituras Passa a assumir o papel masculino
na casa, o que inclui ignorar o ser passivo, no caso, o
marido. A expressão comparativa que se refere à
metamorfose do “homem” em barata, inseto caseiro
por excelência, reflete também a mudança espacial de
João, que passa a ficar preso às fronteiras da casa,
local do confinamento feminino. Não podendo mais
seguir sua odisséia, passa a viver de restos, como a
barata, só que de restos de atenção, de momentos em
que os familiares lembram dele, como quando levam-
no para tomar uma brisa no jardim. Porém essa
pequena atenção é falaz, pois deixam-no só, mesmo
sabendo de sua dependência, o que leva a crer que o
“passeio” é um modo de se livrarem dele, de mantê-lo
à distância, atitude similar ao que era feito com os
leprosos no passado. Como a barata, João é confinado
ao anonimato, pois só é aceito na casa quando não é
percebido pelos moradores dela. Dessa forma, a
vingança da esposa se realiza. Ela faz que ele tenha
uma amostra do que a fez passar. No entanto, a lição
não é captada pelo marido, como prova o desejo de
João de reinar novamente. A sua ânsia de retornar a
ser “homem” pode fazer o leitor inferir que a
experiência de nada valeu e que a vida do casal se
manterá em uma eterna guerra conjugal. q
g
Por sua vez, a alcunha do protagonista, Dadá, tem
uma acepção que se bifurca, correspondendo à
primeira e à segunda parte do conto. Essa bifurcação
serve para reiterar o significado referente a “bebê” de
seu apelido. Na primeira, o sentido de “Dadá” diz
respeito ao pedido constante feito por crianças por
meio do verbo “dar” na forma imperativa e coloquial:
“— Dá, dá”. Como uma criança, Dadá está sempre
querendo algo, mais e mais, não abrindo mão de suas
vontades, desejando todos os prazeres para si, em
uma atitude egocêntrica, típica da fase infantil. Assim, a fantasia carnavalesca de Dadá, um babador
com uma chupeta gigante, não poderia ser mais
significativa. Novamente
ressaltamos,
aqui,
a
necessidade da intervenção criativa do receptor nos
textos daltonianos. O conto é um exemplo daquilo
que Eco chamou de “obra em movimento” (1991:
46), uma vez que tem por base o uso de códigos e de
símbolos para a comunicação do indefinido. A ministória “92” em duas leituras Trata-se de uma estratégia pela qual a mulher obtém
condições
para
expressar
comportamentos
de
punição. Evidentemente, essa possibilidade de
entendimento do texto não está explícita no tecido
textual: tem-se uma interpretação potencial, que só Maringá, v. 26, n. 2, p. 195-200, 2004 198 Lopes e Libanori será percebida quando o fruidor se dispuser a ser um
intérprete da obra. Assim, cada fruidor é um
intérprete e um executante da obra, o que a faz
“reviver dentro de uma perspectiva original” (Eco,
1991: 40). Explicita-se, desse modo, a necessidade da
co-autoria do leitor para a constituição de um objeto
artístico. O leitor traz, para o campo literário, uma
execução individual da obra literária, estabelecendo
sentidos
não
previstos
pelo
autor,
mas
constitutivamente possíveis. mais de um terço da história, dona Cotinha sofre com
as farras de Dadá. Ele, além de mentir, costuma
dormir fora de casa para ficar com as amantes, na
roda de samba e na bebedeira. Quando sua esposa
reclama
sobre
suas
andanças,
Dadá
torna-se
extremamente cínico e sarcástico. O narrador, em terceira pessoa, contrapondo as
atitudes algozes do protagonista, reforça o papel de
vítima da esposa ao se referir a ela como “pobre
moça”, “heroína” e “santa”. Essa acepção de mártir se
encontra também no próprio nome de dona Cotinha,
uma apócope do diminutivo ‘coitadinha’ que, somado
ao título de tratamento ‘dona’, colabora para exprimir
um grau superior de sofrimento. A personagem torna-
se então a “dona Coitadinha”, indicando sua natureza
frágil. Assim, a denominação “dona Cotinha” pode
ser considerada um elemento potencial do texto. Conforme Iser (1979: 103), os elementos potenciais
sugerem linhas de interpretação ao leitor. Este pode
supor ou concluir que dona Cotinha manter-se-á
passiva perante as “reinações” do marido, fazendo-se
uma vítima, uma “coitada”. Porém, considerando que
geralmente Dalton Trevisan utiliza diminutivos como
forma de ironia, dona Cotinha pode vir a revelar uma
face contrária a que seu nome sugere. Assim, o silêncio de João, a impossibilidade de
comunicar-se, tem por contraste a fala imperativa da
esposa, única personagem que tem voz na narrativa. Devido ao estado inerte do marido, ela assume o
papel ativo na casa, o de quem manda, mesmo que as
ordens se restrinjam aos afazeres domésticos. É ela a
quem todos obedecem. Ela não está na posição de
quem está recolhendo a roupa, fechando as janelas e
prendendo o cachorro, e sim na posição de quem
detém as ordens. A ministória “92” em duas leituras Dadá faz
questão de ostentar sua condição de boêmio, debocha
de seu próprio cinismo e não se importa que seu
comportamento leve sua esposa ao sofrimento. Acta Scientiarum. Human and Social Sciences “Última corrida de touros em Curitiba” suas
relações com a “92” Temos, então, que a mulher, enquanto esposa,
corresponde a uma extensão da mãe, pois a ambas
cabe perdoar as reinações do “bebê” (do filho, do
homem e do marido). Ela deve abdicar-se para manter
o casamento, instituição que concede à mulher um
lugar na sociedade. Uma leitura do conto “matriz”, “Última corrida de
touros em Curitiba”, torna-se necessária para
vislumbrarmos a maneira pela qual se constrói a
concomitância da “92”. O texto faz parte do volume
de contos O pássaro de cinco asas (1974) e narra as
reinações de Dadá, homem que não abandonou a
boemia quando se casou com Cotinha. Podemos
dividir sua estrutura em três partes ou atos. Durante Na segunda parte, a acepção da alcunha ocorre
devido à invalidez de Dadá após um derrame. Agora,
ele depende da esposa/“mãe” principalmente para a
higiene pessoal e alimentação, e dona Cotinha faz Maringá, v. 26, n. 2, p. 195-200, 2004 Uma leitura ‘dupla’ do conto “92” 199 amante, depois, o bar. Então, vence a corrida de
hemiplégicos, provando sua potência varonil, sua
força de “touro”, chegando ao topo, de onde ocorre a
queda final do “herói”. Justifica-se, desse modo, o
título “Última corrida de touros em Curitiba”, pois o
touro, símbolo da potência, do vigor viril, metaforiza,
no conto, uma raça de homens dominadores que
realizam uma “última” tentativa de provar um grau
máximo de masculinidade e garantir, assim, seu
reinado. Como o conto tem por intenção destruir o
mito do rei da terra, torna-se essa a “última corrida”,
porque, depois da morte patética de Dadá, cai por
terra a imagem de poder que o “rei” pretende manter,
já que uma simples semente de limão foi capaz de
derrubar o touro para sempre. questão de explicitar para todos a dependência de
Dadá em relação a essas duas necessidades principais. Além disso, Dadá também é tratado como criança
quando a esposa e as visitas se referem a ele
utilizando uma linguagem infantilizada, marcada pelo
uso
de
diminutivos. Assim,
ironicamente,
o
diminutivo em vez de carinhoso, tem pretensões
ofensivas. questão de explicitar para todos a dependência de
Dadá em relação a essas duas necessidades principais. Além disso, Dadá também é tratado como criança
quando a esposa e as visitas se referem a ele
utilizando uma linguagem infantilizada, marcada pelo
uso
de
diminutivos. Assim,
ironicamente,
o
diminutivo em vez de carinhoso, tem pretensões
ofensivas. “Última corrida de touros em Curitiba” suas
relações com a “92” É significativo que o narrador reserve mais da
metade do conto para esse episódio, o da invalidez,
pois é o momento em que ocorre a queda do “rei da
terra”, quando Dadá deixa de ser considerado um
“homem”, não podendo exercer seu papel de varão,
de farrista. Cessam as grandes aventuras do “herói”, e
o que era para se tornar uma tragédia, com Dadá a
delirar de sede pelo fato de não se mover, nem falar,
adquire ares de comédia. Colabora para isso, a
intercalação que o narrador faz entre os descasos da
família e algumas alusões às peripécias de Dadá,
como a visita da amante no hospital, e o fato de o
narrador se referir à dona Cotinha como “megera”,
contrastando,
assim,
sua
posição
anterior
de
considerá-la “heroína” e “santa”. Há, então, uma
inversão de papéis, pois de mártir ela passa a algoz, e
o marido sofre a vingança da esposa, que aproveita o
momento oportuno para retribuir as humilhações
sofridas, revelando uma personagem não tão pacata
quanto seu nome indicava: Considerando as três partes em que o conto
“Última corrida de touros em Curitiba” é organizado,
nota-se que Dalton Trevisan, ao reestruturá-lo para
recompô-lo na “92”, eliminou a primeira, a terceira (o
final do conto), e também a metade da segunda parte,
reduzindo-a e, conseqüentemente, o conto em si, aos
acontecimentos ocorridos no momento em que a
personagem já inválida, agora denominada João,
permanece no jardim. Ainda no que se refere a esse
fragmento, o autor selecionou certas informações que
foram suprimidas. A mais relevante delas é a de que
Dadá/João sente saudade da amante. Como todas as
outras referências explícitas sobre o comportamento
boêmio da personagem estavam nas partes que foram
cortadas, essa era a única informação explícita que
sobraria do conto original sobre o seu passado. Ao se
retirar essa informação, a estrutura narrativa da
ministória permitiu que a primeira leitura aqui
apresentada adquirisse maior coerência __ Dadá, eu bem disse, não facilite - e indiferente ao
clarão de ódio no olho estagnado. - Você bebe
demais. Come demais. Já não é moço. Pensa que
ligava? Dadá, olhe a extravagância. Um dia pode ter
uma coisa. Veja o que aconteceu. Bem eu não disse? (Trevisan, s/d: 184). Ao mesmo tempo, permaneceram signos do
universo literário daltoniano que permitiram a
segunda leitura da “92”, um resgate da leitura do
conto “Última corrida de touros em Curitiba”. Acta Scientiarum. Human and Social Sciences “Última corrida de touros em Curitiba” suas
relações com a “92” Esses
signos, como o substantivo “homem” e a expressão
comparativa
“barata
leprosa
com
caspa
na
sobrancelha”, no processo de reestruturação e de
recomposição, não perderam o significado adquirido
no conjunto de obras do autor, mas se permitiram
novos significados que não têm a mesma sustentação
no conto original. Mesmo o momento de solidão e abandono no
quintal perde grande parte de seu efeito trágico em
razão da saudade que Dadá sente da amante. Desse
modo, a narrativa permite vislumbrar a ironia do
narrador ao assumir um certo tom “épico” em relação
às aventuras e desventuras de Dadá. Esse narrador
busca destruir a imagem que a sociedade, aqui
retratada, tem dos papéis masculino e feminino. Para
isso, inverte a atuação de ambos, ridiculariza a queda
do “herói”, a sua não-redenção e, principalmente, a
sua morte, apresentando-a não como um ato digno e
heróico, mas sim de forma cômica, no limiar do
ridículo: “(...) morreu engasgado com a semente da
batida dupla de limão” (Trevisan, s/d: 40). Assim,
Dadá não morre como um “homem”, tal qual desejou
no momento em que, no jardim, sentiu brotar em si
uma ânsia de vida. O procedimento artístico de Dalton Trevisan,
quando não aumenta o número de lacunas e de vazios
a serem preenchidos pelo leitor, faz que as lacunas e
os
vazios
já
existentes
enriqueçam-se
e,
conseqüentemente,
aumentem
as
possibilidades
significativas do conto. Essas possibilidades de
sentido só se manifestam em decorrência do trabalho
interpretativo do leitor. Assim, cabe ao leitor perceber
as marcas textuais que permitem outras leituras da
“92”, sem restringi-la a significações que só reiterem
a condenação da esposa pelo seu descaso com o
marido. A volta triunfal planejada por ele corresponde ao
ato final do conto. O retorno do “homem” Dadá
ocorre em uma gradação crescente que corresponde
às provas pelas quais deve passar para reassumir seu
“trono”, seu papel de macho. Primeiro, visita a Maringá, v. 26, n. 2, p. 195-200, 2004 200 Lopes e Libanori de touros em Curitiba” obteve ao ser transformado na
“ministória” em questão. Assim, no projeto literário
daltoniano
de
recriar
um
conto
que
é
simultaneamente o mesmo e outro, a concomitância
se deve ao trabalho do autor de eliminar e de deixar
marcas textuais, intencionalmente ou não, que
permitam ao leitor utilizar-se do texto de modo a
retirar dele leituras novas e também resgatar
significações do conto original. “Última corrida de touros em Curitiba” suas
relações com a “92” Com efeito, a estrutura narrativa de “Última
corrida de touros em Curitiba” não autoriza que o
marido seja visto apenas como uma vítima, como a
“92” permite. No entanto, o texto não se limita a essa
interpretação, pois também possibilita mostrar o
marido como responsável pela vingança justificável
da esposa, conforme a segunda leitura aqui
apresentada. O trabalho do leitor, em analisar signos
recuperados pelo autor para construção de seus textos
e também em não restringi-los a significações
petrificadas, é necessário para que a “ministória”
manifeste ligações significativas com o conto original
e, ao mesmo tempo, rompa com algumas delas. Acta Scientiarum. Human and Social Sciences Maringá, v. 26, n. 2, p. 195-200, 2004 Received on May 13, 2004.
Accepted on December 03, 2004. TREVISAN, D. A trombeta do anjo vingador & O pássaro
de cinco asas. São Paulo: Círculo do Livro S.A., s/d. Referências ECO, U. Leitura do texto literário: lector in fabula. Trad. de Mario Brito. Lisboa: Editorial Presença, 1979. ECO, U. Leitura do texto literário: lector in fabula. Trad. de Mario Brito. Lisboa: Editorial Presença, 1979. ECO, U. Obra Aberta. 8. ed. Trad. de Pérola de Carvalho. São Paulo: Perspectiva, 1991. Como buscamos demonstrar no desenvolvimento
desse trabalho, o conhecimento, por parte do leitor,
do projeto literário do autor e dos códigos de
comunicação que permeiam suas obras, é de
fundamental importância para uma leitura que se
aproxime das possibilidades de sentido do texto a que
a ministória teve origem. Por outro lado, o trabalho
de refeitura de Dalton Trevisan é o responsável pela
possibilidade
de
o
leitor
resgatar,
na
“92”,
significações do conto primeiro e também de poder
descortinar novas possíveis interpretações, adquiridas
devido à (des)roupagem que o conto “Última corrida ECO, U. Obra Aberta. 8. ed. Trad. de Pérola de Carvalho. São Paulo: Perspectiva, 1991. ISER, W. A interação do texto com o leitor. Trad. de Luiz
Costa Lima. In: LIMA, L. C. (Coord.). A literatura e o
leitor: textos de estética da recepção. Rio de Janeiro: Paz e
Terra, 1979, cap. 3, p. 83-132. TREVISAN, D. Ah, é? 2. ed. Rio de Janeiro: Record, 1994. TREVISAN, D. A trombeta do anjo vingador & O pássaro
de cinco asas. São Paulo: Círculo do Livro S.A., s/d. Maringá, v. 26, n. 2, p. 195-200, 2004
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Generalised predictive controller (GPC) design on single‐phase full‐bridge inverter with a novel identification method
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IET control theory & applications
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Correspondence Correspondence
Seyyed Morteza Ghamari, Faculty of Electrical And
Computer Engineering, Shahrood University of
Technology. Received: 13 October 2021
Revised: 15 February 2022
Accepted: 19 April 2022 Received: 13 October 2021
Revised: 15 February 2022
Accepted: 19 April 2022 Received: 13 October 2021
Revised: 15 February 2022
Accepted: 19 April 2022
DOI: 10.1049/cth2.12295 IET Control Theory & Applications IET Control Theory & Applications ORIGINAL RESEARCH Generalised predictive controller (GPC) design on single-phase
full-bridge inverter with a novel identification method Mehran Jelodari Memeghani1
Seyyed Morteza Ghamari2
Taha YousefiJouybari3
Hasan Mollaee2
Patrick Wheeler4 Mehran Jelodari Memeghani1
Seyyed Morteza Ghamari2
Taha YousefiJouybari3
Hasan Mollaee2
Patrick Wheeler4 The Authors. IET Control Theory & Applications published by John Wiley & Sons Ltd on behalf of The Institution of Engineering and Technology. wileyonlinelibrary.com/iet-cth
1 IET Control Theory Appl. 2022;1–11. properly cited.
© 2022 The Authors. IET Control Theory & Applications published by John Wiley & Sons Ltd on behalf of The Institution of Engineering and Technology. properly cited.
© 2022 The Authors. IET Control Theory & Applications published by John Wiley & Sons Ltd on behalf of The Institution of Engineering and Technology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in
properly cited. Abstract 1Faculty of Electrical And Computer Engineering,
Semnan University
2Faculty of Electrical And Computer Engineering,
Shahrood University of Technology
3Faculty of Electrical And Computer Engineering,
Tabriz University
4Faculty of Electrical And Control Engineering,
Nottingham University
Correspondence
Seyyed Morteza Ghamari, Faculty of Electrical And
Computer Engineering, Shahrood University of
Technology. Email: mortaza.ghamari@yahoo.com The design of an on-line generalised predictive control (GPC) technique with a novel
identification method is presented in this paper for a single-phase full-bridge inverter in
the presence of different disturbances. The controller uses system discrete-time model to
reach the control variables with a prediction over these values, followed by computing a
cost function for control aims. However, in this controller, the need for the mathematical
model of the system is removed since the black-box identification strategy is used. More-
over, GPC structure has many advantages including low computational complexity, sys-
tematic design procedure, and fixed switching frequency that makes it a good alternative
for practical applications. Various disturbances can have a negative impact on a DC–AC
inverter; thus, considering robust dynamics and ease of implantation, the GPC scheme is
used along with an improved exponential regressive least square identification algorithm
as an adaptive strategy in the controller. Moreover, the prediction horizons of this con-
troller have been increased, resulting in its low steady-state error and better performance. Furthermore, harmonics in the sinusoidal signal can decrease the total efficiency of the sys-
tem; thus, an LC filter is used to reduce the level of total harmonic distortion. However, the
stability of the filter is the most challenging issue in designing a suitable controller. Finally,
the strength of the current controller is verified using experimental and simulations results. 4Faculty of Electrical And Control Engineering,
Nottingham University © 2022 The Authors. IET Control Theory & Applications published by John Wiley & Sons Ltd on behalf of The Institu 1
INTRODUCTION advanced controllers are used based on the switching properties
of Inverters: deadbeat technique [8], sliding-mode control [9,
10], and fuzzy-based structure [11–14]. The most significant
aspect of deadbeat, sliding-mode, and fuzzy-based controllers is
the provision of an output result without overshoot and ringing. On the other hand, the complex structure of deadbeat and inter-
nal model techniques introduces them as unsuitable options in
practical applications. Additionally, sliding-mode control pro-
vides great robustness during transients while providing a suit-
able switching nature in power electronic circuits. Meanwhile, it
highly suffers from chattering phenomena. Nowadays, power inverters are utilised in connecting renewable
energy sources for power generation. Single-phase inverters are
well-known and used in various industrial applications. A DC–
AC H-bridge voltage source inverter (VSI) topology is selected
here to feed different linear loads [1, 2]. The generated sinu-
soidal waveform of an inverter must have a constant amplitude
and frequency with a minimal rate of THD. To limit the THD
level, various filters are suggested including LLCL, L, LC, and
LCL. In fact, characteristics of the LC-type filter, make it can
be the most suitable one for stand-alone structures (load con-
nected) [3–6]. Stability proof of the filter is the primary concern
which can harm system dynamics; thus, two strategies were pre-
sented for this challenge: passive and active damping schemes. In passive damping strategy, the inductor is modelled with a
series resistor, while total system efficiency can be affected. To address these issues, model predictive control (MPC) has
attracted a significant importance over the last decades. The
fundamental theory behind MPC is the horizon scheme which
tries to calculate a control signal by optimising process using
future time steps. As a result, physical constraints applied on
state variables are dealt with in an appropriate behavior. In
refs. [15–19] , a general review is reported based on MPC for
switching power converters. Furthermore, two types of MPCs However, for the second scheme, a closed-loop control
strategies have been introduced [7]. However, the following IET Control Theory Appl. 2022;1–11. 2 MEMEGHANI ET AL. FIGURE 1
The topology of the H-bridge inverter; (a) the first switching
mode and (b) the second switching mode are illustrated as continuous control set MPC (CCS-MPC)
and finite control set MPC (FCS-MPC) approaches. It is pre-
sented that in FCS-MPC, the cost function and system states
are predicted regarding switching conditions driven for the
following period. 1
INTRODUCTION g
g
p
y
p
Different approaches have been presented to tackle online
optimisation issue with a fast calculation time and better pre-
diction horizons [28–30]. These papers provide online optimi-
sation strategies of MPC for different converters with less cal-
culation time and a prediction horizon of around 400 to 600
𝜇s. Despite better results presented by these papers compared
with FCS-MPC or E-MPC alternatives, they are not entirely
satisfying. Generally, these methods are generally introduced
to improve performance of predictive controller by optimis-
ing their cost function and horizon predictions with a robust
dynamic against different disturbances. The method used here
is a generalised predictive control (GPC) which is classified as
CCS-MPC sub type and owns some significant merits in pre-
dictive controllers [31]. Most of the MPC structures mentioned
before suffer from a high computational burden, and a limited
prediction horizon followed by their unpopularity in industrial
applications since the control effort and the cost of implanta-
tion can increase significantly. Furthermore, the number of pre-
diction horizons are raised in this method to improve the per-
formance of the GPC against disturbances [32] . To provide
simpler structures, model-free methods have been introduced,
using system identification to get the system dynamic without
knowing the exact dynamic. ∙The need for a mathematical model of the inverter is removed
resulting in less computational burden and faster dynamics,
particularly in ill-defined systems. ∙This controller does not need a predefined cost function
to be minimised at each sampling time (Ts) which is called
“end-to-end technique”; the number of prediction horizons
are also increased to provide better disturbance rejection
responses. ∙The level of THD has been reduced considerably to about
0.5%. ∙Good parametric estimation is achieved by the system identi-
fication method. ∙The efficiency of the controller is reaffirmed in experimental
and simulation environments. 1
INTRODUCTION FCS-MPC yields fast and robust closed-loop
dynamics; yet, it is limited in prediction horizon based on
the significant growth of prediction horizon length with the
switching combinations in the range of one or two prediction
steps [20–22]. In most cases of CCS-MPC, pulse width modu-
lation (PWM) scheme is used to generate a control signal based
on the duty cycle [23, 24]. On-line optimisation of the system
is the primary concern of this strategy handled by explicit MPC
(EMPC) [25–27]. EMPC produces some feedback control laws
and a tree search to reach the state feedback laws, requiring a
large storing capacity and short prediction horizons. FIGURE 1
The topology of the H-bridge inverter; (a) the first switching
mode and (b) the second switching mode This work’s focuses is based on designing an online GPC
for a H-bridge inverter, which is improved in dynamic perfor-
mance by exponential regressive least-square (IERLS) identifi-
cation technique providing better performances in challenging
conditions. The contributions are presented in paper include: ∙An advanced control method with good performance with
excellent stability. g
g
p
y
p
Different approaches have been presented to tackle online
optimisation issue with a fast calculation time and better pre-
diction horizons [28–30]. These papers provide online optimi-
sation strategies of MPC for different converters with less cal-
culation time and a prediction horizon of around 400 to 600
𝜇s. Despite better results presented by these papers compared
with FCS-MPC or E-MPC alternatives, they are not entirely
satisfying. Generally, these methods are generally introduced
to improve performance of predictive controller by optimis-
ing their cost function and horizon predictions with a robust
dynamic against different disturbances. The method used here
is a generalised predictive control (GPC) which is classified as
CCS-MPC sub type and owns some significant merits in pre-
dictive controllers [31]. Most of the MPC structures mentioned
before suffer from a high computational burden, and a limited
prediction horizon followed by their unpopularity in industrial
applications since the control effort and the cost of implanta-
tion can increase significantly. Furthermore, the number of pre-
diction horizons are raised in this method to improve the per-
formance of the GPC against disturbances [32] . To provide
simpler structures, model-free methods have been introduced,
using system identification to get the system dynamic without
knowing the exact dynamic. 2
SYSTEM MODELLING G(z) = Y (z)
U (z) =
b0z + b1
z2 + a1z + a2z
(4) G(z) = Y (z)
U (z) =
b0z + b1
z2 + a1z + a2z
(4) (4) FIGURE 2
Inverter open loop frequency response Furthermore, to estimate b1, b0, a2, and a1 parameters, an iden-
tification method is used that has been explained in more details
in the following sections. where X =
[
i
vc
]
;
A =
⎡
⎢
⎢
⎢⎣
−R1
L
−1
L
1
C
−1
RC
⎤
⎥
⎥
⎥⎦
C =
[1
0]
; B =
⎡
⎢
⎢⎣
VDC
L1
0
⎤
⎥
⎥⎦
(3) X =
[
i
vc
]
;
A =
⎡
⎢
⎢
⎢⎣
−R1
L
−1
L
1
C
−1
RC
⎤
⎥
⎥
⎥⎦
C =
[1
0]
; B =
⎡
⎢
⎢⎣
VDC
L1
0
⎤
⎥
⎥⎦
(3) 3
GENERALISED MPC The GMPC method is a well-known MPC method since it con-
tains an excellent performance with a great robustness for both
academia and industry. The principle behind GMPC is based on
calculating a signal sequence of future control, while the goal is
to reach a minimum value of a multistage cost function depicted
on a prediction horizon [17]. In addition, the optimisation index
is the expectation of a quadratic functions calculating the differ-
ence between the predicted reference and the predicted system
outputs; also, this reference is obtained on the horizon, where
the control effort is measured by a quadratic function. The gen-
eral block diagram of GMPC is shown in Figure 3. (3) 2
SYSTEM MODELLING TABLE 1
System parameters
Parameters
Definition
Amounts
PWM frequancy
fw
10 KHz
DC voltage
VDC
20v
Filter inductor
L
100 μH
Leakage resistor
R1
1 Ω
Filter capacitor
C
1000 μF
Load
R
10 Ω
FIGURE 2
Inverter open loop frequency response
FIG
rela
tion
illus
this
que
dam
dam
F
tion
driv
Fur TABLE 1
System parameters
Parameters
Definition
Amounts
PWM frequancy
fw
10 KHz
DC voltage
VDC
20v
Filter inductor
L
100 μH
Leakage resistor
R1
1 Ω
Filter capacitor
C
1000 μF
Load
R
10 Ω
FIGURE 2
Inverter open loop frequency response
FIGURE 3
GPC block diagram
related to the zero-pole locations of the system, and these loca-
tions are defined by the amount of filter components. Figure 2
illustrates the impact of the resistor on the stability degree and
this system strongly relates to the rate of the resistor. Subse-
quently, assuming that the amount of this filter is zero, the
damping coefficient will be zero. This procedure provides a no-
damping system of sustained oscillation. For the digital GPC technique, a digital discrete transfer func-
tion of the proposed system is needed, and the zero-order hold
drives this duty. G(z) = Y (z)
U (z) =
b0z + b1
z2 + a1z + a2z
(4)
Furthermore to estimate b1 b0 a2 and a1 parameters an iden- FIGURE 3
GPC block diagram FIGURE 3
GPC block diagram TABLE 1
System parameters
Parameters
Definition
Amounts
PWM frequancy
fw
10 KHz
DC voltage
VDC
20v
Filter inductor
L
100 μH
Leakage resistor
R1
1 Ω
Filter capacitor
C
1000 μF
Load
R
10 Ω FIGURE 3
GPC block diagram related to the zero-pole locations of the system, and these loca-
tions are defined by the amount of filter components. Figure 2
illustrates the impact of the resistor on the stability degree and
this system strongly relates to the rate of the resistor. Subse-
quently, assuming that the amount of this filter is zero, the
damping coefficient will be zero. This procedure provides a no-
damping system of sustained oscillation. For the digital GPC technique, a digital discrete transfer func-
tion of the proposed system is needed, and the zero-order hold
drives this duty. 2
SYSTEM MODELLING To model the system, two operation modes are used and illus-
trated in Figure 1. This inverter consisted of four switches,
Q1–Q4, which turn off or on based on the PWM signal [1]. Additionally, the capacitor and the inductor are shown with C
and L. The coefficients of VDC and R are the source voltage
and the resistive load. When the switches Q4 and Q1 are on, it is
called the first mode as shown in Figure 1(a), while the Q3 and
Q2 are off. The second mode is the exact opposite condition for
the switches. The current controller designed for this topology
has the aim to regulate the current flow in the L. g
y
In refs. [33, 34], ANN-based controllers are proposed for var-
ious applications such as power converters. In these approaches,
the need for a mathematical model is removed since the whole
system is a black-box, and the performance of the controller
is optimised by a better parametric tuning. However, the main
drawback concerned with these types of methodologies is their
dependence on a wide range of training data. In ref. [35], a novel
robust identification is designed for an H-bridge Inverter that
uses the same black-box strategy to estimate the parameters
and produce the controller’s gains. The estimation process is
developed in this paper; meanwhile, providing an undesirable
parametric estimation can strongly influence the controller’s
responses. In addition, The proposed identification scheme in
this work uses the same black-box strategy presenting a sim-
ple structure, more accurate parametric estimation, and robust
dynamics. This estimator can help the controller to operate bet-
ter against disturbances; its larger pre-defined estimation limit
also ensures the stability of the closed-loop structure in chal-
lenging situations. The switching equations are obtained as Equation (1) using
the capacitor’s voltage and the inductor’s current. L(di)
dt
= VDCu −(R1)i + vc
C (dvc)
dt
= i −vcR
(1) (1) In addition, this system can be described by these linear state-
equations: 0 ≤d < 1,
u = 2d −1
̇X = Ax + Bu,
y = Cx
(2) (2) 3 MEMEGHANI ET AL. 4
RLS IDENTIFICATION PROCESS Mean-
while, because the power converters are electrical devices work-
ing in different applications, it is possible that various distur-
bances can have a negative impact on their dynamics. Compen-
sating these disturbing effects can be satisfied with an improve-
ment over the identification algorithm. (8) whereA(z−1)Δ = ˜A(z−1). The polynomials E j and Fj are illus-
trated with degrees j −1 and na , respectively. We obtain them
by dividing 1 by ˜A(z−1) until the remainder can be factorised
asz−1Fj (z−1). Therefore, E j (z−1)and Fj (z−1) are illustrated as
follows: E j
(
z−1)
= e j,0 + e j,1z−1 + ⋯+ e j, j−1z−( j−1)
Fj
(
z−1)
= f j,0 + f j,1z−1 + ⋯+ f j,naz−na
(9) Thus, to adopt this development, a factor called 𝜆is applied
to the RLS estimator capable of expanding the weighing func-
tion. Two main benefits can be provided by this factor: the range
of parametric estimation is widened, resulting in more accurate
results and regular update of parametric estimation considering
the latest alterations. Equation (15) illustrates this algorithm, so-
called the “forgetting factor algorithm”. (9) The best prediction of y(t + j) is y(t + j) = ΔG j (z−1)U ( j + t −1) + Fj (z−1)y(t )
(10) (10) U (t ) represents future horizons of the input signal, and the
term of G j (z−1) is shown as E j (z−1)B(z−1). However, the sum
of the past output term Fj (z−1) is called free response, f , and
system response to future value is force response. One can reach
to the optimal control signal by minimising Equation (10). ˆ𝜃
(
z
)
= [𝜃(z −1)𝜙(z)T −y(z)]K (z) + ˆ𝜃
(
z −1
)
]
K (z) = (𝜆I + 𝜙T (z)P(k −1)𝜙(z))−1𝜙(z)P(k −1)
P(z) = P(k −1)K (k)𝜙T (z) −P(k −1)
(15) The best prediction of y(t + j) is The extra factor of 𝜆is called “time-varying forgetting factor”,
−j The extra factor of 𝜆is called “time-varying forgetting factor”,
which is defined by 𝜆= e
−j
Tf . In this definition, Tf and j are
time constant exponential forgetting and sampling cycle. Equa-
tion (15) shows two functional matrices of P(z)and K (z), while
there are known as adaption matrix and covariance matrix. 4
RLS IDENTIFICATION PROCESS below below y(t )A(z−1) = u(t )B(z−1) + C (z−1)(e(t ))
(Δ)
(6) (6) RLS identification technique is a well-known estimator owing to
its prominent benefits, namely: easy implementation, high effi-
ciency in real-time operations, dynamical adaptability, and low
memory capacity [35]. Equation (12) shows the cost function of
RLS technique. where Δ = 1 −z−1 and for white noise, the C is chosen as 1. The GPC strategy contains of applying a control sequence that
minimises a multistage cost function of the form J = 1
N
N
∑
n=1
eT (z)e(z)
e(z) = y(z) −ˆy(z)
(12) J(N2, N1, Nu) =
Nu
∑
j=N1
𝛿( j)[
ˆy(t + j|t ) −𝜔( j + t )
]2
+
Nu
∑
j=1
[
Δu( j + t −1)]
2𝜆( j)
(7) (12) (7) In Equation (12), e(z) shows the error function where Equa-
tion (13) depicts the discrete digital transfer function. In Equation (16), output system prediction over the optimum j
step is shown asˆy(t + j|t ) , the control horizon is Nu , 𝜆( j), and
𝛿( j) are weighting sequences, N2 and N1 are the maximum and
minimum costing horizons, and 𝜔(t + j) is the future reference
trajectory. Computing the future control sequence ,u(t ), u(t +
1), …, is the purpose of predictive control and must be done as
the future plant output y(t + j) gets close to 𝜔(t + j), which is
carried out by minimising J(N1, N2, Nu). The optimal predic-
tions of j ⩾N1and j ⩽N2are fundamental factors in optimis-
ing the cost function y(t + j). Equation (8) is the diophantine
equation: y(z) = −a1y(z −1) −a2y(z −2)
+ b0u(z −1) + b1u(z −2)
(13) (13) Equation (13) can help to reach the Equation (14). Equation (13) can help to reach the Equation (14). y(z) = 𝜙(z)𝜃(z)
𝜙(z) = [u(z −1), u(z −2), −y(z −2), −y(z −1)]
𝜃(z) = [b1, a1, b0, a2]T
(14) (14) 1 = E j (z−1)˜ΔA
(
z−1)
+ z−1Fj (z−1)
(8) Equation (14) shows two coefficients of 𝜙and 𝜃that are vectors
depicts the gains of estimation and observation. However, the
past data-driven of u and y can be held in the 𝜙. Alternatively,
this estimator tries to update the cost function repeatedly. 2.1
Analysis of the LC filter The sinusoidal current generated with the H-bridge con-
tains some harmful harmonics and oscillation which must be
smoothed for different operations. To compensate this phe-
nomenon, the proposed filter is applied to the inverter to atten-
uate this output. However, some challenges are proposed with
the filter and the most problematic issue is its stability insurance,
while ref. [36] has studied some factors about this issue. Table 1
displays the parameters for the system and the bode diagram of
the open-loop frequency response of the system is plotted by
these values in Figure 2. g
g
The single input single output (SISO) system is shown in
Equation (5). y(t )A(z−1) = u(t )B(z−1) + C (z−1)e(t )
(5) (5) Here A, B, and C are system’s polynomials, while u(t) is input,
y(t) is output and e(t) is noise. Also, this function is controller
auto-regressive moving-average (CARMA) model. Based on the
points discussed in ref. [18], an integrated CARMA model is the
most suitable alternative for many industrial applications with
the presence of non-stationary disturbances. This model is as where R1 is the leakage resistor, with an essential duty of con-
trolling the generated signal damping on the output sinusoidal
current. The operation of a closed-loop controller is strongly 4 MEMEGHANI ET AL. below
4 4
RLS IDENTIFICATION PROCESS The
following clarification is used to better describe the role of 𝜆in 𝜕j
𝜕U = 2(GT G + 𝜆I )U + 2GT ( f −w)𝜆
(11) (11) which is defined by 𝜆= e Tf . In this definition, Tf and j are
time constant exponential forgetting and sampling cycle. Equa-
tion (15) shows two functional matrices of P(z)and K (z), while
there are known as adaption matrix and covariance matrix. The
following clarification is used to better describe the role of 𝜆in where U = (w −f )(GGT + 𝜆I )1GT . In (11), f represents the
free response of the system, 𝜆is the weighting factor and w is
the reference trajectory. 5 MEMEGHANI ET AL. TABLE 2
Estimation results with RLS identification strategy
Parameter
Mathematical amounts
Real-time values
a1
−1.75
−1.68
a2
0.75
0.69
b0
0.019
0.0178
b1
−0.019
−0.0181 Equation (17) demonstrates that, the estimated values of 𝜃with
all eigenvalues of one and P matrix with all eigenvalues of 1
𝜆,
are unstable. Consequently, to clarify this condition, estimator
ending situation, the following assumption is presented: first,
estimation is constant ; if 𝜆< 1, P matrix will rise swiftly; also, a
large variation will occur. Thus, to compensate these outcomes,
a method is introduced known as conditional updating. Further-
more, in this new criterion the estimation regarding covariance
matrices is being updated under actuation. Finally, the estima-
tion procedure is enhanced by this factor that tries to update
the pattern of covariance matrix under the occurrence of Equa-
tion (18), which can hinder this issue [37, 38]. Equation (15): if the term of 1 > 𝜆is reached, the rate of K (z)
and P(z) can be high. The primary benefit of this algorithm is
the constant strength that occurred in tracking and compensat-
ing the variations that appeared in the system dynamics. This
developed algorithm with 𝜆is called “Exponential RLS” algo-
rithm or ERLS. A range is introduced in Equation (16) to dedi-
cate the rate of 𝜆. 𝜙(t )T P(t )𝜙(t ) > 2(−𝜆+ 1)
(18) (18) A novel method is used here to handle this issue which
develops the covariance matrix that is “fixed matrix rejection
method”. Subsequently, applying this method needs bounding
of the coefficients of covariance matrix resulting in robust per-
formances over the ratios of estimator. 4.1
Identification procedure (19) Table 2 depicts the estimated values in the mathematical and
practical environments for both working modes. An assumption of c1 > 0 and 0 ≤c2can be considered. The
parameters are selected as Equation (20): An assumption of c1 > 0 and 0 ≤c2can be considered. The
parameters are selected as Equation (20): It can be seen that there is a minimal difference between these
values which can appeared based on the real-time disturbance
such as components’ tolerance or aging. c1
c2
≈104
𝜙(k)T 𝜙(k)c1 ≫1
(20) (20) 4
RLS IDENTIFICATION PROCESS In addition, exerting this
technique in the estimator can hinder the exponential rise of
covariance matrix when the regressive matrix is constant. Equa-
tion (19) depicts this new improved method. 𝜆(t ) = min[𝜆(t −1)𝜆0 + (−𝜆0 + 1)] , 1 −𝜀2
Σ0
(16) (16) Assuming 𝜆as the initial rate, the following procedure can be
defined for Equation (16): assuming the system is in the steady-
state condition, 𝜀can be low or zero while, this can stimulate
the 𝜆to be one or excess it. On the other hand, the level of 𝜀
goes up when some disturbances appear in the system leading a
significant drop over the 𝜆. This process influences on the K (z)
and P(z) matrices which is followed by an improvement on the
parametric estimation of the identification method. ˆ
𝜃(z) =
[
y(k) −𝜙(k)T 𝜃(−1 + z)
]
K (k) + ˆ𝜃(−1 + k)
K (k) = 𝜙(z)P(−1 + z)[𝜆I + 𝜙T (z)𝜙(z)P(−1 + k)]−1
P(k) = 1
𝜆[P(−1 + k) −(𝜙(z)P(−1 + k)𝜙(k)T P(−1 + k)
I + 𝜙T (z)P(−1 + k)𝜙(z))
P(k) = c1
P(k)
trP(k)
+ c2I
(19) ˆ
𝜃(z) =
[
y(k) −𝜙(k)T 𝜃(−1 + z)
]
K (k) + ˆ𝜃(−1 + k)
K (k) = 𝜙(z)P(−1 + z)[𝜆I + 𝜙T (z)𝜙(z)P(−1 + k)]−1
P(k) = 1
𝜆[P(−1 + k) −(𝜙(z)P(−1 + k)𝜙(k)T P(−1 + k)
I + 𝜙T (z)P(−1 + k)𝜙(z)) P(k) = c1
P(k)
trP(k)
+ c2I
(19) 4.2
Improved ERLS identification method In this section, a robust identification algorithm is suggested
to surpass the undesired influences of disturbances, particularly
noise. As discussed before, since the error scale is powered by
two in the least square formula, it is sensitive to a simple big
error. This characteristic of RLS provides an important condi-
tion: a rapid error rise will happen if the value of error becomes
large, which diverts estimator from correct parametric values. Thus, a developed identification algorithm has been introduced
to make the system robust against disturbances. The boundary
suggested for this process is working as anti-saturation proce-
dure that keeps the estimated parameters in the desired limit
appropriate for wide range of disturbances. The term of 𝜆, time variable feature, added to the RLS algorithm
performs as a speed convergence adjuster in the dynamical per-
formances. However, if the value of 𝜆is minimal, the paramet-
ric estimation can be fast; meanwhile, the negative impact of a
significantly large disturbance on the system can increase con-
sistently over the longer operations. The algorithm updated by
𝜆is Exponential RLS or “ERLS” method. Another issue asso-
ciated with this technique is its poor performance in unactuated
conditions. In this condition, the regressive matrix is zero and
the following estimation relationships are reached: P(t + 1) = 1
𝜆P(t )
𝜃(t + 1) = 𝜃(t )
(17) P(t + 1) = 1
𝜆P(t )
𝜃(t + 1) = 𝜃(t )
(17) A general comparison is done in Figure 4 based on the per-
formance of these identification techniques in different scenar-
ios to observe their outcomes in challenging conditions. (17) 𝜃(t + 1) = 𝜃(t ) 6 6 MEMEGHANI ET AL. FIGURE 4
Wide comparison between the proposed identification methods; (a) convergence to estimated parameter with the value of −1.745, (b) tracking
results under parametric variation, (c) tracking results under 0.01 variance noise FIGURE 4
Wide comparison between the proposed identification methods; (a) convergence to estimated parameter with the value of −1.745, (b) tracking
results under parametric variation, (c) tracking results under 0.01 variance noise Considering Figure 4(a) all the proposed techniques are
showing an excellent convergance to the value of 0.91 ,while the
RLS depicts a minimal digression that cannot be problematic. The second case is under sudden parametric variation which in
the IERLS and ERLS strategies have fast and accurate tracking
,but the RLS technique shows total weakness in compensating
this disturbance. 4.2
Improved ERLS identification method In a practical application the negative impact
of noise is inevitable that is tested in Figure 4(c). In this situa-
tion, a 0.01 variance noise is injected to the structure, while the
performance of the RLS is completely unacceptable and the
ERLS tries to reach the ideal value in a longer period of time
that can be a serious issue for the controller to generate signal
for the GPC block. On the other hand, the IERLS technique is
performing with robust dynamics resulting in suitable estima-
tions that propose it as a proper alternative for industrial usages
in the presence of various harmful disturbances. operations. It should be noted that the tracking speed in IERLS
method is another considerable criterion that has been depicted
vividly in Figure 4 for different cases as a significant factor to
illustrate its robustness. 5
EXPERIMENTAL AND SIMULATION
RESULTS It should be noted that zero-order hold (ZOH) technique is
utilised to discrete the controller. Also, the PWM scheme is used
to fire the converter switches. To clarify this technique for bet-
ter understanding, one can assume that the PWM is a block that
gets the control signal and compare it with a triangular signal,
then a square wave is generated that has the role of actuating
the switches. In Figure 5a hardware real-time implementation
of converter is shown consisting of the power and control real-
isation, simultaneously. To better depict the validity of the proposed identification
strategies, a numerical convergence analysis is demonstrated in
the Table 3 considering different scenarios. Based on this detailed comparison in this table, RLS method
is not an appropriate alternative for challenging and practical
usages; however, ERLS is showing more robust dynamics with
better converging behaviour under disturbing conditions but its
tracking differences can result in some issues for the controller
in generating control signal. Consequently, IERLS method has
the most significant manner with the best possible convergence
results that make it a perfect option for practical and industrial 5.1 The Prototype Machine shown in Figure 5 was implanted to
analyse the accuracy of the GMPC for the topology. In addi-
tion, different segments and devices are included: Arduino
micro-processor (DUE type) is the core of this structure which
generates the control signal, Inverter is built based on the 7 7 MEMEGHANI ET AL. TABLE 3
Convergence analysis between the proposed identification schemes for the mathematical value of a1 = −1.75
Methods
No disturbance
Parametric
variation
Noise with
0.01 variance
Noise with
0.1 variance
Noise with
0.5 variance
Supply voltage
variation
RLS
−1.68
−1.32
−1.02
−0.86
−0.46
−0.94
ERLS
−1.73
−1.702
−1.55
−1.02
−0.98
−1.21
IERLS
−1.742
−1.739
−1.733
−1.7105
−1.69
−1.725 TABLE 3
Convergence analysis between the proposed identification schemes for the mathematical value of a1 = −1.75 The results are tested by the LC filter for the predictive con-
troller. Considering Figure 6(a), an excellent convergence to the
reference signal can be seen with the presented control method,
while the negative impact of sudden parametric variation is
totally compensated. Besides, Figure 6(b) illustrates the amount
of THD to be approximately 0.2% that is in a very low limit. As
noted earlier, a predictive controller uses prediction horizons to
track the reference signal. To illustrate this process, four predic-
tion horizons are shown for the output and the control signals
in Figure 6(c,b). FIGURE 5
The whole structure consisted of the topology and the
micro-processor GPC can work perfectly with an excellent convergence to
the reference input (Figure 6); however, the dynamics is not
fast enough and takes time to track the reference. On the other
hand, since this is electronic hardware, they are the possibilities
of load alterations and connection issues for the system. Hence,
GPC must be able to compensate physical and dynamical con-
strains. In Figure 7, the GPC scheme faced some load variations;
the experimental and simulation results depict a perfect perfor-
mance of GPC in overcoming load uncertainties. FIGURE 5
The whole structure consisted of the topology and the
micro-processor components listed in Table 3 with various loads to verify its per-
formance under challenging dynamical variations. 5.1 Furthermore,
a current sensor (LI25-P) is attached to the load to transfer the
output to the micro-processor to make the control signal by the
aid of Matlab∖Simulink; also, the model of diodes and switches
are IRF9630 and UF4007 and the switching frequency is in the
range of 20 KHz . The micro-processor tries to convert the
current measured by the sensor through its analogue-to-digital
block, then the average value generated in this process will be
used as the input of the control loop. 5.1.1
Results in different loads TABLE 4
Various loads
Loads
THD
Resistive load
%0.2
Resistive–inductive load
%0.38
Resistive–capacitive load
%0.41
TABLE 5
Estimated values using IERLS-GPC under 0.1 variance noise
Parameters
Mathematical
parameters
Real-time
parameters
a1
−1.75
−1.71
a2
0.75
0.72
b0
0.019
0.0175
b1
−0.0196
−0.0184 TABLE 5
Estimated values using IERLS-GPC under 0.1 variance noise
Parameters
Mathematical
parameters
Real-time
parameters
a1
−1.75
−1.71
a2
0.75
0.72
b0
0.019
0.0175
b1
−0.0196
−0.0184 TABLE 5
Estimated values using IERLS-GPC under 0.1 variance noise results reached by GPC, the level of THD in the output current
is significantly low with a nearly pure sinusoidal waveform. TABLE 6
A detailed analysis of output signal (R = 10 Ω)
Parameters
Simulation
Experiment
Peak–peak
9.98
10.2
Settling time
0.08s
1.45 s
RMS
3.57
3.42
Crest factor
2.65
2.9
Average THD
0.2%
0.28% TABLE 6
A detailed analysis of output signal (R = 10 Ω) Figure 8 justifies the robust performance of the GPC under
different loads. Meanwhile, a slight tracking difference is seen
for both loads that can be the result of DC bias on the system,
but the operation of the controller is still appropriate. Further-
more, the THD level of output current for linear loads is shown
in Table 4, verifying a very low value of THD. Another disturbance that has a harmful influence on the sys-
tem is noise, which is an inevitable factor. To observe the track-
ing results of control strategies in noisy situations, a 0.01 vari-
ance noise is injected in Figure 9. Observing the results of Figure 9(a,b), the impact of 0.01 vari-
ance noise can lift the amplitude upward, which cannot make
difficulties for applications. Meanwhile, an increase in the vari-
ance of noise to 0.1 can result in a minor digress in Figure 9(c,d). This noise increases the converging amplitude of the control
loop which is capable of making serious issues for the structure. Table 4 displays the estimated parameters by an estimator under
influence of the noise. To better examine the merits of this work
compared to other methods, an analytic review is reported in the
next section (Table 5). The values in Table 6 are showing that the generated signal
has good range of additional criteria considering the operation
of the controller. However, power efficiency is another effective
parameter that is discussed in the following section. 5.1.1
Results in different loads Different linear loads are connected to the Inverter, and
Figure 8 shows the output current connected to the Resistive–
capacitive load and the Resistive–inductive load. Regarding the FIGURE 6
Controller’s results connected to the inverter a resistance load; (a) GPC performance with additive parametric variations, (b) THD rate, (c) four
prediction horizons of the control signal, (d) four prediction horizons of the output signal FIGURE 6
Controller’s results connected to the inverter a resistance load; (a) GPC performance with additive parametric variations, (b) THD rate, (c) four
prediction horizons of the control signal, (d) four prediction horizons of the output signal 8 MEMEGHANI ET AL. FIGURE 7
The performance of the GPC in load disconnections (10 Ω); (a) simulation results under the sudden load disconnection and connection with load
variations, (b) experimental result for sudden load removal, (c) experimental result for sudden load connection, (d) experimental result for load variation of 30 Ω, (e)
experimental result for load variation of 50 Ω FIGURE 7
The performance of the GPC in load disconnections (10 Ω); (a) simulation results under the sudden load disconnection and connection with load
variations, (b) experimental result for sudden load removal, (c) experimental result for sudden load connection, (d) experimental result for load variation of 30 Ω, (e)
experimental result for load variation of 50 Ω FIGURE 8
Simulation and experimental outputs with GPC for different loads. (a) Simulation result in resistive-inductive load (R = 30 Ω, L = 10 μH), (b)
experimental result in resistive-inductive load (R = 30 Ω, L = 10μH), (c) simulation result in resistive-capacitive load (R = 50 Ω, C = 100μF), (d) experimental
result in resistive-capacitive load (R = 50 Ω,C = 100 μF) FIGURE 8
Simulation and experimental outputs with GPC for different loads. (a) Simulation result in resistive-inductive load (R = 30 Ω, L = 10 μH), (b)
experimental result in resistive-inductive load (R = 30 Ω, L = 10μH), (c) simulation result in resistive-capacitive load (R = 50 Ω, C = 100μF), (d) experimental
result in resistive-capacitive load (R = 50 Ω,C = 100 μF) 9 MEMEGHANI ET AL. ORCID SeyyedMortezaGhamari
https://orcid.org/0000-0001-5082-
820X SeyyedMortezaGhamari
https://orcid.org/0000-0001-5082-
820X The design of a GPC on single-phase full-bridge inverter is pre-
sented by this work using an LC filter to control the current feed
to the load. The efficiency of the controller has been tested by
simulation and experimental results. This predictive algorithm
is classified as CCS-MPC, which needs an accurate parametric
estimation. Moreover, to fulfill this requirement, a novel identi-
fication technique is used called IERLS method showing more
robust dynamics under disturbances. Another primary concern
about inverters is to minimise THD on the output signal, which
is done here in a perfect way to around 0.5%. This structure
was built using hardware and real-time components, demon-
strating the robust and perfect performance of the controller DATA AVAILABILITY STATEMENT
Data available on request from the authors DATA AVAILABILITY STATEMENT
Data available on request from the authors 5.1.2
Power efficiency Control
technique
Under load
disturbances
Under
Noise
THD
%
Optimisation
[8]
SMC
VG
−
3.8
Of
[9]
SMC
G
−
2.5
Of
[10]
DBC
VG
−
3.96
Of
[11]
IMC
VG
−
1.65
Of
[12]
IMC
G
−
1.04
Of
[13]
MPC
VG
−
1.7
On
[16]
MPC
VG
−
2.08
Of
[17]
FCS-MPC
VG
−
3.25
Of
[18]
FCS-MPC
G
−
1.04
Of
[19]
FCS-MPC
VG
−
2.3
Of
[20]
FCS-MPC
VG
−
2.98
On
[21]
FCS-MPC
VG
−
1.21
On
[22]
OSV-MPC
VG
−
3.11
Of
[23]
CCS-MPC
VG
−
1.32
Of
[25]
CCS-MPC
VG
−
1.8
Of
[28]
CCS-MPC
G
−
1.49
Of
[29]
GPC
VG
−
3.79
On
[30]
NNC
VG
−
4.1
On
[32]
NNC
G
−
5.5
On
[33]
STR-AC
VG
G
1.14
On
This
Work
GPC
VG
VG
0.5
On TABLE 7
The efficiency measured based on the structure
Iout
Measured
efficiency (%)
Calculated
efficiency (%)
1A
95.3
96.64
2A
95.11
96.32
3A
94.21
94.81
4A
92.71
93.81 TABLE 7
The efficiency measured based on the structure TABLE 8
Comparison. (Good=G, Very Good=VG, On-line=On,
Off-line=Of) where pL is inductor conduction loss, prDS is MOSFET con-
duction loss, and prC is loss of power of the capacitor, while the
details about these factors are given by ref. [39]. Based on the
output power of system, one can reach the total efficiency of
the structure with the presented equation: 𝜂=
Po
Po + PTotal lossess
(22) Considering the factors given in these equations, the level of
efficiency of this structure is given in Table 7. It should be
noted that as the level of duty cycle increases, the total efficiency
decreases, respectively. 5.2
Comparison with the latest approaches To reaffirm the better results of the designed control technique,
Table 8 shows a comparison which is done between the pro-
posed controller and conventional approaches. Consequently,
three main aims are highlighted to be considered in this com-
parison as follows: THD value, robustness against disturbances,
and robustness against noise. under harmful disturbances. Overall, the proposed GPC pro-
vides many benefits in the area of MPCs, with a simple struc-
ture and robust dynamics under vaporous disturbances can be
the most significant ones. As it is depicted in Table 8, it is vivid that robustness and
THD rate for most of the literature are in a perfect perform-
ing limit; however, the proposed strategy has the least amount
of THD, and it has been tested in noise situations showing a
significant result. In refs. [40–42], the IEEE standard for THD
rate is studied thoroughly and it is clear that the THD rate for
different load in this work is in a very good limit of 0.4%. CONFLICT OF INTEREST The authors declare no conflict of interest. The authors declare no conflict of interest. 5.1.2
Power efficiency The overall losses of the converter can be calculated by the pro-
posed equation: Also, a comparison table is depicted here based on the output
sinusoidal signal of the structure feed to resistive load to better
identify the superiority of this method: (21) PTotal losses = PL + PrDS + PrC + other losses
(21) FIGURE 9
Outputs under the negative impact of noise (10 Ω). (a) GPC simulation result with applied 0.01 noise variance, (b) GPC experimental result with
applied 0.01 noise variance, (c) GPC simulation result with applied 0.1 noise variance, and (d) GPC experimental result with applied 0.1 noise variance FIGURE 9
Outputs under the negative impact of noise (10 Ω). (a) GPC simulation result with applied 0.01 noise variance, (b) GPC experimental result with
applied 0.01 noise variance, (c) GPC simulation result with applied 0.1 noise variance, and (d) GPC experimental result with applied 0.1 noise variance 10 10 MEMEGHANI ET AL. TABLE 7
The efficiency measured based on the structure
Iout
Measured
efficiency (%)
Calculated
efficiency (%)
1A
95.3
96.64
2A
95.11
96.32
3A
94.21
94.81
4A
92.71
93.81
where pL is inductor conduction loss, prDS is MOSFET con-
duction loss, and prC is loss of power of the capacitor, while the
details about these factors are given by ref. [39]. Based on the
output power of system, one can reach the total efficiency of
the structure with the presented equation:
𝜂=
Po
Po + PTotal lossess
(22)
Considering the factors given in these equations, the level of
efficiency of this structure is given in Table 7. It should be
noted that as the level of duty cycle increases, the total efficiency
decreases, respectively. 5.2
Comparison with the latest approaches
To reaffirm the better results of the designed control technique,
Table 8 shows a comparison which is done between the pro-
posed controller and conventional approaches Consequently
TABLE 8
Comparison. (Good=G, Very Good=VG, On-line=On,
Off-line=Of)
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Powered single hip joint exoskeletons for gait rehabilitation: a systematic review and Meta-analysis
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Open Access Open Access Powered single hip joint exoskeletons
for gait rehabilitation: a systematic review
and Meta‑analysis Mahla Daliri1 , Mohammad Ghorbani1,2 , Alireza Akbarzadeh3 , Hossein Negahban1,4, Mohammad
H Ebrahimzadeh1, Elham Rahmanipour1,2 and Ali Moradi1* Mahla Daliri1 , Mohammad Ghorbani1,2 , Alireza Akbarzadeh3 , Hossein Negahban1,4, Mohammad
H Ebrahimzadeh1, Elham Rahmanipour1,2 and Ali Moradi1* © The Author(s) 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://creativecom-
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background Gait disorders and as a consequence, robotic rehabilitation techniques are becoming increasingly
prevalent as the population ages. In the area of rehabilitation robotics, using lightweight single hip joint exoskeletons
are of significance. Considering no prior systematic review article on clinical outcomes, we aim to systematically
review powered hip exoskeletons in terms of gait parameters and metabolic expenditure effects. Methods Three databases of PubMed, Scopus, and Web of science were searched for clinical articles comparing
outcomes of gait rehabilitation using hip motorized exoskeleton with conventional methods, on patients with gait
disorder or healthy individuals. Of total number of 37 reviewed articles, 14 trials were quantitatively analyzed. Analyses
performed in terms of gait spatiotemporal parameters like speed (self-speed and maximum speed), step length, stride
length, cadence, and oxygen consumption. Results Improved clinical outcomes of gait spatiotemporal parameters with hip joint exoskeletons are what our
review’s findings show. In terms of gait values, meta-analysis indicates that rehabilitation with single hip joint exo-
skeleton enhanced parameters of maximum speed by 0.13 m/s (0.10–0.17) and step length by 0.06 m (0.05–0.07). For the remaining investigated gait parameters, no statistically significant difference was observed. Regarding
metabolic parameters, oxygen consumption was lower in individuals treated with hip exoskeleton (− 1.23 ml/min/kg;
range − 2.13 to − 0.32). Conclusion Although the analysis demonstrated improvement with just specific gait measures utilizing powered hip
exoskeletons, the lack of improvement in all parameters is likely caused by the high patient condition heterogeneity
among the evaluated articles. We also noted in patients who rehabilitated with the hip exoskeleton, the oxygen cost
was lower. More randomized controlled trials are needed to verify both the short- and long-term clinical outcomes,
including patient-reported measures. Level of evidence Level I (systematic review and meta-analysis). Keywords Hip, Powered, Exoskeleton, Gait, Rehabilitation, Clinical outcomes *Correspondence:
Ali Moradi Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80
https://doi.org/10.1186/s12891-024-07189-4 Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80
https://doi.org/10.1186/s12891-024-07189-4 BMC Musculoskeletal
Disorders Protocolh This systematic review and meta-analysis was conducted
based on the Preferred Reporting Items for Systematic
Reviews and Meta-Analysis: the PRISMA Statement [25]. © The Author(s) 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://creativecom-
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 2 of 15 Daliri et al. BMC Musculoskeletal Disorders Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Introduction human walking lowers the risk of strokes and coronary
heart disease, improving both psychological and physical
well-being [20]. Gait disorders become more common as population
ages, increasing from 30% of adults 60 years and older to
more than 60% of people over the age of 80 [1, 2]. Gait
disorders may originate from neurologic problems (e.g. sensory or motor impairments like stroke or spinal cord
injuries), orthopedic disorders (e.g. osteoarthritis or skel-
etal deformities) or other general medical conditions (e.g. cardiovascular diseases, pulmonary diseases, obesity or
psychiatric disorders) [3–5]. Gait and balance problems
contribute to poor quality of life and increased morbidity
and mortality in the elderly [2]. Although there are medi-
cal and surgical managements for gait disorders, ambu-
latory devices are the only option left in most cases [6],
and previous studies supported that continues locomotor
activity improves patients’ condition [7, 8]. Today, the exoskeletons’ technology progress have been
studied and reviewed comprehensively [21–23]. Regard-
ing powered hip robots, there are some reviews in terms
of design and control [22, 24]. However, we found no
comprehensive review in terms of clinical gait outcomes
with powered single hip joint exoskeletons (PSHJE). For a
better comprehension of this technology practical appli-
cations, we extensively summarize the 1) clinical out-
comes including gait spatiotemporal parameters and 2)
metabolic cost on walking-assist hip exoskeletons in this
systematic review. Searching strategy
l An electronic search was conducted in the databases
PubMed/MEDLINE, Scopus and ISI Web of Science
from inception until August 31th, 2022. The search state-
ment was: ((“robot*” OR “exoskelet*” OR “automatic
orthos*” OR “powered orthos*”) AND (“hip” OR “coxa*”
OR “acetabul*”) AND (“gait” OR “walk” OR “rehab*” OR
“mobil*”). The references of retrieved articles were also
checked to find possibly relevant studies. We refined our
search by available English language abstract and article
document type. Other filters were not applied. Grey lit-
erature was not checked in this study. Materials and methods
Protocolh Exoskeletons are external devices worn with the aim
of rehabilitation or replacement for lost physical func-
tions and integrate the human intelligence with robotic
power [9]. In the past decade, several lower limb exoskel-
etons (LLEs) have been developed such as ReWalk [10],
Honda Walking Assist® (HWA) [11], Stride Manage-
ment Assist (SMA), Gait Enhancing Mechatronic System
(GEMS), Hybrid Assistive Limb (HAL) [12] and Vander-
bilt [13]. However, there is still a lot to do in development
and application of LEEs, such as control, actuators, and
humane-machine interface (HMI) optimization. By pro-
viding almost consistent torque profiles throughout the
rehabilitation process, which cannot be accomplished
by manual assistance or verbal feedback from the physi-
otherapist, powered exoskeletons help increase treatment
reproducibility. Additionally, by integrating individual-
ized parameters, the device power can be adjusted to
target particular gait impairments in a regulated man-
ner. Using these devices relieves therapists of huge bur-
den while providing various personalized task-specific
practice in novel dynamic environments, and they allow
continuous monitoring of patients’ performance and pro-
gression [14]. The employment of light single-joint exo-
skeletons for gait training has recently become a trend
in the field of rehabilitation robotics [15]. Motorized hip
exoskeletons appear to favorably enhance the rehabilita-
tion of stroke survivors and other patients with restricted
movement capacities by enhancing gait spatiotempo-
ral parameters, metabolic economy, and biomechanical
quality [16, 17]. Additionally, patients with neurologi-
cal conditions brought on by disorders or trauma like a
stroke or spinal cord injury frequently have weak mus-
cles, which could result in inadequate force or torque
at the hip joints during limb movements [18], specially
that due to the different muscle properties, the hip joint
has a higher metabolic expenditure for the generation of
equivalent mechanical joint power [19]. Fully functioning Data extraction and quality assessmenth Three reviewers separately collected data from full texts
of included studies using a pre-designed Excel form. Results were compared and double checked by the same
reviewers. The data extracted included: author, year,
study design, sample size, disorder, mean age, sex ratio,
disease type, robot name, self-speed (self-selected com-
fortable speed), max speed, cadence, step length, stride
length and metabolic cost. The methodological quality
and validity of each included study was evaluated using
the JBI critical appraisal checklists for clinical trials and
single group studies (Tables 1 and 2). When rehabilitating with Honda Walking Assist (HWA),
patients with walking difficulty showed augmented
motivation as measured by Intrinsic Motivation Inven-
tory (IMI) [29]. Implementing on 50 chronic stroke
patients, powered hip exoskeleton could enhance gait
clinical outcomes (endurance (P .033), balance (P 0.036),
step count (P 0.013) etc.), compared with the functional
training [17]. Patient-reported outcomes of balance con-
fidence and falls efficacy also improved with hip exo-
skeleton [17]. Assistance timing increases, step length,
cadence, and walk ratio (i.e. step length/cadence ratio)
all increase with Gait Enhancing Mechatronic System
(GEMS) [30]. Healthy elderly patients showed no dif-
ference in stair climbing cadence while using GEMS or
not [31]. Studying hip exoskeleton in comparison with Eligibility criteria and study selection
Th The present systematic review screened all studies that
met the PICOD protocol requirements: P (Problem):
gait disorder; I (Intervention): rehabilitation with PSHJE;
C (Comparison): conventional rehabilitation; O (Out-
comes): gait spatiotemporal parameters; and D (design):
controlled or pre-post clinical trial. All the search results
were imported to Endnote X9 citation manager and
duplicate studies were removed. Two reviewers (M. D,
M. Gh) independently screened the titles and abstracts
of studies to select relevant ones. Disagreements were
resolved by consensus. Studies evaluating the outcomes
of using powered single hip joint exoskeleton (PSHJE)
robots (Fig. 1) in patients with gait disorders (stroke,
amputees, joint arthroplasty, elderly, cerebral palsy, etc.)
were included in this review. All abstracts, review arti-
cles, case-reports, case-series, letters to editors, pro-
totypes and animal studies were excluded. Additional
exclusion criteria were studies dealing with powered Page 3 of 15 Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Daliri et al. BMC Musculoskeletal Disorders Fig. 1 The powered single hip joint exoskeleton (PSHJE) robots
developed in FUM CARE, named HEXA. The robot is portable
and weighs less than 4 kg and muscle activity. Forest plots were depicted to assess
for heterogeneity and calculate the pooled weighted
mean difference (WMD) with corresponding 95% confi-
dence intervals for visual inspection across studies. Due
to conceptual heterogeneity, a random-effects meta-anal-
ysis was conducted to account for the heterogeneity of
the study populations. Pooled estimates with their corre-
sponding 95% CIs were calculated using inverse-variance
weights methods [26]. The I2 statistics was used to assess
the heterogeneity across studies (I2 = 0% indicates no
observed heterogeneity and I2 ≥ 50% indicates substan-
tial heterogeneity). Cochran’s Q statistic was also used to
analyze the statistical significance of heterogeneity [27]. Sensitivity analysis was performed to determine which
study (if any) had the largest impact on the heterogeneity
and to assess the robustness of pooled estimates. WMD
was plotted against the inverse of the square of the stand-
ard error. All statistical tests were two-tailed, and the
significance level was set at less than 0.05 for all, except
for the heterogeneity test. Statistical analyses were per-
formed using Stata version 17.0 (Stata Corp., College Sta-
tion, Texas, USA). Fig. 1 The powered single hip joint exoskeleton (PSHJE) robots
developed in FUM CARE, named HEXA. The robot is portable
and weighs less than 4 kg Study characteristics Finally, a systematic review and meta-analysis of 13 com-
parative clinical research, including 7 clinical trials and
7 single group trials was conducted. Four publications
assessed GEMS, four SMA, two HWA, and two Active
Pelvis Orthosis (APO). Each of the following was exam-
ined in a single paper: ALEX II exoskeleton, HAL lum-
bar type, and Robotic Assisted Rehabilitation Trainer
(RART). One study focused on pediatric patients [28],
while others investigated adults and the elderly (Table 3). exoskeletons on joints other than hip or whole lower
body robots, passive hip exoskeletons, and non-clinical
studies (Supplementary Figure). Whenever several stud-
ies were published from a trial with the same population,
only the results and data of the last study (provided that it
has the largest sample size, mentions accurate and com-
plete information of patients in depth, and a complete/
correct reporting of the outcomes) would meet the crite-
ria for our systematic review. Statistical analysis Our primary focus are clinical outcomes in terms of gait
spatiotemporal parameters. As the secondary outcomes
we reviewed metabolic and oxygen cost, hip joint angle, Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 4 of 15 Table 1 Quality assessment checklist for randomized controlled trials (Critical Appraisal tools for use in JBI Systematic Reviews)
N
Criteria
Jayaraman (2018) Ji (2018) Kawasaki
(2014)
Martini (2019) Tanaka (2017) Buesing (2015) Hwang-Jae
Lee (2019)
1
Was true randomization used
for assignment of participants
to treatment groups? Yes
Yes
Yes
Yes
Yes
Yes
Yes
2
Was allocation to treatment groups
concealed? Unclear
Unclear
No
Unclear
No
No
Unclear
3
Were treatment groups similar
at the baseline? Yes
Yes
NA
No
Yes
Yes
Yes
4
Were participants blind to treat-
ment assignment? No
No
No
No
No
No
No
5
Were those delivering treatment
blind to treatment assignment? No
Unclear
No
Unclear
No
No
Unclear
6
Were outcomes assessors blind
to treatment assignment? Yes
Unclear
No
Unclear
No
Yes
Unclear
7
Were treatment groups treated
identically other than the interven-
tion of interest? Yes
Yes
Yes
Yes
Yes
Yes
Yes
8
Was follow up complete and if not,
were differences between groups
in terms of their follow up ade-
quately described and analyzed? Yes
Yes
Yes
Yes
Yes
Yes
NA
9
Were participants analyzed
in the groups to which they were
randomized? Yes
Yes
Yes
Yes
Yes
Yes
Yes
10 Were outcomes measured
in the same way for treatment
groups? Yes
Yes
Yes
Yes
Yes
Yes
Yes
11 Were outcomes measured in a reli-
able way? Yes
Yes
Yes
Yes
Yes
Yes
Yes
12 Was appropriate statistical analysis
used? Yes
Yes
Yes
Yes
Yes
Yes
Yes
13 Was the trial design appropriate,
and any deviations from the stand-
ard RCT design (individual randomi-
zation, parallel groups) accounted
Yes
Unclear
No
Yes
Yes
Yes
Yes Table 1 Quality assessment checklist for randomized controlled trials (Critical Appraisal tools for use in JBI Systematic Reviews) Table 1 Quality assessment checklist for randomized controlled trials (Critical Appraisal tools for use in JBI Systematic Reviews) 11 Were outcomes measured in a reli-
able way? Yes 12 Was appropriate statistical analysis
used? Yes NA Not applicable enabled the production of joint moments that were closer
to the normal values (ICC = 0.80) [33]. Statistical analysis During a rand-
omized trial, the robotic trainer to control hip motions,
improved gait parameters of speed, cadence, and balance
in hemiplegic patients [34]. Koseki et al. conducted three
studies on Honda Walking Assistive device® (HWA) and
showed increased gait parameters (speed, step length and
cadence) in one subject with hip osteoarthritis and two
subjects with transfemoral amputation [11, 35]. They also
noted early gait improvement when waring HWA dur-
ing a clinical trial and a case report on total knee arthro-
plasty patients [36, 37]. HWA outcomes were similar on
one patient with spinal cord injury [38]. Elderly people
in the GEMS group showed enhanced gait performance,
reduced muscle effort, and lower metabolic expenditure
[39, 40], as well as patients with chronic stroke patients
[41]. Miura et al. reported enhanced balancing function treadmill on children with cerebral palsy (CP), showed
notable increase in walking speed (P < 0.05) [28]. In one
patient with spinal cord injury, powered hip exoskeleton
increased walking speed (0.24 to 0.31 m/s), step length
(0.38 to 0.41 m), cadence (37.5 to 44.81 step/min), and
decreased compensatory movements, when compared
with Isocentric Reciprocating Gait Orthosis (IRGO)
[32]. With relation to integrating orthotics along with
functional electrical stimulation (FES), reciprocal gait
orthosis (RGO) with a variable constraint hip mechanism
(VCHM) compared with IRGO on five able-bodied indi-
viduals, and stated that when hip controller was active,
hip kinematics was more similar to normal hip joints
(ICC = 0.96). There was no difference in terms of step
length, but IRGO resulted in walking speed closer to the
norms [33]. Compared to the IRGO (intraclass correla-
tion coefficient = .68), the VCHM with controller active treadmill on children with cerebral palsy (CP), showed
notable increase in walking speed (P < 0.05) [28]. In one
patient with spinal cord injury, powered hip exoskeleton
increased walking speed (0.24 to 0.31 m/s), step length
(0.38 to 0.41 m), cadence (37.5 to 44.81 step/min), and
decreased compensatory movements, when compared
with Isocentric Reciprocating Gait Orthosis (IRGO)
[32]. With relation to integrating orthotics along with
functional electrical stimulation (FES), reciprocal gait
orthosis (RGO) with a variable constraint hip mechanism
(VCHM) compared with IRGO on five able-bodied indi-
viduals, and stated that when hip controller was active,
hip kinematics was more similar to normal hip joints
(ICC = 0.96). NA Not applicable Statistical analysis There was no difference in terms of step
length, but IRGO resulted in walking speed closer to the
norms [33]. Compared to the IRGO (intraclass correla-
tion coefficient = .68), the VCHM with controller active Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 5 of 15 Table 2 Quality assessment checklist for single group and non-randomized clinical trials (Critical Appraisal tools for use in JBI
Systematic Reviews)
NA Not applicable
N
Criteria
Kitatani (2014) Koseki (2021) Kim (2018) Hwang-
Jae Lee
(2016)
Su-Hyun
Lee
(2017)
Lenzi (2013)
1
Were there clear criteria for inclusion in the case series? Yes
Yes
Yes
Yes
Yes
No
2
Was the condition measured in a standard, reliable way
for all participants included in the case series? NA
Yes
Yes
Yes
Yes
NA
3
Were valid methods used for identification of the condi-
tion for all participants included in the case series? NA
Yes
Yes
Yes
Yes
NA
4
Did the case series have consecutive inclusion of partici-
pants? Yes
Yes
Yes
Yes
Yes
No
5
Did the case series have complete inclusion of partici-
pants? Yes
Yes
Yes
Yes
Yes
No
6
Was there clear reporting of the demographics
of the participants in the study? Yes
Yes
Yes
Yes
Yes
No
7
Was there clear reporting of clinical information
of the participants? NA
Yes
Yes
Yes
Yes
No
8
Were the outcomes or follow up results of cases clearly
reported? Yes
Yes
Yes
Yes
Yes
Yes
9
Was there clear reporting of the presenting site(s)/
clinic(s) demographic information? Yes
Yes
Yes
Yes
Yes
Yes
10 Was statistical analysis appropriate? Yes
Yes
Yes
Yes
Yes
Yes Table 2 Quality assessment checklist for single group and non-randomized clinical trials (Critical Appraisal tools for use in JBI
Systematic Reviews) controller active enabled greater hip flexion compared to
the IRGO and provided smooth control of the hip joints
via context-dependent coupling [33]. A case report study
revealed that pneumatic artificial muscle (PAM) pow-
ered hip orthosis, operated via a voluntary activation
algorithm relies on the hip joint’s angular characteristics,
could be adjusted to provide a satisfying and comfort-
able application during the gait cycle and improved left
step transposition on the patient with polio [46]. Peak hip
and knee flexion angles improved (reduced) with robotic
trainer to control hip motions [34]. Muscle activity When adding hip joint paretic side corrective force to the
robotic treadmill on 15 post stroke subjects, increased
muscle activity and more symmetric hip movements
were observed [44]. Corticomotor excitability (CME)
corresponding to rectus femoris muscle in patients with
chronic stroke augmented with hip exoskeleton com-
pared with functional training (P 0.010). primary senso-
rimotor cortex (SMC) showed augmented activation in
patients with stroke, as revealed by infrared spectroscopy
[45]. Statistical analysis Limb symmetry and
maximum hip angle associate with hip exoskeleton assis-
tance and timing in children with spastic CP [28]. Maxi-
mum hip flexion decreased from 45.7 to 34.4 with HWA
in a patient underwent total hip arthroplasty (THA) [35]
but increased from 24 to 30 in two amputees [11]. There
is the report stating powered or unpowered conditions
when using hip exoskeleton are similar in terms of hip
moment pattern and despite having different hip joint
angles for a particular walking pace, people follow similar
joint moment patterns when walking [47]. variables, despite the fact that mobility function met-
rics like the 10MWT did not significantly improve after
Hybrid Assistive Limb (HAL) physiotherapy in patients
with locomotive syndrome [42]. Unlike control group,
applying stride management assist (SMA) with exoskel-
eton resulted in notable improvement post-training in
terms of maximum gait speed, paralysis-side step length,
symmetry, and cadence in subacute stroke [43]. At vari-
ous times, SMA group showed further advancements
across gait metrics [16]. Hip joint angles Physiologic cost index (PCI) decreased 20.5% while wear-
ing hip exoskeleton by an above knee amputee (P < 0.01)
[48]. The association between assistant timing of GEMS
hip exoskeleton and metabolic cost has shown maximum
21% reduction at 0% assistance timing compared with no
exoskeleton walking [30]. GEMS was studied on healthy Comparing powered hip exoskeleton with Isocentric
Reciprocating Gait Orthosis (IRGO), hip angles were
comparable to those shown by normal walking motions
while wearing this orthosis, but reduced compared to
normal gait with both orthoses [32]. The VCHM with Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 6 of 15 Table 3 Included studies’ basic characteristics
F/U follow up, RCT randomized clinical trial, NRCT non-randomized clinical trial, PSHJE powered single hip joints exoskeleton, SMA Stride Management Assist, HWA
Honda Walking Assist, RART Robotic Assisted Rehabilitation Trainer, GEMS Gait Enhancing and Motivating System, RAGT Robot-assisted gait training, APO Active Pelvis
Orthosis, HAL Hybrid Assistive Limb, NI Not indicated
N
Author (year)
Study type
Sample size
Condition
F/U (month)
Group (N)
Robot
Age
Sex ratio
(female
%)
1
Jayaraman (2018)
RCT
50
Chronic stroke
3
PSHJE (25)
SMA
59.5 ± 9.7
36
No PSHJE (25)
61.6 ± 12.6
32
2
Kitatani (2014)
Single group clini-
cal trial
10
Healthy
0
PSHJE
SMA
24.4 ± 3.5
50
No PSHJE
3
Koseki (2021)
NRCT
22
Knee arthroplasty
2
PSHJE (11)
HWA
71.8 ± 6.2
100
No PSHJE (11)
75.9 ± 6.9
90.9
4
Ji (2018)
RCT
16
Hemiplegic
2
PSHJE (8)
RART
48. Hip joint angles The motorized hip exoskeleton’s interface design
optimization and its effect on metabolic cost has been
studied, recently [50]. Studying the Active Pelvis Ortho-
sis (APO) on elderly, oxygen (4.24 ± 2.57%) and meta-
bolic (− 26.6 ± 16.1%) consumption reduced post-training
notably more than control group [51]. On patients with
lower limb amputation, motorized hip exoskeleton could elderly individuals walking stairs and revealed notable
decrease in oxygen consumption per unit mass (P 0.013),
metabolic power per unit mass (P 0.001) and metabolic
equivalents (P < 0.05) values [31]. When assisting hip
flexion and extension of healthy individuals, oxygen con-
sumption and heart rate reduced [49]. At a self-selected
speed, GEMS resulted in a 7 and 6.6% reduction in
oxygen consumption per unit and energy expenditure,
respectively (p 0.05) [39], and the net cardiopulmonary
metabolic energy cost was also decreased by 14.71% fol-
lowing the intervention in patients with chronic stroke
[41]. The motorized hip exoskeleton’s interface design
optimization and its effect on metabolic cost has been
studied, recently [50]. Studying the Active Pelvis Ortho-
sis (APO) on elderly, oxygen (4.24 ± 2.57%) and meta-
bolic (− 26.6 ± 16.1%) consumption reduced post-training
notably more than control group [51]. On patients with
lower limb amputation, motorized hip exoskeleton could reduce 15.6% of metabolic cost [52]. When studying the
effect of placing actuators on lower extremity joints, the
motors at the hip were mostly responsible for the lower-
ing of metabolic costs [53]. Hip exoskeleton from Sam-
sung GEMSv2 decreased metabolic cost by 13.5, 15.5 and
9.8%. (31.9, 51.6 and 45.6 W) at, 0, 5, and 10% surface gra-
dient, respectively [54]. Hip flexion and extension meta-
bolic costs were lowered by 9.7 and 10.3%, respectively,
in the optimized powered condition compared to the
unpowered condition [55]. Hip joint angles 85 ± 20.9
43.75
No PSHJE (8)
5
Kim (2018)
Single group clini-
cal trial
15
Healthy elderly
0
PSHJE
GEMS
74.33 ± 4.56
60
No PSHJE
6
Kawasaki (2020)
Cross-over RCT
10
Spastic CP chil-
dren
0
PSHJE
HWA (RAGT)
11.1 ± 2.3
60
No PSHJE
7
Hwang-Jae Lee
(2016)
Single group clini-
cal trial
30
Healthy elderly
0
PSHJE
GEMS
74.07 ± 4.14
50
No PSHJE
8
Su-Hyun Lee
(2017)
Single group clini-
cal trial
30
Healthy elderly
0
PSHJE
GEMS
74.10 ± 4.18
53.3
No PSHJE
9
Hwang-Jae Lee
(2019)
RCT
26
Chronic stroke
0
PSHJE (14)
GEMS
61.85 ± 7.87
50
No PSHJE (12)
62.25 ± 6.36
41.6
10
Lenzi (2013)
Single group clini-
cal trial
10
Healthy
0
PSHJE
ALEX II
NI
NI
No PSHJE
11
Martini (2019)
RCT
20
Elderly
1
PSHJE (10)
APO
70 ± 5
20
No PSHJE (10)
70 ± 4
70
12
Tanaka (2017)
RCT
41
subacute
stroke patients
with hemiplegia
0
PSHJE (21)
SMA
64.9 ± 12.2
38
No PSHJE (20)
62.3 ± 9.3
30
13
Buesing (2015)
RCT
50
Chronic stroke
3
PSHJE (25)
SMA
60 ± 2
32
No PSHJE (25)
62 ± 3
36 F/U follow up, RCT randomized clinical trial, NRCT non-randomized clinical trial, PSHJE powered single hip joints exoskeleton, SMA Stride Management Assist, HWA
Honda Walking Assist, RART Robotic Assisted Rehabilitation Trainer, GEMS Gait Enhancing and Motivating System, RAGT Robot-assisted gait training, APO Active Pelvis
Orthosis, HAL Hybrid Assistive Limb, NI Not indicated F/U follow up, RCT randomized clinical trial, NRCT non-randomized clinical trial, PSHJE powered single hip joints exoskeleton, SMA Stride Management Assist, HWA
Honda Walking Assist, RART Robotic Assisted Rehabilitation Trainer, GEMS Gait Enhancing and Motivating System, RAGT Robot-assisted gait training, APO Active Pelvis
Orthosis, HAL Hybrid Assistive Limb, NI Not indicated elderly individuals walking stairs and revealed notable
decrease in oxygen consumption per unit mass (P 0.013),
metabolic power per unit mass (P 0.001) and metabolic
equivalents (P < 0.05) values [31]. When assisting hip
flexion and extension of healthy individuals, oxygen con-
sumption and heart rate reduced [49]. At a self-selected
speed, GEMS resulted in a 7 and 6.6% reduction in
oxygen consumption per unit and energy expenditure,
respectively (p 0.05) [39], and the net cardiopulmonary
metabolic energy cost was also decreased by 14.71% fol-
lowing the intervention in patients with chronic stroke
[41]. Quantitative results (meta‑analysis)
Gait self‑speed (m/s) Analyzing six studies, gait self -speed increased 0.07 m/s
(− 0.01–0.15) on average, among patients rehabilitated
by PSHJE (Fig. 2A). Sensitivity analysis showed the
mean change of gait self -speed was consistent (range
of summary WMDs: 0.05–0.09), indicating that the Page 7 of 15 Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Daliri et al. BMC Musculoskeletal Disorders Table 4 Included studies’ outcomes regarding gait parameters
SS self-speed, MaxS maximum speed, PSHJE powered single hip joints exoskeleton
N
Author (year)
Group (N)
Speed (m/s) SS
Speed (m/s)
MaxS
Step length (m)
Stride length
(cm)
Cadence (step/
min)
Fugl-Meyer
(balance)
Metabolic (W/
kg)
Oxygen (ml/min/
kg)
1
Jayaraman (2018)
PSHJE (25)
0.95 ± 0.06
1.24 ± 0.05
21.4 ± 16.7
No PSHJE (25)
0.85 ± 0.04
1.12 ± 0.05
11.8 ± 16.6
2
Kitatani (2014)
PSHJE
1.06 ± 0.16
1.51 ± 0.15
0.13 ± 0.01
No PSHJE
1.07 ± 0.15
1.52 ± 0.13
0.15 ± 0.015
3
Koseki (2021)
PSHJE (11)
0.96 ± 0.17
1.20 ± 0.21
0.55 ± 0.08
104.1 ± 8.47
No PSHJE (11)
0.7 ± 0.29
0.90 ± 0.35
0.46 ± 0.11
87.53 ± 23.99
4
Ji et al. (2018)
PSHJE (8)
0.23 ± 0.11
52.75 ± 14.65
24 (18–32)
No PSHJE (8)
0.29 ± 0.10
55.63 ± 5.97
18 (16–22)
5
Kim et al. (2018)
PSHJE
53.25 ± 4.56
6.10 ± 1.15
17.43 ± 4.09
No PSHJE
53.92 ± 5.82
6.79 ± 1.18
19.07 ± 4.00
6
Kawasaki et al. (2020)
PSHJE
0.60 ± 0.38
No PSHJE
0.41 ± 0.19
7
Hwang-Jae Lee
et al. (2016)
PSHJE
0.040 ± 0.001
117.76 ± 12.95
111.32 ± 9.43
4.48 (3.58–5.6)
13.5 (10.5–16)
No PSHJE
0.035 ± 0.005
107.23 ± 15.56
105.61 ± 5.46
5.04 (3.92–6-16)
14.5 (11.5–17)
8
Su-Hyun Lee et al. (2017)
PSHJE
1.10 ± 0.13
117.76 ± 12.95
113.36 ± 6.92
No PSHJE
0.97 ± 0.15
107.23 ± 15.56
107.90 ± 5.80
9
Hwang-Jae Lee
et al. (2019)
PSHJE (14)
0.91 ± 0.15
92.64 ± 15.46
106.88 ± 29.48
16 ± 1.2
No PSHJE (12)
0.78 ± 0.11
80.64 ± 16.44
88.01 ± 22.82
17.5 ± 1.5
10
Lenzi et al. (2013)
PSHJE
101.4 ± 0.46
No PSHJE
102.7 ± 0.48
11
Martini et al. (2019)
PSHJE (10)
3.45 ± 0.64
10.06 ± 1.88
No PSHJE (10)
3.58 ± 0.72
10.54 ± 2.11
12
Tanaka et al. (Seei gu e o
e t page.)
Fig. 2 Forest plot comparing gait self-speed (m/s) between PSHJE and no PSHJE groups (A). Forest plot of subgroup analysis among patients
with chronic stroke, comparing gait self-speed (m/s) between PSHJE and no PSHJE groups (B). Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies Oxygen (ml/min/kg) Pooled estimation of the two RCTs, comparing PSHJE
rehabilitation with control group, oxygen cost decreased
− 1.23 ml/min/kg (− 2.13 to − 0.32) on average, among
patients rehabilitated by PSHJE (Fig. 6). I square showed
low heterogeneity among reported data for oxygen cost
(I2: 0%, P = 0.33). Cadence (step/min)i Analyzing five clinical trials, cadence increased 4.74 step/
min (− 3.52–13.00) on average, among patients reha-
bilitated by PSHJE (Fig. 5A). I square showed high het-
erogeneity among reported data for cadence (I2: 61%,
P = 0.035). Sensitivity analysis showed the mean change
of cadence was not consistent (range of summary
WMDs: 1.1–8.5), indicating that the meta-analysis model
was not robust. Step length (m) The present review investigated the association of the
motorized hip-assist robots’ application with gait and
metabolic parameters in patients with gait disorders, as
well as healthy individuals. The main meta-analysis find-
ing was that two of the five gait spatiotemporal values for
max speed and stride length improve significantly after
rehabilitation with single joint hip powered robots com-
pared with conventional methods. Additionally, oxygen
consumption was lower in individuals used hip robots. Gait values of self-peed and step length were also differ-
ent between two groups but not statistically significantly
(P = 0.09 and 0.12, respectively). Analyzing three studies, step length increased 0.06 m
(0.05–0.07) on average, among patients rehabilitated by
PSHJE. Sensitivity analysis showed the mean change of
step length was consistent (range of summary WMDs:
0.05–0.06), indicating that the meta-analysis model
was robust. I square showed low heterogeneity among
reported data for step length (I2: 0%, P = 0.48) (Fig. 4). Gait max speed (m/s) Gait max speed (m/s)
Analyzing four studies, gait max speed increased
0.13 m/s (0.10–0.17) on average, among patients reha-
bilitated by PSHJE. Sensitivity analysis showed the mean
change of gait max speed was consistent (range of sum-
mary WMDs: 0.13–0.15), indicating that the meta-analy-
sis model was robust. I square showed low heterogeneity
among reported data for gait max speed (I2: 22%, P = 0.28)
(Fig. 3). Quantitative results (meta‑analysis)
Gait self‑speed (m/s) (2017)
PSHJE (21)
1.08 ± 0.43
0.53 ± 0.14
116.56 ± 24.66
No PSHJE (20)
1.06 ± 0.61
0.52 ± 0.19
108.80 ± 32.78
13
Buesing et al. (2015)
PSHJE (25)
0.87 ± 0.05
1.23 ± 0.07
0.58 ± 0.03
110 ± 6
95.67 ± 3.69
No PSHJE (25)
0.88 ± 0.07
1.08 ± 0.07
0.52 ± 0.02
97 ± 5
97.37 ± 5.08 Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 8 of 15 Subgroup analysis: among healthy elderly, three sin-
gle group pre-post studies were analyzed. The results of
subgroup meta-analyses show that cadence increased
3.53 step/min (− 0.48–7.55) on average (Fig. 5B). I square
showed high heterogeneity among reported data for
cadence (I2: 73%, P = 0.025). Sensitivity analysis showed
the mean change of cadence was not consistent (range
of summary WMDs: 2.5–5.5), indicating that the meta-
analysis model was not robust. meta-analysis model was robust. I square showed high
heterogeneity among reported data for gait self –speed
(I2: 86%, P < 0.001). Subgroup analysis: among patients
with chronic stroke, three clinical trials were analyzed. The results of subgroup meta-analyses show that gait
self -speed increased 0.07 m/s (− 0.02–0.16) on average. I square also showed high heterogeneity in subgroup
meta-analysis (I2: 92%, P < 0.001) (Fig. 2B). Limitationh The included studies typically have a small sample size,
and limited follow-up period. Therefore, until more
consistent results are revealed in bigger RCTs, no clini-
cal advice can be made to use hip exoskeletons in people
with gait disability. The patient’s condition heterogene-
ity among analyzed articles are significant. However, this
does not seem to be the source of heterogeneity (high I2)
in the gait self-speed and cadence, because the subgroup
analyses on the homogenous population (chronic stroke
for gait self-speed and healthy elderly individuals for
cadence) did not affect the I2 value. Analyzing four single group pre-post studies, cadence
increased 2.12 step/min (− 1.93–6.17) on average, among
patients rehabilitated by PSHJE (Fig. 5A). I square
showed high heterogeneity among reported data for
cadence (I2: 89%, P < 0.001). Sensitivity analysis showed
the mean change of cadence was not consistent (range
of summary WMDs: 0.94–3.5), indicating that the meta-
analysis model was not robust. Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Daliri et al. Limitationh BMC Musculoskeletal Disorders Page 9 of 15 NOTE: Weights are from random effects analysis
Overall (I-squared = 86.1%, p = 0.000)
Jayaraman (2018), RCT
ID
Kawasaki (2020), Cross-over RCT
Lee (2019), RCT
Study
Ji (2018), RCT
Koseki (2021), NRCT
Buesing (2015), RCT
0.07 (-0.01, 0.15)
0.10 (0.07, 0.13)
WMD (95% CI)
0.19 (-0.07, 0.45)
0.13 (0.03, 0.23)
-0.06 (-0.16, 0.04)
0.26 (0.06, 0.46)
-0.01 (-0.04, 0.02)
100.00
24.74
Weight
6.32
17.75
%
17.45
9.35
24.39
0.07 (-0.01, 0.15)
0.10 (0.07, 0.13)
WMD (95% CI)
0.19 (-0.07, 0.45)
0.13 (0.03, 0.23)
-0.06 (-0.16, 0.04)
0.26 (0.06, 0.46)
-0.01 (-0.04, 0.02)
100.00
24.74
Weight
6.32
17.75
%
17.45
9.35
24.39
0
-.459
0
.459 NOTE: Weights are from random effects analysis
Overall (I-squared = 86.1%, p = 0.000)
Jayaraman (2018), RCT
ID
Kawasaki (2020), Cross-over RCT
Lee (2019), RCT
Study
Ji (2018), RCT
Koseki (2021), NRCT
Buesing (2015), RCT
0.07 (-0.01, 0.15)
0.10 (0.07, 0.13)
WMD (95% CI)
0.19 (-0.07, 0.45)
0.13 (0.03, 0.23)
-0.06 (-0.16, 0.04)
0.26 (0.06, 0.46)
-0.01 (-0.04, 0.02)
100.00
24.74
Weight
6.32
17.75
%
17.45
9.35
24.39
0.07 (-0.01, 0.15)
0.10 (0.07, 0.13)
WMD (95% CI)
0.19 (-0.07, 0.45)
0.13 (0.03, 0.23)
-0.06 (-0.16, 0.04)
0.26 (0.06, 0.46)
-0.01 (-0.04, 0.02)
100.00
24.74
Weight
6.32
17.75
%
17.45
9.35
24.39
0
-.459
0
.459
NOTE: Weights are from random effects analysis
Overall (I-squared = 92.3%, p = 0.000)
ID
Lee (2019), RCT
Study
Jayaraman (2018), RCT
Buesing (2015), RCT
0.07 (-0.02, 0.16)
WMD (95% CI)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
Weight
25.99
%
37.30
36.71
0.07 (-0.02, 0.16)
WMD (95% CI)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
Weight
25.99
%
37.30
36.71
0
-.23
0
.23
2 (See legend on previous page.) NOTE: Weights are from random effects analysis
Overall (I-squared = 86.1%, p = 0.000)
Jayaraman (2018), RCT
ID
Kawasaki (2020), Cross-over RCT
Lee (2019), RCT
Study
Ji (2018), RCT
Koseki (2021), NRCT
Buesing (2015), RCT
0.07 (-0.01, 0.15)
0.10 (0.07, 0.13)
WMD (95% CI)
0.19 (-0.07, 0.45)
0.13 (0.03, 0.23)
-0.06 (-0.16, 0.04)
0.26 (0.06, 0.46)
-0.01 (-0.04, 0.02)
100.00
24.74
Weight
6.32
17.75
%
17.45
9.35
24.39
0.07 (-0.01, 0.15)
0.10 (0.07, 0.13)
WMD (95% CI)
0.19 (-0.07, 0.45)
0.13 (0.03, 0.23)
-0.06 (-0.16, 0.04)
0.26 (0.06, 0.46)
-0.01 (-0.04, 0.02)
100.00
24.74
Weight
6.32
17.75
%
17.45
9.35
24.39
0
-.459
0
.459 ID NOTE: Weights are from random effects analysis NOTE: Weights are from random effects analysis
Overall (I-squared = 92.3%, p = 0.000)
ID
Lee (2019), RCT
Study
Jayaraman (2018), RCT
Buesing (2015), RCT
0.07 (-0.02, 0.16)
WMD (95% CI)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
Weight
25.99
%
37.30
36.71
0.07 (-0.02, 0.16)
WMD (95% CI)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
Weight
25.99
%
37.30
36.71
0
-.23
0
.23
Fig. Limitationh I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies NOTE: Weights are from random effects analysis
Overall (I-squared = 22.3%, p = 0.277)
Koseki (2021), NRCT
Tanaka (2017), RCT
Jayaraman (2018), RCT
Buesing (2015), RCT
0.13 (0.10, 0.17)
0.30 (0.06, 0.54)
0.02 (-0.30, 0.34)
0.12 (0.09, 0.15)
0.15 (0.11, 0.19)
100.00
1.65
0.92
57.38
40.05
0.13 (0.10, 0.17)
0.30 (0.06, 0.54)
0.02 (-0.30, 0.34)
0.12 (0.09, 0.15)
0.15 (0.11, 0.19)
100.00
1.65
0.92
57.38
40.05
0
-.541
0
.541
Fig. 3 Forest plot comparing gait maximum-speed (m/s) between PSHJE and no PSHJE. Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies Fig. 3 Forest plot comparing gait maximum-speed (m/s) between PSHJE and no PSHJE. Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies Limitationh 2 (See legend on previous page.) NOTE: Weights are from random effects analysis
Overall (I-squared = 92.3%, p = 0.000)
ID
Lee (2019), RCT
Study
Jayaraman (2018), RCT
Buesing (2015), RCT
0.07 (-0.02, 0.16)
WMD (95% CI)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
Weight
25.99
%
37.30
36.71
0.07 (-0.02, 0.16)
WMD (95% CI)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
Weight
25.99
%
37.30
36.71
0
-.23
0
.23
2 (See legend on previous page.) ID NOTE: Weights are from random effects analysis
Overall (I-squared = 92.3%, p = 0.000)
Lee (2019), RCT
Jayaraman (2018), RCT
Buesing (2015), RCT
0.07 (-0.02, 0.16)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
25.99
37.30
36.71
0.07 (-0.02, 0.16)
0.13 (0.03, 0.23)
0.10 (0.07, 0.13)
-0.01 (-0.04, 0.02)
100.00
25.99
37.30
36.71
0
-.23
0
.23
g. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 10 of 15 Daliri et al. BMC Musculoskeletal Disorders Study ID NOTE: Weights are from random effects analysis
Overall (I-squared = 22.3%, p = 0.277)
ID
Koseki (2021), NRCT
Tanaka (2017), RCT
Jayaraman (2018), RCT
Buesing (2015), RCT
0.13 (0.10, 0.17)
WMD (95% CI)
0.30 (0.06, 0.54)
0.02 (-0.30, 0.34)
0.12 (0.09, 0.15)
0.15 (0.11, 0.19)
100.00
Weight
1.65
0.92
57.38
40.05
0.13 (0.10, 0.17)
WMD (95% CI)
0.30 (0.06, 0.54)
0.02 (-0.30, 0.34)
0.12 (0.09, 0.15)
0.15 (0.11, 0.19)
100.00
Weight
1.65
0.92
57.38
40.05
0
-.541
0
.541
Fig. 3 Forest plot comparing gait maximum-speed (m/s) between PSHJE and no PSHJE. Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. Discussion BMC Musculoskeletal Disorders NOTE: Weights are from random effects analysis
Overall (I-squared = 0.0%, p = 0.485)
ID
Study
Tanaka (2017), RCT
Buesing (2015), RCT
Koseki (2021), NRCT
0.06 (0.05, 0.07)
WMD (95% CI)
0.01 (-0.09, 0.11)
0.06 (0.05, 0.07)
0.09 (0.01, 0.17)
100.00
Weight
%
1.81
95.25
2.94
0.06 (0.05, 0.07)
WMD (95% CI)
0.01 (-0.09, 0.11)
0.06 (0.05, 0.07)
0.09 (0.01, 0.17)
100.00
Weight
%
1.81
95.25
2.94
0
-.17
0
.17
Fig. 4 Forest plot comparing step length (m) between PSHJE and no PSHJE groups. Diamond represents the weighted mean difference (pooled
WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point reflects
the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies ID Buesing (2015), RCT NOTE: Weights are from random effects analysis Fig. 4 Forest plot comparing step length (m) between PSHJE and no PSHJE groups. Diamond represents the weighted mean difference (pooled
WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point reflects
the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies (weak) leg during the corrective sessions, the diseased
muscles become substantially more active [64, 65]. improve if they remove their exoskeleton. Neverthe-
less, after its discontinuation, there is a chance that
gait pattern goes back to how it was. Therefore, to con-
duct a more robust homogeny meta-analysis on clinical
outcomes, more randomized controlled trials must be
carried out on each of the gait disorder types. These tri-
als might confirm both the short- and long-term clini-
cal outcomes, including patient-reported satisfaction,
quality of life, and fall prevention on individuals with
gait abnormalities. Evaluating post-intervention sat-
isfaction and its association with gait outcomes would
be an interesting topic to explore in future studies. Fig. 5 Forest plot comparing cadence (step/min) between PSHJE and no PSHJE groups (A). Forest plot of subgroup analysis among healthy
elderly individuals, comparing cadence (step/min) between PSHJE and no PSHJE groups (B). Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies
(See figure on next page.) Discussion brain connections through movement [59]. In patients
with gait disorders, brain neurophysiology and organi-
zation are altered, leading to distinct brain activity pat-
terns from those of healthy people [60]. Humans have a
central pattern generator (CPG) that uses supra-spinal
control of cerebral neural networks to enable rhyth-
mic and repeated locomotor patterns [61]. Specific
abnormalities in human movement are brought on by
damage to specific supra-spinal structures, as seen in
people who have had brain injuries such strokes [62]. To encourage the restoration of a movement pattern,
the exoskeleton should provide flexibility in lower limb
dynamics through sufficient degrees of freedom in all
three motion planes [63]. Multisensory stimulation
has been shown to be beneficial for brain reconfigura-
tion. The reconfiguration is provided by the combina-
tion of personalized support and progression, real-time
monitoring and instruction, and motor exercises that
test balance control and coordination. Furthermore,
because of the greater weight-shift to the affected Gait speed (n = 12) and cadence (n = 10) were the most
often reported gait parameters, even though almost all
included studies examined at least one temporal gait
measure. Gait speed is significant for out-door everyday
life activities, as well as enrolling in a rehabilitation pro-
gram. Unfortunately, there is a lack of agreement regard-
ing the best biomechanical gait measurements to utilize
when examining motor coordination and the quality of
walking patterns [56–58]. Although quantitative analysis
in this study showed some gait values were not signifi-
cantly different between the two groups, hip exoskeleton
provides the opportunity for the patient to walk on his
own and correct gait motor errors, and therefore increase
their self-confidence, motivation, and improve psycho-
logical status.h The neuroplasticity phenomenon is the primary
mechanism in which exoskeletons correct gait pattern. A well-designed robot should be able to produce com-
plex, controlled multimodal stimulation from the bot-
tom up and top down in order to alter the plasticity of Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 11 of 15 Daliri et al. Discussion By
assessing patient satisfaction, we can gain insights into Future research should focus on improving methods
for determining each patient’s specific power needs
while moving through a powered single joint hip exo-
skeleton and matching those needs to a practical and
production of smaller powered actuators for this use. As a result, patients’ compliance toward lower weight
exoskeletons would increase. According on the user’s
physical conditions, the rehabilitation process for peo-
ple with gait abnormalities using powered hip exoskel-
etons can be separated into multiple stages. After a
prolonged period of use, the user’s gait function may Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 12 of 15 Daliri et al. BMC Musculoskeletal Disorders NOTE: Weights are from random effects analysis
. . Double group
Koseki (2021), NRCT
Ji (2018), RCT
Lee (2019), RCT
Tanaka (2017), RCT
Buesing (2015), RCT
Subtotal (I-squared = 61.3%, p = 0.035)
Single group
Kim (2018)
Lee (2016)
Lee (2017)
Lenzi (2013)
Subtotal (I-squared = 89.5%, p = 0.000)
ID
Study
16.57 (1.54, 31.60)
-2.88 (-13.84, 8.08)
18.87 (-1.26, 39.00)
7.76 (-10.06, 25.58)
-1.70 (-4.16, 0.76)
4.74 (-3.52, 13.00)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
5.46 (2.23, 8.69)
-1.30 (-1.71, -0.89)
2.12 (-1.93, 6.17)
WMD (95% CI)
16.62
22.40
11.57
13.59
35.81
100.00
23.35
22.96
24.60
29.09
100.00
Weight
%
16.57 (1.54, 31.60)
-2.88 (-13.84, 8.08)
18.87 (-1.26, 39.00)
7.76 (-10.06, 25.58)
-1.70 (-4.16, 0.76)
4.74 (-3.52, 13.00)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
5.46 (2.23, 8.69)
-1.30 (-1.71, -0.89)
2.12 (-1.93, 6.17)
WMD (95% CI)
16.62
22.40
11.57
13.59
35.81
100.00
23.35
22.96
24.60
29.09
100.00
Weight
%
0
-39
0
39
NOTE: Weights are from random effects analysis
Overall (I-squared = 73.0%, p = 0.025)
Lee (2017), pre-post
Kim (2018), pre-post
Study
Lee (2016), pre-post
ID
3.53 (-0.48, 7.55)
5.46 (2.23, 8.69)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
WMD (95% CI)
100.00
35.29
32.74
%
31.96
Weight
3.53 (-0.48, 7.55)
5.46 (2.23, 8.69)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
WMD (95% CI)
100.00
35.29
32.74
%
31.96
Weight
0
-9.61
0
9.61
See legend on previous page.) NOTE: Weights are from random effects analysis
. . Discussion Double group
Koseki (2021), NRCT
Ji (2018), RCT
Lee (2019), RCT
Tanaka (2017), RCT
Buesing (2015), RCT
Subtotal (I-squared = 61.3%, p = 0.035)
Single group
Kim (2018)
Lee (2016)
Lee (2017)
Lenzi (2013)
Subtotal (I-squared = 89.5%, p = 0.000)
ID
Study
16.57 (1.54, 31.60)
-2.88 (-13.84, 8.08)
18.87 (-1.26, 39.00)
7.76 (-10.06, 25.58)
-1.70 (-4.16, 0.76)
4.74 (-3.52, 13.00)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
5.46 (2.23, 8.69)
-1.30 (-1.71, -0.89)
2.12 (-1.93, 6.17)
WMD (95% CI)
16.62
22.40
11.57
13.59
35.81
100.00
23.35
22.96
24.60
29.09
100.00
Weight
%
16.57 (1.54, 31.60)
-2.88 (-13.84, 8.08)
18.87 (-1.26, 39.00)
7.76 (-10.06, 25.58)
-1.70 (-4.16, 0.76)
4.74 (-3.52, 13.00)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
5.46 (2.23, 8.69)
-1.30 (-1.71, -0.89)
2.12 (-1.93, 6.17)
WMD (95% CI)
16.62
22.40
11.57
13.59
35.81
100.00
23.35
22.96
24.60
29.09
100.00
Weight
% NOTE: Weights are from random effects analysis
. . Double group
Koseki (2021), NRCT
Ji (2018), RCT
Lee (2019), RCT
Tanaka (2017), RCT
Buesing (2015), RCT
Subtotal (I-squared = 61.3%, p = 0.035)
Single group
Kim (2018)
Lee (2016)
Lee (2017)
Lenzi (2013)
Subtotal (I-squared = 89.5%, p = 0.000)
ID
Study
16.57 (1.54, 31.60)
-2.88 (-13.84, 8.08)
18.87 (-1.26, 39.00)
7.76 (-10.06, 25.58)
-1.70 (-4.16, 0.76)
4.74 (-3.52, 13.00)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
5.46 (2.23, 8.69)
-1.30 (-1.71, -0.89)
2.12 (-1.93, 6.17)
WMD (95% CI)
16.62
22.40
11.57
13.59
35.81
100.00
23.35
22.96
24.60
29.09
100.00
Weight
%
16.57 (1.54, 31.60)
-2.88 (-13.84, 8.08)
18.87 (-1.26, 39.00)
7.76 (-10.06, 25.58)
-1.70 (-4.16, 0.76)
4.74 (-3.52, 13.00)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
5.46 (2.23, 8.69)
-1.30 (-1.71, -0.89)
2.12 (-1.93, 6.17)
WMD (95% CI)
16.62
22.40
11.57
13.59
35.81
100.00
23.35
22.96
24.60
29.09
100.00
Weight
%
0
-39
0
39 NOTE: Weights are from random effects analysis
Overall (I-squared = 73.0%, p = 0.025)
Lee (2017), pre-post
Kim (2018), pre-post
Study
Lee (2016), pre-post
ID
3.53 (-0.48, 7.55)
5.46 (2.23, 8.69)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
WMD (95% CI)
100.00
35.29
32.74
%
31.96
Weight
3.53 (-0.48, 7.55)
5.46 (2.23, 8.69)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
WMD (95% CI)
100.00
35.29
32.74
%
31.96
Weight
0
-9.61
0
9.61
Fig. Conclusion Analyzing current literature on powered hip exoskel-
eton, gait max speed and stride length were different
between the two groups (P < 0.05). Regarding metabolic
cost, patients who rehabilitated with the hip exoskel-
eton have consumed lower amounts of oxygen. More
randomized controlled trials must be carried out to
verify both the short- and long-term clinical outcomes,
including patient-reported measures. The considera-
tion of population demographic and ethnic diversity
among the included articles is an important limitation
to be acknowledged. Statement of the location This work was performed in Mashhad University of Medical Sciences (MUMS)
and Ferdowsi University of Mashhad, Center of Advanced Rehabilitation and
Robotics Research (FUM CARE). Acknowledgements g
The hip exoskeleton (HEXA) developed by Center of Advanced Rehabilita-
tion and Robotics Research (FUM Care) was supported as a research project
financed by the National Institute for Medical Research Development
(NIMAD). Discussion I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies NOTE: Weights are from random effects analysis
Overall (I-squared = 0.0%, p = 0.328)
Lee (2019), RCT
Martini (2019), RCT
-1.23 (-2.13, -0.32)
-1.50 (-2.56, -0.44)
-0.48 (-2.23, 1.27)
100.00
73.34
26.66
-1.23 (-2.13, -0.32)
-1.50 (-2.56, -0.44)
-0.48 (-2.23, 1.27)
100.00
73.34
26.66
0
-2.56
0
2.56
Fig. 6 Forest plot comparing oxygen cost (ml/min/kg) between PSHJE and no PSHJE groups. Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies Fig. 6 Forest plot comparing oxygen cost (ml/min/kg) between PSHJE and no PSHJE groups. Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies Supplementary Information their subjective experiences and perceptions of the
robotic rehabilitation interventions. This information
can provide valuable supplementary data to further
understand the effectiveness and acceptability of these
interventions. The online version contains supplementary material available at https://doi. org/10.1186/s12891-024-07189-4. Discussion 5 (See legend on previous page.) NOTE: Weights are from random effects analysis
Overall (I-squared = 73.0%, p = 0.025)
Lee (2017), pre-post
Kim (2018), pre-post
Study
Lee (2016), pre-post
ID
3.53 (-0.48, 7.55)
5.46 (2.23, 8.69)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
WMD (95% CI)
100.00
35.29
32.74
%
31.96
Weight
3.53 (-0.48, 7.55)
5.46 (2.23, 8.69)
-0.67 (-4.41, 3.07)
5.71 (1.81, 9.61)
WMD (95% CI)
100.00
35.29
32.74
%
31.96
Weight
0
-9.61
0
9.61
(See legend on previous page.) ID Fig. 5 (See legend on previous page.) Fig. 5 (See legend on previous page.) Daliri et al. BMC Musculoskeletal Disorders (2024) 25:80 Page 13 of 15 Page 13 of 15 Daliri et al. BMC Musculoskeletal Disorders NOTE: Weights are from random effects analysis
Overall (I-squared = 0.0%, p = 0.328)
ID
Lee (2019), RCT
Martini (2019), RCT
Study
-1.23 (-2.13, -0.32)
WMD (95% CI)
-1.50 (-2.56, -0.44)
-0.48 (-2.23, 1.27)
100.00
Weight
73.34
26.66
%
-1.23 (-2.13, -0.32)
WMD (95% CI)
-1.50 (-2.56, -0.44)
-0.48 (-2.23, 1.27)
100.00
Weight
73.34
26.66
%
0
-2.56
0
2.56
Fig. 6 Forest plot comparing oxygen cost (ml/min/kg) between PSHJE and no PSHJE groups. Diamond represents the weighted mean differenc
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% C
of the study. I2 test and Cochran’s Q statistic were used to assessing the statistical heterogeneity (P < 0.10) across studies NOTE: Weights are from random effects analysis
Overall (I-squared = 0.0%, p = 0.328)
Lee (2019), RCT
Martini (2019), RCT
-1.23 (-2.13, -0.32)
-1.50 (-2.56, -0.44)
-0.48 (-2.23, 1.27)
100.00
73.34
26.66
-1.23 (-2.13, -0.32)
-1.50 (-2.56, -0.44)
-0.48 (-2.23, 1.27)
100.00
73.34
26.66
0
-2.56
0
2.56
Fig. 6 Forest plot comparing oxygen cost (ml/min/kg) between PSHJE and no PSHJE groups. Diamond represents the weighted mean difference
(pooled WMD) estimate and its width shows corresponding 95% CI with random effects estimate. The size of the square and its central point
reflects the study specific statistical weight (inverse of variance) and point estimate of the WMD and horizontal line reflects corresponding 95% CI
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Mashhad, Iran. 2 Health Policy Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran. 3 Mechanical Engineering Department, Ferdowsi Uni-
versity of Mashhad, Mashhad, Iran. 4 Department of Physical Therapy, School
of Paramedical and Rehabilitation Sciences, Mashhad University of Medical
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Tilburg University Tilburg University The role of performance pressure, loneliness and sense of belonging in predicting
burnout symptoms in students in higher education
Dopmeijer, Jolien M.; Schutgens, Christine A. E.; Kappe, F. Rutger; Gubbels, Nikkie;
Visscher, Tommy L. S.; Jongen, Ellen M. M.; Bovens, Rob H. L. M.; De Jonge, Jannet M.;
Bos, Arjan E. R.; Wiers, Reinout W. Published in:
PLOS ONE The role of performance pressure, loneliness and sense of belonging in predicting
burnout symptoms in students in higher education
Dopmeijer, Jolien M.; Schutgens, Christine A. E.; Kappe, F. Rutger; Gubbels, Nikkie;
Visscher, Tommy L. S.; Jongen, Ellen M. M.; Bovens, Rob H. L. M.; De Jonge, Jannet M.;
Bos, Arjan E. R.; Wiers, Reinout W. Published in:
PLOS ONE Document Version
Publisher's PDF, also known as Version of record Link to publication in Tilburg University Research Portal Citation for published version (APA):
Dopmeijer, J. M., Schutgens, C. A. E., Kappe, F. R., Gubbels, N., Visscher, T. L. S., Jongen, E. M. M., Bovens,
R. H. L. M., De Jonge, J. M., Bos, A. E. R., & Wiers, R. W. (2023). The role of performance pressure, loneliness
and sense of belonging in predicting burnout symptoms in students in higher education. PLOS ONE, 17(12),
Article e0267175. https://doi.org/10.1371/journal.pone.0267175 General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners
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Dopmeijer, J. M., Schutgens, C. A. E., Kappe, F. R., Gubbels, N., Visscher, T. L. S., Jongen, E. M. M., Bovens,
R. H. L. M., De Jonge, J. M., Bos, A. E. R., & Wiers, R. W. (2023). The role of performance pressure, loneliness
and sense of belonging in predicting burnout symptoms in students in higher education. PLOS ONE, 17(12),
Article e0267175. https://doi.org/10.1371/journal.pone.0267175 Download date: 24. Oct. 2024 General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners
and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. OPEN ACCESS Citation: Dopmeijer JM, Schutgens CAE, Kappe
FR, Gubbels N, Visscher TLS, Jongen EMM, et al. (2022) The role of performance pressure,
loneliness and sense of belonging in predicting
burnout symptoms in students in higher education. PLoS ONE 17(12): e0267175. https://doi.org/
10.1371/journal.pone.0267175 Editor: Sergio A. Useche, Universitat de Valencia,
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ur claim Download date: 24. Oct. 2024 PLOS ONE RESEARCH ARTICLE Introduction For many students, their time at university is stressful, marked with many new experiences,
challenges and life events [1]. Perhaps stronger than previously, student life is about meeting
high expectations: getting good grades, building a resume and a good career start next to a rich
social life [2]. Recent reports show an increase in perceived performance pressure among stu-
dents leading to psychosocial problems, particularly burnout symptoms [2,3]. Accordingly,
several studies have shown that experiencing burnout symptoms has become a common prob-
lem among students in higher education, with the reported prevalence ranging between 45
and 71% [1,4–6]. Burnout symptoms often lead to students feeling less engaged in their
studies, poor academic performance, study delay and drop-out [1,7]. Over the past years,
researchers have found that those students who report feelings of burnout are more likely to The role of performance pressure, loneliness
and sense of belonging in predicting burnout
symptoms in students in higher education Jolien M. DopmeijerID1,2,3☯*, Christine A. E. Schutgens4,5☯, F. Rutger Kappe6,
Nikkie Gubbels7, Tommy L. S. Visscher8, Ellen M. M. Jongen5, Rob H. L. M. Bovens9,
Jannet M. de Jonge10, Arjan E. R. Bos5, Reinout W. Wiers2 Jolien M. DopmeijerID1,2,3☯*, Christine A. E. Schutgens4,5☯, F. Rutger Kappe6,
Nikkie Gubbels7, Tommy L. S. Visscher8, Ellen M. M. Jongen5, Rob H. L. M. Bovens9,
Jannet M. de Jonge10, Arjan E. R. Bos5, Reinout W. Wiers2 1 Trimbos-instituut, Netherlands Institute of Mental Health and Addiction, Utrecht, The Netherlands,
2 Department of Psychology, Addiction Development and Psychopathology (ADAPT-)lab, Centre for Urban
Mental Health, University of Amsterdam, The Netherlands, 3 Teachers College, Windesheim University of
Applied Sciences, Zwolle, The Netherlands, 4 Institute of Sport and Exercise Studies, HAN University of
Applied Sciences, Nijmegen, The Netherlands, 5 Faculty of Psychology, Open University, Heerlen, The
Netherlands, 6 Department of Education and Innovation, Study Success, Inholland University of Applied
Sciences, Haarlem, The Netherlands, 7 Quality Assurance Department, Codarts University of the Arts,
Rotterdam, The Netherlands, 8 Staff Office Education and Research, Hanze University of Applied Sciences,
Groningen, The Netherlands, 9 Tranzo, Scientific Center for Care and Wellbeing, University of Tilburg,
Tilburg, The Netherlands, 10 Faculty of Health, ACHIEVE, Centre of Applied Research, Amsterdam
University of Applied Sciences, Amsterdam, the Netherlands a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ☯These authors contributed equally to this work. * jm.dopmeijer@windesheim.nl ☯These authors contributed equally to this work. Abstract Student burnout is raising an increasing amount of concern. Burnout often leads to psycho-
social problems and drop-out. In this study multiple regression analysis was used to exam-
ine the impact of performance pressure, loneliness, and sense of belonging on the
underlying dimensions of burnout in 3,134 university students in the Netherlands. Results
suggest that sense of belonging could be targeted as a way to enhance student wellbeing,
in order to improve the ability to cope with the high demands in student life and the preven-
tion of burnout. Editor: Sergio A. Useche, Universitat de Valencia,
SPAIN Data Availability Statement: All relevant data are
within the paper and its Supporting information
files. Data Availability Statement: All relevant data are
within the paper and its Supporting information
files. Funding: Funding was provided to Jolien M. Dopmeijer by the Netherlands Organization for
Scientific Research (NWO) under grant number
023.004.118. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 1 / 11 PLOS ONE Predicting burnout symptoms in students in higher education experience poor physical health and cardio-vascular disease [8], psychological distress such as
depression and even suicidal ideation [4]. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Burnout refers to a state of psychological exhaustion and is originally known as a work-
related phenomenon defined as ‘a psychological syndrome as reaction to chronic interpersonal
stressors at work’ [9]. This reaction consists of feeling exhausted, experiencing depersonaliza-
tion, and feeling incompetent. Although the concept of burnout has originally been defined in
the context of work-related stress, the traditional concept and scope have recently been broad-
ened to include study-related problems [10]. Indeed, research on burnout symptoms among
students in higher education is upcoming, showing that burnout symptoms among students
seem even more prevalent than among the working population [5,6,11,12]. Burnout symptoms
among students are similar to burnout symptoms among professionals, they refer to feelings
of exhaustion (because of high study demands), having a cynical and detached attitude toward
one’s study and experiencing a reduced sense of accomplishment as a student [10]. The development of burnout is explained by Job Demands-Resources model for Burnout
and Engagement (JD-R Model). The model assumes that high demands lead to stress reactions
and exhaustion, while having many resources (sources of energy), like rewards or social sup-
port, leads to higher productivity, which is a motivational process [13,14]. Burnout symptoms
are a result of insufficient balance between experienced demands and resources [14]. While
the JD-R model finds its origin in workplace settings, the model has also shown to be valid in
the educational setting according to a study by Robins et al (2015) [15]. This study showed that
mediation effects of study demands and (personal) resources (such as emotional stability and
belonging) with psychological flexibility were found. Competing interests: The authors have declared
that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 Data Availability Statement: All relevant data are
within the paper and its Supporting information
files. The study found that the JD-R model
contributed to the knowledge about burnout and engagement in university students, especially
with regard to personal resources. The relationship between performance pressure and burn-
out symptoms is relevant to investigate. The increase of both perceived performance pressure
and burnout symptoms makes it plausible that performance pressure could be a demand that
contributes to the development of burnout symptoms among students and therefore could be
a predictor of burnout symptoms. Together with the increase in studies regarding burnout symptoms among students, there
is a growing interest in loneliness among students. Loneliness can be defined as ‘the subjective
experience of an unpleasant or unacceptable lack of (quality of) certain social relationships’ [16]. Several studies show that loneliness is a common problem among students and that loneliness
and experienced study stress appear to explain depressive symptoms [17]. Another study
shows that there also appears to be a relationship between loneliness and (lack of) study prog-
ress: lonely students experience more stress, are less successful in their final exams and are less
involved in their studies [18]. An overview study shows loneliness to be related to cardiovascu-
lar diseases, the functioning of the immune system, premature death, fatigue, depression and
anxiety [19]. Both loneliness and burnout are related to negative psychological health. The
relationship between these concepts was investigated by Lin and Huang among a sample of
Taiwanese university students and by Stoliker and Lafreniere among a sample of Canadian
undergraduate students. These studies show that there’s a relationship between loneliness and
burnout symptoms among students [1,20] and that loneliness is also a predictor of students’
engagement in their studies [1]. According to Ponzetti [21], students with feelings of loneliness
may also be experiencing increased levels of social detachment, which is one of the dimensions
of burnout, called depersonalization in the JD-R Model. Given the results of these previous
studies, it is likely that loneliness plays a role in the development of burnout symptoms. It’s
likely that the loneliness can be seen as a demand. The other related concept to study resources is sense of belonging, which refers to ‘feelings
of safety and comfort that arise from the idea of being part of a community, organization or PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 2 / 11 PLOS ONE Predicting burnout symptoms in students in higher education institution’ [22]. Data Availability Statement: All relevant data are
within the paper and its Supporting information
files. The most important dimensions of sense of belonging are the feeling of being
appreciated and the feeling of fitting within the group. Sense of belonging is a widely studied
concept within the educational setting, primarily focused on the relationship with academic
performance and motivation. It is seen as an important condition for learning, since learning
is generally not considered as an individual, but social process that takes place through collabo-
ration. Students with a higher sense of belonging are more motivated, more committed to
their studies and have a greater intention to continue studying [23–26]. On a smaller scale,
research has been done regarding the relationship between sense of belonging and mental
health of students. A higher sense of belonging was found to be associated with fewer study
problems and fewer depression symptoms [27], where another study shows that a lower sense
of belonging explained the significantly higher degree of psychosocial problems experienced
by children of immigrants, who were the first to go to university [28]. Although little research
has been done regarding the relationship between sense of belonging and burnout symptoms,
a Norwegian study shows a negative relationship between sense of belonging and one of the
dimensions of burnout, exhaustion [29]. For a long time, the concepts of loneliness and sense of belonging were seen as two
sides of the same coin, as a result of which they’ve rarely been investigated together. Although both concepts stem from the need for connectedness, they differ from each
other. Loneliness arises from a discrepancy between the desired and the perceived con-
nectedness with others, which depends on someone’s expectations of friendship or inti-
mate relationships and what they deliver. Sense of belonging depends on the degree of
being involved and valued on the one hand, and on the other having the feeling of fitting
into a system or environment. Relationships are part of that, but not the only thing that
makes someone feel fitting in [22]. Another difference is that sense of belonging is context
specific and loneliness is not [22]. Because burnout is also context specific and the feeling
of fitting into a school environment could be a resource that is more comprehensive than
just the satisfaction about social relationships, unlike loneliness, sense of belonging is
likely to be a resource and may be a specific and important protective factor for burnout
symptoms. Data Availability Statement: All relevant data are
within the paper and its Supporting information
files. It is clear that studies show that burnout symptoms have severe consequences for students,
what makes it of great importance to gain insight into the factors that are related to the devel-
opment of these symptoms and the key principles that can contribute to its prevention. Although several studies have shown that there are indications that performance pressure,
loneliness and sense of belonging are predictive of burnout symptoms in general, little is
known about what predicts burnout among students. The main aim of this study is to examine
if there is a relationship between performance pressure, loneliness and sense of belonging on
the one hand and the three dimensions of burnout (i.e., emotional exhaustion, depersonaliza-
tion and reduced sense of personal accomplishment) on the other hand among students in
higher education. Measures Outcomes. Burnout was assessed with the validated Dutch version of the Maslach
Burnout Inventory General Survey (MBI-GS), the Utrecht Burnout Scale General Version
(UBOS-A) [30], that was adapted for use in our student sample. For instance, the item “I feel
mentally drained from my job” was rephrased in “I feel mentally drained from my studies”. The
UBOS-A questionnaire consists of 15 items that constitute 3 subscales (or dimensions) that
together measure burnout symptoms: Emotional Exhaustion (EE; Cronbach’s alpha .87 in the
present study), Depersonalization (DP; Cronbach’s alpha .87 in the present study) and a
reduced sense of Personal Accomplishment (PA; Cronbach’s alpha.73 in the present study,
obtained by removal of the item “I just want to do my studies and not be bothered”, which
made the alpha considerably higher, i.e., from .60 to .73). All 14 items are scored on a 7-point
Likert scale from 1 (never) to 7 (always). The average scale scores of the three UBOS-A scales
are calculated by adding up the scores and dividing by the number of items in the scale of ques-
tion. High scores on Emotional Exhaustion (EE, mean score 2.39) and Depersonalization
(DP, mean score 2.25) and low scores on Personal Accomplishment (PA, mean score .49)
are indicative for burnout [7]. Predictors. Performance pressure was assessed with the question: ‘To what extent do you
feel that you have to perform or achieve?’ The item was scored on a 5-point Likert scale from 1
(never) to 5 (very often). The higher the score, the more performance pressure was experi-
enced. This scale yielded an average of 2.85 (SD = .88). Loneliness was assessed with the De Jong-Gierveld Loneliness Scale with a Cronbach’s alpha
of .93 in the present study [16]. This scale consists of the emotional loneliness subscale (5
items) and the social loneliness subscale (6 items) which together measure loneliness. An
example from the emotional loneliness subscale is: “I experience a general sense of emptiness”
and an example of the social loneliness subscale is: “There are plenty of people I can rely on
when I have problems”. All items were scored from 1 (Yes! Totally agree) to 5 (No! Totally dis-
agree). First, the emotional loneliness score is computed by counting all neutral and positive
answers on the negatively formulated items and second, the social loneliness score is computed
by counting all neutral and negative answers on positively formulated items. Procedure Cross-sectional data were used from the Study environment, Health and Study
Success online survey, carried out between December 2017 and March 2018 in a large uni-
versity of applied sciences in the Netherlands. Fulltime students were invited by email
(N = 18,163). They were informed upfront about the objective and procedure of the study
and participated voluntarily. The consent of the participants was obtained by virtue of sur-
vey completion. 3 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 PLOS ONE Predicting burnout symptoms in students in higher education Measures The total loneli-
ness score (0 to 11) is computed by taken the sum of the emotional loneliness score and the
social loneliness score. This scale yielded an average of 23.41 (SD = 8.29) and a Cronbach’s
alpha of .93 in the present study. Sense of belonging was assessed by the Sense of Belonging questionnaire [25], consisting of 6
items, for example: “I feel at home at this university of applied sciences.” All items are rated on
a 5-point Likert scale from 1 (not true at all) to 5 (completely true). An average score of the
items was calculated for each participant. The higher the total score, the more the respondent
experienced a sense of belonging. This scale yielded an average of 3.81 (SD = .60) and a Cron-
bach’s alpha of .77 in the present study. Covariates. Covariates age, year of study and gender were obtained from data of the Stu-
dent Administration of the university. Age was expressed in years, and gender was coded
dichotomously (1 = female, 2 = male). The students’ year of study was either 1st year (= 1), 2nd
year (= 2), 3rd year (= 3), 4th year (= 4) or higher than 4th year (= 5). Living situation was
assessed as a self-reported item and was also coded dichotomously (1 = living independently,
2 = living with parents). PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 Statistical analysis Sociodemographic characteristics and descriptive values for all variables were calculated using
IBM SPSS Statistics, version 23 [31]. Multiple regression analysis using Muthe´n and Muthe´n’s PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 4 / 11 PLOS ONE Predicting burnout symptoms in students in higher education (1998–2011) software MPLUS version 8.0 [32], was used to test the predictive role of perfor-
mance pressure, sense of belonging and loneliness in burnout symptoms. All three predictors
in the model, i.e. emotional exhaustion, depersonalization and (lack of) personal accomplish-
ment, which together predict burnout symptoms, were analyzed together to control for their
shared variance in burnout symptoms. Main effects were analyzed while controlling for demo-
graphic covariates (age, gender, living situation and year of study). Maximum likelihood with robust standard error (MLR) was used as estimation method in
order to control for possible non-normality’s of the data. Preliminary analyses were performed
and it was ensured that there were no violations of the assumption of normality, linearity, mul-
ticollinearity and homoscedasticity. PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 PLOS ONE PLOS ONE Table 2. Correlations (N = 3,141). Emotional exhaustion
Depersonalization
Personal accomplishment
Loneliness
Performance pressure
Sense of belonging
Emotional exhaustion
1
-
Depersonalization
.57
1
Personal accomplishment
-.30
-.45
1
Loneliness
.32
.27
-.28
1
Performance pressure
.37
.15
-.09
.15
1
Sense of belonging
-.35
-.55
.39
-.29
-.07
1
p < .01. Table 2. Correlations (N = 3,141). https://doi.org/10.1371/journal.pone.0267175.t002 often to very often. Almost half of the participants experienced loneliness (43.3%) and the
average score for sense of belonging was 3.81 (SD = .60). Results of the correlations analyses are shown in Table 2. This table shows that although
some variables appeared to be correlated, no high intercorrelations occurred among variables
of our multiple regression model. This shows that there were no violations of the assumption
of normality, linearity, multicollinearity and homoscedasticity. Results of the multiple regression analysis are shown in Table 3. Performance pressure,
loneliness and sense of belonging were significantly associated with the three dimensions of
burnout: emotional exhaustion, depersonalization, and personal accomplishment, adjust-
ing for gender, age, living situation and year of study. All these associations reached statisti-
cal significance, except one: the association between performance pressure and personal
accomplishment. Results A total of 3,141 students completed the “Study Environment, Health and Study Success” sur-
vey (response rate 17.3%), 60% of whom were female students (N = 1886) and 40% of whom
were male students (N = 1255) (Table 1). Their average age was 21.8 years old (SD = 3.45). Of
all students 70.6% were living with their parents and 29.4% were living independently. A total
of 28.1% of the participants were freshman (first year) and 71.9% were students in later years. Almost two-third (68.9%) of the participants said that they experienced performance pressure Table 1. Sample characteristics (N = 3,141). Variable
% Participants (or mean with SD)
Gender
Male
40.0
Female
60.0
Age (mean, SD)
21.8 (3.45)
Ethnicity
Non-Dutch
11.6
Dutch
88.4
Living situation
Living with parents
70.9
Living independently
29.4
Year of study
1st
28.1
2nd
21.3
3rd
21.8
4th
18.4
Higher than 4th
10.4
Burnout
Emotional Exhaustion subscale (high-very high)
60.2
Depersonalization subscale (high-very high)
34.4
Personal accomplishment scale (very low-low)
53.2
Burned out
44.2
Performance pressure
Often or very often
68.9
Loneliness
Not
56.7
Moderate to very severe
43.3
Sense of belonging (mean, SD)
3.81 (0.60)
https://doi.org/10.1371/journal.pone.0267175.t001 Table 1. Sample characteristics (N = 3,141). 5 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 Predicting burnout symptoms in students in higher education Emotional exhaustion Table 2 shows that performance pressure, loneliness and sense of belonging explained 28.8%
of the variance in emotional exhaustion, while adjusting for gender, age, living situation and
year of study. Performance pressure, loneliness and sense of belonging were all significantly
predicting emotional exhaustion independently from each other (all p-values < .01). Back-
ground variables that contributed significantly (p < .05) to the prediction of emotional exhaus-
tion were gender, age, and year of study. Exhaustion was more present among younger, female
students and students that are longer on campus. Living situation did not explain emotional
exhaustion significantly. Table 3. Emotional exhaustion, depersonalization and personal accomplishment explained by multiple regression analysis (N = 3,141). Emotional Exhaustion
Depersonalization
Personal Accomplishment
Variable
Standardized β Standard
Error
p-value Standardized β Standard
Error
p-value Standardized β Standard
Error
p-value
Predictors
Performance
pressure
.29
.02
.000
.10
.02
.000
.00
.02
.817
Loneliness
.19
.02
.000
.12
.02
.000
-.15
.02
.000
Sense of belonging
-.28
.02
.000
-.47
.02
.000
.23
.02
.000
Covariates
Gender
-.11
.02
.000
.04
.02
.006
.08
.02
.000
Age
-.07
.02
.000
-.05
.02
.003
.02
.02
.338
Living situation
.02
.02
.242
-.02
.02
.299
-.02
.02
.445
Year of study
.07
.02
.000
.21
.02
.000
-.08
.02
.000
R2 = .288
R2 = .365
R2 = .107 onal exhaustion, depersonalization and personal accomplishment explained by multiple regression analysis (N = 3,141). PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 6 / 11 PLOS ONE Predicting burnout symptoms in students in higher education Depersonalization Regarding depersonalization, Table 2 shows that performance pressure, loneliness and sense
of belonging explained 36.5% of its variance, while adjusting for gender, age, living situation
and year of study. Performance pressure, loneliness and sense of belonging were all signifi-
cantly predicting depersonalization independently from each other (all p-values for
beta < .01). Comparing the standardized regression coefficients (beta’s) shows that sense of
belonging (β = -.47) appeared to be the strongest predictor of depersonalization compared to
performance pressure (β = .10) and loneliness (β = .12). Background variables that contributed
significantly (p < .05) to the prediction of depersonalization (p < .05) were age, gender and
year of study. Living situation did not contribute significantly. Depersonalization was more
present among students that were longer on campus. Personal accomplishment Regarding (the lack of) Personal Accomplishment, Table 2 shows that performance pressure,
loneliness and sense of belonging explained 10.7% of its variance, while adjusting for gender,
age, living situation and year of study. Loneliness and sense of belonging were both signifi-
cantly predicting personal accomplishment independently from each other (all p-values for
beta < .01). Performance pressure did not significantly predict personal accomplishment. Background variables that contributed significantly (p < .05) to the prediction of personal
accomplishment (p < 05) were gender and year of study A sense of reduced personal accom Regarding (the lack of) Personal Accomplishment, Table 2 shows that performance pressure,
loneliness and sense of belonging explained 10.7% of its variance, while adjusting for gender,
age, living situation and year of study. Loneliness and sense of belonging were both signifi-
cantly predicting personal accomplishment independently from each other (all p-values for
beta < .01). Performance pressure did not significantly predict personal accomplishment. Background variables that contributed significantly (p < .05) to the prediction of personal
accomplishment (p < .05) were gender and year of study. A sense of reduced personal accom-
plishment was more present among male students and students that were longer on campus. Age and living situation did not contribute significantly. PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 Key results Our results show that performance pressure, loneliness and sense of belonging are significant
predictors of burnout symptoms among students, especially of the facets relating to emotional
exhaustion and depersonalization. Sense of belonging appeared to be the strongest predictor
of all burnout symptoms. Our results are in line with the JD-R model, explaining that burnout
symptoms are caused by an imbalance between demands and resources, where high demands
lead to emotional exhaustion and low resources ensure depersonalization [14]. A study by Maslach, Schaufeli & Leiter [9] shows that work pressure plays an important
role in the development of burnout symptoms. Jacobs & Dodd [33] show that this also applies
to performance pressure among students, just like in the present study. Both studies show that
performance pressure is most strongly related to the development of emotional exhaustion
and less to depersonalization and personal accomplishment. It is likely that students who expe-
rience performance pressure work harder. Eventually, for most students it leads to symptoms
of exhaustion, which affects their functioning. The present study shows that exhaustion and depersonalization were more present among
students that are longer on campus and that exhaustion was more present among female stu-
dents, where depersonalization and a sense of reduced personal accomplishment were more
present among male students. This is in line with previous studies according to a meta-analysis
by Purvana and Muros [34] that states that women are more likely to report emotional exhaus-
tion and men are more likely to report depersonalization, but these gender differences lay
in the close-to-zero effect size range. Our findings regarding the higher presence of exhaustion
and depersonalization among students that are longer on campus support findings of a study
by Salmela-Aro & Read [35]. They state that burnout symptoms increase along with the PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 7 / 11 PLOS ONE Predicting burnout symptoms in students in higher education numbers of years of study. We believe that further study into the issue of gender, age and years
on campus differences is necessary. The appeared predictive value of sense of belonging confirms the important role of the
university context in the development of burnout symptoms. Hence, sense of belonging is
mostly about feeling at home at university. Students’ sense of belonging increases as they feel a
stronger connection with their university and the people there [22]. Limitations The measurement of performance pressure is debatable. Because performance pressure among
students is a fairly new concept, no reliable and valid questionnaire was available. This also
might have led to limitations regarding the estimation of perceived performance pressure in
students. However, our findings were in line with previous studies that are firmly rooted in the
theory of the JD-R model. Furthermore, because cross-sectional data were used, causal interpretations require cau-
tion. Students’ experiences are subject to change over time. As a result, it is unknown how
burnout symptoms develop over time. Longitudinal data are needed to confirm that students’
experiences predict burnout and that changes in students’ experiences predict changes in
burnout levels. Future studies should also focus on the occurrence and consequences of burn-
out and performance pressure among a broader sample of several universities, based on a new
definition of both burnout and performance pressure specifically for students. Finally, the results of the present study are based on a Dutch sample. An advantage of the
present study is the large sample that was found to be representative for the total population
of the university where the study was conducted in terms of the mean age and distribution of
students’ gender, faculties and year of study, which contributes to validated conclusions. The
extent to which burnout symptoms exist may differ between universities, however, there’s no
reason to assume that the associations found in the present study will differ greatly between
universities. Key results Thus, the feeling of not
belonging within the university context increases the risk of emotional exhaustion and deper-
sonalization: symptoms of burnout. Loneliness appeared to be a weaker predictor of burnout
symptoms than sense of belonging was. Loneliness can arise without being related to the study
context. This could explain why students can report low levels of sense of belonging and high
level of burnout symptoms in their studies, while not reporting loneliness. In addition to previous findings of Bakker, Demerouti, & Euwema [36], the present study
also shows a relationship between resources and emotional exhaustion. Sense of belonging
and social support appear to be important resources here that provide energy and reduce the
chance of getting emotional exhausted. Possibly, when students feel connected to the study
program and at the same time feel supported during this program, study tasks cost less energy
and exhaustion, therefore burnout symptoms, are prevented. Implications and conclusions This study highlights the importance of including sense of belonging, loneliness and perfor-
mance pressure in future studies of student burnout. There’s a need for a better understanding
of what underlies performance pressure, loneliness and burnout and how we can adequately
address these factors. There are indications that performance pressure, loneliness and burnout
symptoms are not only rooted within the educational environment, but also in a broader socie-
tal context [2,3]. In order to address these problems efficiently, it is important to examine the
origin of performance pressure, loneliness and burnout symptoms and the factors that may be
of influence within the educational setting thoroughly. 8 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 PLOS ONE Predicting burnout symptoms in students in higher education Our findings show that performance pressure and loneliness as demands, and sense of
belonging as a resource predict burnout symptoms in students. Performance pressure, as one
of the greatest concerns of this time regarding student wellbeing, is strongly associated with
mainly emotional exhaustion, but also with depersonalization. Performance pressure can
therefore be seen as an important demand during student life. There is a need to examine the
association between performance pressure, loneliness and burnout symptoms on the one hand
and academic performance on the other hand in more extended longitudinal studies, with a
starting point in the first year when students have no academic experience yet. Our findings increase the understanding of burnout development among students from a
more contextual perspective. They show the importance of the study environment as protec-
tive factor for the development of burnout symptoms. A lack of sense of belonging showed the
highest, significantly increased risk for all three dimensions of burnout. Therefore, sense of
belonging appears to be essential as a resource that leads to the prevention of burnout symp-
toms. This is not surprising, since the association between sense of belonging and positive
mental health is well demonstrated [28]. Several studies suggest that sense of belonging is a sig-
nificant predictor of both academic success and student mental health [37–40]. Students who
report a high sense of belonging adjust better to university life [39] and report lower levels of
depression and loneliness [38]. Bakker, Hakanen, Demerouti, & Xanthopoulou [41] found
that resources like social support and appreciation enhance the sense of belonging and miti-
gate the negative effect of high performance pressure. Acknowledgments We would like to thank all students and employees of Windesheim University of Applied
Sciences and HAN University of Applied Sciences who made a contribution to this study
and who supported the development of the web-based questionnaires. We would like to
thank Margot Peeters of Utrecht University for her contribution to the multiple regression
analysis. Implications and conclusions We therefore suggest to make sense of
belonging a priority in the enhancement of student wellbeing, as this resource could improve
the ability of coping with the high demands in student life. What happens to student wellbeing and academic performance if you enhance resources
like social support and a supportive study environment? With a better understanding of both
the course and consequences of burnout and possible other psychosocial problems and protec-
tive factors that make prevention of these problems possible, we will be able to promote the
wellbeing and student success of students in higher education. Investigation: Jolien M. Dopmeijer. Investigation: Jolien M. Dopmeijer. Methodology: Jolien M. Dopmeijer, Christine A. E. Schutgens, Ellen M. M. Jongen, Jannet M. de Jonge, Arjan E. R. Bos, Reinout W. Wiers. Project administration: Jolien M. Dopmeijer. Project administration: Jolien M. Dopmeijer. Resources: Jolien M. Dopmeijer. Resources: Jolien M. Dopmeijer. Supervision: Tommy L. S. Visscher, Ellen M. M. Jongen, Rob H. L. M. Bovens, Jannet M. de
Jonge, Arjan E. R. Bos, Reinout W. Wiers. Validation: Jolien M. Dopmeijer. Validation: Jolien M. Dopmeijer. Writing – original draft: Jolien M. Dopmeijer, Christine A. E. Schutgens. Writing – review & editing: Jolien M. Dopmeijer, Christine A. E. Schutgens, F. Rutger Kappe,
Nikkie Gubbels, Tommy L. S. Visscher, Ellen M. M. Jongen, Rob H. L. M. Bovens, Jannet
M. de Jonge, Arjan E. R. Bos, Reinout W. Wiers. Author Contributions Conceptualization: Jolien M. Dopmeijer, Christine A. E. Schutgens, Ellen M. M. Jongen, Jan-
net M. de Jonge, Arjan E. R. Bos. Data curation: Jolien M. Dopmeijer, Christine A. E. Schutgens. Data curation: Jolien M. Dopmeijer, Christine A. E. Schutgens. Formal analysis: Jolien M. Dopmeijer, Christine A. E. Schutgens, Ellen M. M. Jongen, Arjan
E. R. Bos. Funding acquisition: Jolien M. Dopmeijer. Funding acquisition: Jolien M. Dopmeijer. 9 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
December 21, 2022 PLOS ONE Predicting burnout symptoms in students in higher education Investigation: Jolien M. Dopmeijer. References 10 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0267175
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Correction: A New Strategy for Enhancing the Oral Bioavailability of Drugs with Poor Water-Solubility and Low Liposolubility Based on Phospholipid Complex and Supersaturated SEDDS
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Correction Correction: A New Strategy for Enhancing the Oral
Bioavailability of Drugs with Poor Water-Solubility and
Low Liposolubility Based on Phospholipid Complex and
Supersaturated SEDDS The PLOS ONE Staff The legends for Figure 3 and 4 are swapped. Please see the
corrected legends for Figure 3 and Figure 4 here. Figure 4. DSC thermograms of phospholipid, physical mixture,
SPC and scutellarin powder. All samples were heated from 50 uC to
300 uC at a rate of 10 uC/min. doi:10.1371/journal.pone.0084530.g004 Reference
1. Zhou H, Wan J, Wu L, Yi T, Liu W, et al. (2013) A New Strategy for Enhancing
the Oral Bioavailability of Drugs with Poor Water-Solubility and Low
Liposolubility Based on Phospholipid Complex and Supersaturated SEDDS. PLoS ONE 8(12): e84530. doi:10.1371/journal.pone.0084530
Figure 3. X-ray diffraction patterns of phospholipid, physical
mixture, SPC and scutellarin powder. All samples were scanned
over a range of 2 h angles from 3u to 65u with an angular increment of
0.02u per second. doi:10.1371/journal.pone.0084530.g003
Figure 4. DSC thermograms of phospholipid, physical mixture,
SPC and scutellarin powder. All samples were heated from 50 uC to
300 uC at a rate of 10 uC/min. doi:10.1371/journal.pone.0084530.g004 Figure 3. X-ray diffraction patterns of phospholipid, physical
mixture, SPC and scutellarin powder. All samples were scanned
over a range of 2 h angles from 3u to 65u with an angular increment of
0.02u per second. doi:10.1371/journal.pone.0084530.g003 Figure 4. DSC thermograms of phospholipid, physical mixture,
SPC and scutellarin powder. All samples were heated from 50 uC to
300 uC at a rate of 10 uC/min. doi:10.1371/journal.pone.0084530.g004 p
doi:10.1371/journal.pone.0084530.g003 Reference Citation: The PLOS ONE Staff (2014) Correction: A New Strategy for Enhancing
the Oral Bioavailability of Drugs with Poor Water-Solubility and Low Liposolubility
Based on Phospholipid Complex and Supersaturated SEDDS. PLoS ONE 9(2):
e91605. doi:10.1371/journal.pone.0091605 Published February 27, 2014 Copyright: 2014 The PLOS ONE Staff. This is an open-access article
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Strategi Implementasi Pendekatan Scientivic pada Pembelajaran Al-Qur’an Hadis untuk Meningkatkan Keterampilan Berpikir Kritis dan Sikap Ilmiah Siswa MAN di Provinsi Aceh
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Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk M. Chalis1* Muthmainnah2 Sulaiman3 1,2Universitas Islam Negeri Ar-Raniry Banda Aceh, Indonesia
3STAI-PTIQ Aceh dan Universitas Islam Negeri Ar-Raniry Banda Aceh
*1Email: m.chalis@ar-raniry.ac.id,
2,3Email: muthmainnah.ismail@ar-raniry.ac.id, sulaiman.man684@gmail.com 1,2Universitas Islam Negeri Ar-Raniry Banda Aceh, Indonesia
3STAI-PTIQ Aceh dan Universitas Islam Negeri Ar-Raniry Banda Aceh
*1Email: m.chalis@ar-raniry.ac.id,
2 3 1,2Universitas Islam Negeri Ar-Raniry Banda Aceh, Indonesia
3STAI-PTIQ Aceh dan Universitas Islam Negeri Ar-Raniry Banda Aceh
*1Email: m.chalis@ar-raniry.ac.id,
2,3Email: m thmainnah ismail@ar ranir ac id s laiman man684@gmail com 2,3Email: muthmainnah.ismail@ar-raniry.ac.id, sulaiman.man684@gmail.com Abstract This study aims to implement a scientific approach to Qur'an hadith lessons to
improve students' critical thinking and scientific skills at the Public Islamic
Schools (Madrasah Aliyah) in Aceh province. The research methods used were
qualitative, with 13 participants, including: three supervisors and ten Qur'an
hadith teachers who served in three Public Islamic Schools in Aceh province,
selected purposively. Data was obtained using interviews, observations, and
documentation. The result shows; (1) the strategy of implementing a scientific
approach to the learning of the Qur'an hadith at the Public Islamic Schools in
Aceh province begins with the preparation of a scientific-based learning
implementation plan and the application of scientific steps, and (2) The
application of a scientific approach has implications for improving students'
critical thinking with indicators of students actively engage in learning activities,
such as asking and discussing. In addition, it also has implications for improving
scientific attitudes, indicated by an increase in student curiosity about learning
the Qur'an hadith, respect (objective and honest), thinking critically, creative,
openness in group collaboration, perseverance, and care for each other. Keywords: scientific; strategy implementation; thinking skill; scientific skill Strategi Implementasi Pendekatan Scientivic pada Pembelajaran Al-
Qur’an Hadis untuk Meningkatkan Keterampilan Berpikir Kritis dan
Sikap Ilmiah Siswa MAN di Provinsi Aceh M. Chalis1* Muthmainnah2 Sulaiman3 Strategi Implementasi Pendekatan
M. Chalis, dkk Abstrak Penelitian ini bertujuan untuk mengetahui implementasi pendekatan scientivic
pada pembelajaran Al-Qur’an hadis untuk meningkatkan keterampilan berpikir
kritis dan sikap ilmiah siswa Madrasah Aliyah Negeri di provinsi Aceh. Motode
penelitian yang digunakan kualitatif dengan subjek 13 orang, meliputi; 3 kepala 648 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 dan 10 orang guru Al-Qur’an hadis yang bertugas pada 3 Madrasah Aliyah
Negeri di provinsi Aceh yang ditentukan secara purposive. Pengumpulan data
menggunakan teknik; wawancara, observasi, dan dokumentasi. Temuan hasil
penelitian; (1) strategi implementasi pendekatan saintifik pada pembelajaran Al-
Qur’an hadis pada Madrasah Aliyah Negeri di provinsi Aceh diawali dengan
penyusunan perencanaan pelaksanaan pembelajaran berbasis saintifik dan
penerapan langkah-langkah saintifik, dan (2) Penerapan pendekatan saintifik
berimplikasi terhadap peningkatan berpikir kritis siswa dengan indikator siswa
aktif melakukan aktivitas belajar, seperti bertanya dan berdiskusi. Selain itu,
berimplikasi juga terhadap peningkatan sikap ilmiah dengan indicator, sikap;
ingin tahu siswa dalam proses pembelajaran Al-Qur’an hadis meningkat, respek
(objektif dan jujur), berpikir kritis, kreatif, terbuka dalam kerjasama kelompok,
tekun, dan peduli antar sesama. p
Kata Kunci: saintifik; strategi implementasi; keterampilan berpikir; sikap ilmiah PENDAHULUAN Implementasi pendekatan santifik pada kurikulum 2013 bertujuan untuk
melibatkan peserta didik secara aktif dalam pembelajaran. Implementasi
kurikulum 2013 menuntut perubahan yang sangat mendasar dalam proses
pembelajaran, berupa pembelajaran yang menitikberatkan pada pembelajaran
aktif (Fitrah, A., Yantoro, Y., & Hayati, S. 2022; Yusuf, M., & Darimi, I. 2021). Sesuai dengan Permendikbud No. 65 tahun 2013 tentang Standar Proses,
kurikulum
2013
menggunakan
pendekatan
ilmiah
(saintifik)
sebagai
pendekatan pokok yang perlu diperkuat dengan pembelajaran berbasis
penyingkapan (discovery learning), pembelajaran berbasis penelitian (inquiry
learning),
dan
pembelajaran
berbasis
proyek
(project
based
learning). Keberhasilan pelaksanaan Kurikulum 2013 sangat ditentukan oleh keberhasilan
guru dalam mengembangkan pembelajaran berdasarkan pendekatan atau
model pembelajaran aktif tersebut. Partisipatif peserta didik secara aktif dalam pembelajaran Al-Qur’an dan
Hadis dapat dilakukan melalui penerapan pendekatan saintifik, tujuannya agar
peserta didik dapat melakukan aktivitas belajar secara optimal. Keterlibatan
peserta didik penting dan menentukan keberhasilan pembelajaran (Mulyasa,
2014). Peran aktif peserta didik dalam pembelajaran Al-Qur’an dan Hadis agar
dapat dikelola secara baik oleh guru untuk mengembangkan pembelajaran
yang optimal di sekolah. 649 Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk M. Chalis, dkk Implementasi pendekatan saintifik berimplikasi terhadap meningkatkan
kreativitas belajar dan sikap ilmiah siswa dalam pembelajaran Al-Qur’an dan
Hadis. Adapun indikator Keterampilan Berpikir Kritis (KBK) menurut Ennis
(1985), meliputi; 1) Menganalisis argumen (KBK 1). 2) Bertanya dan menjawab pertanyaan klarifikasi dan pertanyaan yang
menantang (KBK 2). 3) Mengeduksi dan mempertimbangkan hasil deduksi, (KBK 3). 4) Memutuskan suatu tindakan (KBK 4) (Saminan, N. F., Gani, A., & Safitri, R. 2016). Ciredes, A (2014) dalam penelitiannya terdapat tiga indikator kreativitas
siswa, mencakupi; (1) Kemampuan rasa ingin tahu siswa, (2) Kemampuan
siswa menentukan solusi dan jawaban, dan (3) Kemampuan siswa menentukan
kesimpulan. Sementara sikap ilmiah dalam penelitian Lestari, I. L., et al (2015)
mencakupi; (1) sikap ingin tahu, (2) luwes, (3) sikap kritis, (4) sikap jujur, dan
(5) ketelitian. Sedangkan menurut Sa'adah, H., & Kusasi, M. (2017) sikap ilmiah
dalam penelitiannya, meliputi; (1) Antusias mencari jawaban, (2) Menanyakan
setiap langkah kegiatan, (3) Tidak memanipulasi data, (4) Mengambil
keputusan sesuai fakta, (5) Menghargai pendapat/temuan orang lain, dan (6)
Berpartisipasi aktif dalam kelompok. Sejumlah
hasil
penelitian
membuktikan
bahwa
implementasi
pendekatan saintifik pada pembelajaran Al-Quran dan Hadis berimplikasi
positif terhadap meningkatkan hasil belajar, keterampilan berpikir kritis dan
sikap ilmiah siswa. Khakim, L., et al. PENDAHULUAN (2020) menjelaskan, penerapan
pendekatan saintifik dapat meningkatkan kemampuan berpikir kritis siswa
dalam pembelajaran, siswa aktif berdiskusi dan melakukan aktifitas belajar
melalui mengamati, menanya, menalar, mengolah informasi/mencoba, dan
mengkomunikasikannya. Pejelasan hampir sama juga disampikan oleh Manan,
A., & Imron, M. (2020) dalam artikelnya bahwa pendekatan saintifik 650 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 berkontribusi positif terhadap pengembangan atau peningkatan kemampuan
berpikir kritis peserta didik dalam pembelajaran Al-Qur’an hadis. berkontribusi positif terhadap pengembangan atau peningkatan kemampuan
berpikir kritis peserta didik dalam pembelajaran Al-Qur’an hadis. Raudah, R. (2018) dalam hasil penelitiannya menguraikan kemampuan
berpikir kritis dan sikap ilmiah siswa dipengaruhi oleh berapa faktor,
diantaranya penerapan pendekatan saintifik dalam pembelajaran rumpun
pendidikan agama Islam. Hidayah, N. S (2017), dalam hasil penelitiannya
pendekatan saintifik 93% relevan diterapkan dalam pembelajaran Al-Qur’an
Hadis dan berimplikasi postif terhadap pengembangan sikap ilmiah siswa
berupa antusias siswa mencari jawaban, aktif bertanya, Mengambil keputusan
sesuai fakta, dan berpartisipasi aktif dalam belajar kelompok. Dasar beberapa penelitian tersebut menunjukkan pendekatan saintifik
dalam pembelajaran Al-Qur’an hadis efektif digunakan atau diterapkan untuk
pengembangan sikap ilmiah dan keterampilan berpikir kritis pada siswa. Namun berdasarkan fakta penelitian awal yang dilakukan wawancara dengan
kepada Madrasah Aliyah Negeri di provinsi Aceh 2021 penerapan pendekatan
saintifik jarang digunakan guru dan sebagian guru masih menggunakan
pendekatan tradisonal berupa pendekatan pembelajaran yang berorintasi pada
guru dalam pembelajaran Al-Qur’an Hadis. Dasar permasalahan tersebut, peneliti tertarik untuk meneliti lebih
mendalam
terkait
dengan
implementasi
pendekatan
scientivic
pada
pembelajaran Al-Qur’an hadis untuk meningkatkan keterampilan berpikir
kritis dan sikap ilmiah siswa Madrasah Aliyah Negeri Di Provinsi Aceh. Penerapan Pendekatan Pembelajaran Saintifik dalam Pembelajaran Al-
Qur’an Hadis Pendekatan ilmiah atau scientific aproach merupakan pendekatan
pembelajaran yang menuntut terhadap keaktifan siswa dalam menemukan
pengalaman belajarnya melalui metode dan langkah-langkah ilmiah. Konsep
pembelajaran dengan pendekatan saintifik (scientific approach) dikenal juga
dengan proses pembelajaran yang menuntut siswa beraktivitas sebagaimana 651 Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk seorang ahli sains. Dalam praktiknya siswa diharuskan melakukan serangkaian
aktivitas layaknya langkah-langkah dalam metode ilmiah. Serangkaian
aktivitas yang dimaksud meliputi; (1) merumuskan masalah, (2) mengajukan
hipotesis, (3) mengumpulkan data, (4) mengolah dan menganalisa data, dan (5)
membuat kesimpulan (Machin, A. 2017). Penerapan pendekatan saintifik dalam pembalajaran Al-Qur’an hadis
berupa proses pembelajaran yang dilakukan dengan menggukanan metode
ilmiah (scientific teaching) atau cara kerja ilmiah yang melibatkan siswa secara
aktif
mencari
tahu
dalam
rangka
pengembangan
pengalaman
dan
pengetahuannya. Implementasi pendekatan saintifik pada pembelajaran Al-Qur’an hadis
memiliki bertujuan untuk meningkatkan kemampuan berpikir dan nalar siswa
serta dapat meningkatkan sikap ilmiah pada siswa. Terdapat enam tujuan
pembelajaran dengan pendekatan saintifik didasarkan pada keunggulan
pendekatan tersebut, antara lain: 1. Meningkatkan kemampuan intelek, khususnya kemampuan berpikir
tingkat tinggi. 2. Untuk membentuk kemampuan siswa dalam menyelesaikan suatu
masalah secara sistematik. 3. Terciptanya kondisi pembelajaran di mana siswa merasa bahwa
belajar itu merupakan suatu kebutuhan. 4. Memperoleh hasil belajar yang tinggi. 5. Untuk melatih siswa dalam mengomunikasikan ide-ide, khususnya
dalam menulis artikel ilmiah, dan 6. Untuk mengembangkan karakter siswa (Machin, A. 2014). Dasar keunggulan tersebut, penerapan pendekatan saintifik dalam
pembelajaran Al-Qur’an Hadis berkontribusi positif terhadap peningkatan
kualitas pembelajaran melalui proses pembelajaran yang melibatkan partisipasi
keaktifan siswa melalui kolaborasi dan kerja sama. Implementasi pendekatan 652 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 saintifik dapat juga dikaloborasi dengan metode inquiri dan model
pembelajaran kooperatif. saintifik dapat juga dikaloborasi dengan metode inquiri dan model
pembelajaran kooperatif. Langkah-Langkah Implementasi Pendekatan Saintifik dalam Pembelajaran
Al-Qur’an Hadis Secara
operasional,
sesuai
dengan
penerapan
Kurikulum
2013
pendekatan yang digunakan adalah pendekatan saintifik (scientific approach)
atau pendekatan berbasis keilmuan, di mana dalam proses pembelajaran
dengan pendekatan ini siswa difasilitasi guru agar dapat melakukan dan
mampu melaksanakan lima langkah pembelajaran. Adapun lima langkah pembelajaran dalam pendekatan saintifik sebagai
berikut: 1. Observing (mengamati) 2. Questioning (mempertanyakan) 3. Experimenting (mencoba) 4. Associating (menalar) 4. Associating (menalar) 5. Networking (membentuk Jejaring) untuk semua mata pelajaran
(Hanafiah, N., & Herlina, L. (2019). 5. Networking (membentuk Jejaring) untuk semua mata pelajaran
(Hanafiah, N., & Herlina, L. (2019). Lima langkah tersebut sesuai dengan Permendikbud Nomor 65 tahun
2013 tentang Standar Proses Pendidikan Dasar dan Menengah bahwa
pendekatan saintifik dilaksanakan dengan langkah-langkah sebagai berikut: (1)
mengamati, (2) menanyakan, (3) mencoba, (4) mengasosiasi, dan (5)
mengomunikasikan. Langkah-langkah tersebut dalam berbagai artikel oleh para penulis
sering digambarkan sebagai berikut yang menunjukkan langkah atau tahapan
yang harus dilakukan dalam penerapan pendekatan saintifik dalam proses
pembelajaram. 653 Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk Guru Al-Qur’an Hadis diharapkan mampu menerapkan pendekatan ini
dengan baik dalam memfasilitasi akativitas belajar siswa. Supaya lima langkah
tersebut dapat dilakukan dengan tepat oleh siswa maka harus didukung
desain perencanaan pembelajaran Al-Qur’an Hadis sehingga setiap langkah
tersebut dapat terlaksana dengan tepat dan benar serta relevan dengan
tuntutan Kurikulum 2013. Meningkatkan Keterampilan Berpikir Kritis Berpikir merupakan proses mental, para ahli memberikan pendapat
yang bermacam-macam mengenai berpikir. Ahli-ahli psikologi asosiasi
menganggap bahwa berpikir adalah kelangsungan tanggapan-tanggapan di
mana subjek yang berpikir pasif (Suryabrata, S. 2008). Sementara berpikir kritis
adalah aktivitas kognitif yang terkait dengan penggunaan pikiran. Berpikir
dengan cara kritis analitis dan evaluatif berarti menggunakan proses mental
seperti perhatian, kategorisasi, seleksi, dan penilaian (Cottrell, S. 2005). Sedangkan indikator seseorang memiliki keterampilan berpikir kritis menurut
Glaser dalam Fisher, A (2001), menjelaskan bahwa seseorang dikatakan
mempunyai kemampuan berpikir kritis sebagai: (1) An attitude of being disposed to consider in a thoughtful way the problems
and subjects that come within the range of one’s experience. (2) Knowledge of the methods of logical enquiry and reasoning. (3) Some skill in applying those method. Kemampuan berpikir kritis atau berpikir kreatif merupakan kemampuan
yang penting harus dikembangkan pada siswa dalam pembelajaran Al-Qur’a
hadis. Kemampuan berpikir kritis tersebut memili manfaat yang besar dan
dapat melatih kemampuan berpikir siswa dalam pemecahan persoalan hidup
dalam sehari-hari. Oleh sebab itu, pengembangan keterampilan berpikir kritis
urgen diterapkan dalam pembelajaran Al-Qur’an Hadis. Meningkatkan Sikap Ilmiah Uraian tentang sikap ilmiah sangat bervariasi menurut masing-masing
ahli, misalnya Anni dalam Ulfa, S. W. (2016) mengemukakan sikap merupakan 654 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 kombinasi dari konsep, informasi dan emosi yang dihasilkan di dalam
predisposisi untuk merespon orang, kelompok, gagasan, peristiwa, atau objek
tertentu secara menyenangkan atau tidak menyenangkan. Sejalan dengan penjelasan tersebut, Jasin, M (2010) menjelaskan bahwa
sikap ilmiah merupakan sikap yang perlu dimiliki oleh ilmuan, mencakupi
sembilan sikap: (1) Memiliki rasa ingin tahu yang tinggi dan kemampuan
belajar yang besar, (2) Tidak dapat menerima kebenaran tanpa bukti, (3) Jujur,
(4) Terbuka, (5) Toleran, (6) Skeptis, (7) Optimis, (8) Pemberani, (9) Kreatif atau
swadaya. Sikap ilmiah tersebut diperoleh para ilmuan melalui usaha yang
sungguh-sungguh dan kerja keras. Beberapa percobaan mereka lakukan
menumbuhkan sikap ilmiah tersebut. Kunci sukses dalam pembelajaran Al-Qur’an dan Hadis bagaimana guru
dapat memfasilitas proses pembelajaran dengan efektif dan mengembangkan
atau meningkatkan rasa ingin tahu sehingga muncul kemauan siswa untuk
mencari tahu dan menggunakan kemampuan berpikir untuk menalar. Aspek
inilah yang penting dikembangkan pada siswa pada pembelajaran Al-Qur’an
Hadis sehingga juga tidak terkesan proses pembelajaran Al-Quran Hadis
cenderung menoton dan membosankan. Upaya Meningkatkan Keterampilan Berpikir dan Sikap Ilmiah dalam
Pembelajaran Al-Qur’an Hadis Al-Qur’an hadis merupakan mata pelajaran rumpun Pendidikan Agama
Islam (PAI) yang menekankan pada kemampuan baca tulis yang baik dan
benar, memahami makna secara tekstual dan konstekstual, serta mengamalkan
kandunganya dalam kehidupan sehari-hari (Achmad, S. 2019). Mata pelajaran
ini pada Madrasah Aliyah dapat disebutkan sebagai lanjutan dari yang sudah
dipelajari siswa pada MTs. Namun mata pelajaran Al-Qur’ah hadis pada
Madrasah Aliyah lebih pada peningkatan yang dilakukan dengan cara
mengkaji, pelajari, dan memperkaya kajian Al-Qur’an hadis sebagai persiapan
untuk melanjutkan pendidikan pada jenjang pendidikan lebih tinggi. 655 Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk Pembelajaran Al-Qur’an hadis bagi sebagian siswa sulit untuk
mempelajarinya sehingga menimbulkan kebosanan dan siswa tidak bisa
berpikir kritis dengan baik seperti pada mata pelajaran lain yang dianggap
lebih mudah. Bila hal ini dialami oleh siswa maka guru harus melakukan
terobosan mengajar yang bersifat inovatif. Salah satu terobosan yang dapat
dilakukan guru Al-Qur’an hadis berupa penerapan pendekatan saintifik
sebagai
upaya
untuk
mengatasi
kebosanan
dan
bermanfaat
untuk
meningkatkan keterampilan berpikir kritis dan sikap ilmiah pada siswa. Sama halnya juga terhadap peningkatan keterampilan berpikir kritis
siswa dalam pembelajaran Al-Qur’an hadis guru dapat menerapkan
pendekatan saintifik (Fahrurrozi, M. 2021). Selanjutnya upaya peningkatan
keterampilan berpikir kritis dan sikap ilmiah pada siswa dalam pembelajaran
Al-Qur’an hadis melalui penerapan pendekatan saintifik dapat juga dipadu
dengan menggunakan pembelajaran kooperatif yang menekankan pada aspek
keaktifan siswa dalam proses pembelajaran. Strategi Implementasi Pendekatan Saintifik dalam Pembelajaran Al-Qur’an Strategi Implementasi Pendekatan Saintifik dalam Pembelajaran Al-Qur’an METODE PENELITIAN Penelitian ini menggunakan metode penelitian kualitatif. Menurut
Denzin & Lincoln (1994) menyatakan bahwa penelitian kualitatif merupakan
penelitian yang menggunakan latar alamiah dengan maksud menafsirkan
fenomena yang terjadi dan dilakukan dengan jalan melibatkan berbagai metode
yang ada. Erickson (1968) menyatakan bahwa penelitian kualitatif berusaha
untuk menemukan dan menggambarkan secara naratif kegiatan yang
dilakukan dan dampak dari tindakan yang dilakukan terhadap mereka
(Anggito, A., & Setiawan, J. 2018) Lokasi penelitian ini dilakukan tiga Madrasah Aliyah Negeri (MAN) di
provinsi Aceh, berupa; MAN 1 Kota Banda Aceh, MAN 1 Pidie Jaya, dan MAN
6 Kota Baro Aceh Besar dengan subjek penelitian 3 kepala Madrasah atau wakil
kepala madrasah dan 10 guru Al-Qur’an hadis pada tiga MAN yang ditentukan
secara purposive. Purposive adalah teknik subjek yang digunakan oleh peneliti 656 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 jika peneliti mempunyai pertimbangan-pertimbangan tertentu di dalam
pengambilan sampelnya (Arikunto, S. 2003). Adapun pertimbangannya subjek
berupa orang atau guru yang memahami tentang kondisi yang sedang kaji
dalam penelitian ini sehingga dapat memberikan data tepat. Teknik pengumpulan data melalui wawancara, dokumentasi, dan
observasi. Wawancara dilakukan dengan dengan kepala MAN dan guru Al-
Qur’an hadis terkait penerapan pendekatan siantifik, implikasi, peningkatan
keterampilan berpikir kritis, dan sikap ilmiah siswa pada Madrasah Aliyah
Negeri di provinsi Aceh. Selanjutnya dokumentasi yang diambil pada lokasi
penelitian berupa; proses pembelajaran dengan menggunakan pendekatan
saintifik dan suasana pembelajaran Al-Qur’an hadis pada Madrasah Aliyah
Negeri provinsi Aceh. Sedangkan observasi digunakan untuk melihat secara
langsung terhadap penerapan pendekatan saintifik dan partisipasi siswa dalam
pembelajaran Al-Qur’an hadis pada Madrasah Aliyah di provinsi Aceh. Berikutnya, analisis data dilakukan dengan menggunakan tiga langkah, yaitu;
reduksi data, penyajian data, dan verifikasi data. Hadis pada MAN di Provinsi Aceh Pendekatan saintifik merupakan pendekatan pembelajaran yang
diimplementasikan dalam proses pembelajaran sejalan dengan penerapan
kurikulum 2013 (Imron, M. (2016). Pendekatan saintifik pada masa sekarang
sudah sangat populer bagi kalangan pendidik atau guru dan siswa karena
dalam proses implementasinya berorientasi pada siswa dan siswa diarahkan
untuk lebih aktif dalam proses pembelajaran. Pendekatan saintifik juga relatif sering digunakan dalam pembelajaran
Al-Qur’an hadits pada Madrasah Aliyah di provinsi Aceh. Hal ini sebagaimana
data wawancara dengan guru yang bertugas pada Madrasah Aliyah Negeri
yang menjadi objek penelitian ini. Salah seorang guru menjelaskan 657 Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk M. Chalis, dkk implementasi pendekatan saintifik pada pembelajaran Al-Qur’an hadis
memiliki dampak positif untuk mendorong siswa lebih giat dalam melakukan
aktivitas belajarnya (AM. 2022). Guru lain memberikan data lebih lanjut terhadap kelebihan penerapan
pendekatan saintifik dalam pembelajaran Al-Qur’an hadis, sebagai guru mata
pelajaran Al-Qur’an hadis tentu kami melakukan langkah-langkah strategis
untuk membantu siswa agar cepat menguasai materi dalam proses
pembelajaran. Langkah strategis tersebut berupa menerapkan berbagai metode
dan pendekatan yang relevan untuk mempercepat siswa dalam belajar,
termasuk
menggunakan
pendekatan
saintifik
yang
dianggap
banyak
keunggulan (SR. 2022). Data tersebut didukung dengan data wawancara dengan kepala
Madrasah Aliyah Negeri 1 Kota Banda Aceh bahwa pendekatan saintifik
banyak keunggulan dan dapat melatih kemampuan berpikir kritis siswa
sehingga atas pertimbangan tersebut guru pelajaran Al-Qur’an hadis pada
madrasah ini sering menerapkan pendekatan saintifik (NS. 2022). Data yang
sama juga disampaikan oleh kepala Madrasah Aliyah lain. Lebih lanjut terkait strategi implementasi pendekatan saintifik dalam
pembelajaran Al-Qur’an hadis pada Madrasah Aliyah Negeri di provinsi Aceh
dilakukan dengan persiapan perencanaan berbasis saintifik dan mengikuti
langkah-langkah saintifik. Dua strategi ini diuraikan lebih lanjut sebagai
berikut. Perencanaan pembelajaran Al-Qur’an hadis berbasis saintifik Perencanaan Pelaksanaan Pembelajaran (RPP) merupakan rencana yang
menggambarkan prosedur dan manajemen pembelajaran untuk mencapai satu
atau lebih kompetensi dasar yang diterapkan dalam standar isi dan dijabarkan
lebih lanjut dalam silabus. RPP pada hakikatnya merupakan perencanaan
jangka pendek untuk memperkirakan atau memproyeksikan apa yang akan
dilakukan dalam pembelajaran (Mulyasa, 2007). 658 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 Data wawancara dengan guru Al-Qur’an hadis yang bertugas pada
Madrasah Aliyah Negeri di Aceh, umumnya guru sudah mempersiapkan
perencanaan silabus dan rencana pelaksanaan pembelajaran berbasis pada
pendekatan saintifik sebagaimana penerapan kurikulum 2013 pada madrasah. Data ini didukung dengan wawancara dengan kepala Madrasah Aliyah Negeri
di Aceh. Kepala MAN 1 Pidie Jaya menegaskan silabus dan RPP selalu
dimintakan untuk disusun oleh guru sebelum masa sekolah aktif dan saat
proses pembelajaran aktif mulai pada awal semester, silabus dan RPP tersebut
akan mintakan kepada guru sehingga bila terdapat guru yang belum
mempersiapkan RPP tidak dibolehkan melanjutkan proses mengajar hingga
memiliki RPP. Ia menambahkan, berdasarkan supervisi yang dilakukan guru
Al-Qur’an hadis sudah mempersiapkan dan memiliki silabus dan RPP yang
berbasis pada pendekatan saintifik (SD. 2022). Kepala MAN lain juga
membenarkan bahwa guru Al-Qur’an hadis mempersiapkan RPP berbasis
saintifik. Data wawancara dengan guru dan kepala madrasah sebagaimana uraian
tersebut didukung juga dengan data dokumentasi berupa silabus dan
perencanaan pelaksanaan pembelajaran mata pelajaran Al-Qur’an hadis yang
terdapat pada Madrasah Aliyah Negeri di Aceh bahwa silabus dan RPP yang
disusun oleh guru sudah memuat pendekatan saintifik didalamnya dan sudah
sesuai dengan penerapan kurikulum 2013. Berdasarkan berbagai data tersebut menunjukan guru Al-Qur’an hadis
yang bertugas pada Madrasah Aliyah Negeri di Aceh sudah mempersiapkan
perencanaan pembelajaran berbasis pendekatan saintifik. Perencanaan tersebut
telah dilakukan guru sejak awal masuk semester baru tujuannya agar
pelaksanaan pembelajaran Al-Qur’an hadits sesuai dengan perencanaan yang
sudah ditetapkan dalam silabus dan RPP. Silabus dan perencanaan pelaksanaan pembelajaran Al-Qur;an hadis
yang disusun guru pada Madrasah Aliyah Negeri di Aceh sudah memuat 659 Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk unsur-unsur saintifik yang memberikan penjelasan tahapan-tahapan saintfik. Perencanaan pembelajaran Al-Qur’an hadis berbasis saintifik Secara keseluruhan, perencanaan pembelajaran Al-Qur’an hadis, baik silabus
dan RPP sudah menggunakan pendekatan saintifik dan sesuai dengan
penerapan kurikulum 2013. Qur’an hadis Strategi implementasi pendekatan saintifik dalam pembelajaran Al-
Qur’an hadis yang dilakukan guru pada Madrasah Aliyah Negeri di provinsi
Aceh mengacu pada 5 langkah pendekatan saintifik yang lebih populer dengan
sebutan 5M. Penjabaran hasil penelitian lebih lanjut dapat dibaca pada uraian
hasil penelitian di bawah ini. Pertama, mengamati merupakan langkah pertama dalam penerapan
pendekatan saintifik. Guru Al-Qur’an Hadis pada Madrasah Aliyah Negeri
provinsi Aceh sudah mengimplementasikan pendekatan saintifik dengan
strategi mengikuti langkah pertama dalam pendekatan saintifik berupa
mengamati (observasi). Dalam kegiatan mengamati, guru memfasilitasi dan
memberikan kesempatan belajar kepada siswa untuk melakukan aktivitas;
melihat, membaca, mendengar, dan menyimak. Melalui kegiatan mengamati
dalam pembelajaran Al-Qur’an hadis dapat mengembangkan keterampilan
siswa dalam menggali pengetahuan baru atau informasi masi dan dapat juga
melatih ketelitian siswa. Inilah bagian kompetensi yang dapat dikebangkan
pada siswa melalui kegiatan mengamati dalam implementasi pendekatan
saintifik pada mata pelajaran Al-Qur’an hadis. Langkah kedua adalah menanya. Menanya dapat diartikan sebagai
kegiatan aktif siswa bertanya atau mengajukan pertanyaan dalam proses
pembelajaran. Siswa diberikan kesempatan untuk bertanya hal-hal yang terkait
dengan materi yang sedang dikaji. Kegiatan aktif siswa dalam bertanya harus
difasilitasi guru dengan baik karena aspek ini merupakan salah satu
kompetensi siswa yang harus dikembangkan, khususnya dalam pembelajaran
Al-Qur’an Hadis. Hasil penelitian menunjukkan, kegiatan bertanya siswa 660 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 pembelajaran Al-Qur’an hadis terlihat aktif. Aspek ini memiliki sasaran yang
bagus untuk melatih keterampilan berpikir kritis pada siswa. Oleh karena itu,
guru Al-Qur’an hadis pada Madrasah Aliyah Negeri di Aceh memfasilitasi dan
membimbing siswa dalam bidang bertanya dengan strategi implementasi
pendekatan saintifik. Lengkah ketiga, mengumpulkan data atau eksperimen/eksplorasi dalam
implementasi
pendekatan
saintifik
yang
menekankan
pada
aspek
pengumpulan data atau siswa diarahkan untuk mengumpulkan informasi
melalui berbagai proses belajar. Kegiatan mengumpulkan data adalah
melakukan eksperimen, membaca berbagai sumber informasi lainnya selain
yang terdapat pada buku teks, mengamati objek, mengamati kejadian,
melakukan
aktivitas
tertentu,
hingga
berwawancara
dengan
seorang
narasumber. Kompetensi yang ingin dikembangkan antara lain: peserta didik
akan mengembangkan sikap teliti, jujur, sopan, menghargai pendapat orang
lain,
memiliki
kemampuan
berkomunikasi,
memiliki
kemampuan
mengumpulkan informasi dengan berbagai cara, mengembangkan kebiasaan
belajar, hingga menjadi seorang pebelajar sepanjang hayat (life long learner)
(Musfiqon, 2015). Dasar data penelitian yang dihimpun pada Madrasah Aliyah di Provinsi
Aceh menunjukkan siswa terlibat aktif melakukan aktivitas belajar dengan
implementasi pendekatan saintifik. Qur’an hadis Siswa terlibat langsung dalam melakukan
eksperimen, membaca, mengamati, dan melakukan wawancara dengan teman
kelas atau guru. Aktivitas yang dilakukan siswa tersebut merupakan wujud
keaktifan siswa dalam pembelajaran Al-Qur’an hadis sangat bermanfaat untuk
melatih peningkatan keterampilan berpikir kritis siswa. Lengkah keempat, mengasosiasikan (mengolah informasi). Mengelolah
informasi wujud keaktifan belajar siswa dalam pembelajaran Al-Qur’an hadis
yang
harus
mendapat
perhatian
dari
guru
dan
memfasilitasi
pengembangannya melalui pendekatan pendekatan saintifik. Data penelitian
bahwa kegiatan siswa dalam mengoleh atau mengasosiasikan berimplikasi 661 Strategi Implementasi Pendekatan
M. Chalis, dkk Strategi Implementasi Pendekatan
M. Chalis, dkk terhadapat keterampilan berpikir, menganalisa, dan membentuk sikap jujur,
disiplin, dan teliti dalam mengolah informasi, data, dan materi yang dipelajari. Pengembangan keterampilan siswa dalam bidang pengolahan informasi
dilakukan secara berkelanjutan dalam proses pembelajaran Al-Qur’an hadis. Berikutnya langkah kelima berupa, mengkomunikasikan. Kegiatan
belajar mengkomunikasikan yang dilakukan siswa dalam proses pembelajaran
Al-Qur’an hadis berupa menyampaikan informasi atau pengetahuan yang
diperoleh dari hasil kegiatan membaca atau kegiatan belajar lain. Data
penelitian menunjukkan penerapan pendekatan saintifik dapat meningkatkan
keterampilan siswa dalam menyampaikan pengetahuan dan hasil belajarnya
kepada sesama siswa pada saat belajar Al-Qur’an hadis. Oleh karena itu,
pendekatan saintifik efektik diterapkan untuk meningkatkan keterampilan
berpikir kritis siswa. Demikian data tambahan dari guru Al-Qur’an hadis pada
MAN di provinsi Aceh. Implikasi
Penerapan
Pendekatan
Saintifik
terhadap
Peningkatan
Keterampilan Berpikir Kritis Siswa dalam Pembelajaran Al-Qur’an Hadis
pada MAN di Provinsi Aceh Implikasi
Penerapan
Pendekatan
Saintifik
terhadap
Peningkatan
Keterampilan Berpikir Kritis Siswa dalam Pembelajaran Al-Qur’an Hadis
pada MAN di Provinsi Aceh p
Pembelajaran saintifik merupakan pembelajaran yang mengadopsi
langkah-langkah saintis dalam membangun pengetahuan melalui model
ilmiah. Model pembelajaran yang diinginkan berupa memungkinkan untuk
peningkatan kecakapan berpikir sains, berkembangnya sense of inquiry, dan
kemampuan berpikir kreatif siswa (Satria, T. G. 2017). Penjelasan ini
memberikan mengisyaraktan bahwa pendekatan saintifik efetif diterapakan
untuk meningkatkan kemampuan berpikir kritis siswa. Sasaran utama penerapan pendekatan saintifik dalam pembelajaran Al-
Qur’an hadis bagi siswa bukan hanya belajar sekedar menghafal akan tetapi
dapat
mengidentifikasi
hadis-hadis
dan
mempraktekkan
atau
mengimplementasikan pesan hadis dalam kelas sesama teman, guru, dan
masyarakat sebagai bentuk keterampilan yang diperoleh peserta didik dari
proses pembelajaran yang dilakukan. 662 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 Keterampilan berpikir kritis siswa dalam proses pembelajaran Al-Quran
hadis penting ditingkatkan untuk mendukung mencapai tujuan pembelajaran
dan peningkatan kualitas pembelajaran. Keterkaitan mengapa peningkatan
berpikir kritis penting karena berpikir kritis merupakan sebuah proses yang
terarah dan jelas yang digunakan dalam kegiatan mental seperti memecahkan
masalah, mengambil keputusan, membujuk, menganalisis asumsi, dan
melakukan penelitian ilmiah (Johnson, 2009 dalam Sukroyanti, B. A., & Sufianti,
I. 2017). Senada dengan pendapat tersebut, berpikir kritis disebut juga sebagai
proses mental untuk menganalisis atau mengevaluasi informasi. Dengan
demikian, berpikir kritis dapat melatih siswa untuk merumuskan dan
mengevaluasi keyakinan dan pendapat mereka sendiri maupun pendapat
orang lain melalui serangkaian proses sistematis (Fahruddin Faiz 2012 dalam
Sukroyanti, B. A., & Sufianti, I. 2017). Data penelitian menunjukkan bahwa implementasi pendekatan saintifik
dalam proses pembelajaran Al-Qur’an hadits pada MAdrasah Aliyah Negeri di
Aceh berimplikasi positif terhadap peningkatan keterampilan berpikir kritis
siswa, meskipun masih terdapat sebagian kecil siswa masih kurang mampu
dalam berpikir kritis. Implikasi positif tersebut sebagaimana dapat dilihat pada
table berikut. Table. Indikator dan implikasi keterampilan berpikir kritis siswa dalam
pembelajaran Al-Qur’an hadis pada MAN di Aceh e. Indikator dan implikasi keterampilan berpikir kritis siswa dalam
pembelajaran Al-Qur’an hadis pada MAN di Aceh Table. Indikator dan implikasi keterampilan berpikir kritis siswa dalam
pembelajaran Al-Qur’an hadis pada MAN di Aceh
No
Indikator
Keterangan Implikasi Terhadap Keterampilan
Berpikir Kritis Siswa
1
Penjelasan/pertany
aan sederhana
(elementary
classification)
● Siswa mampu memberikan penjelasan atau
memberikan argumen sederhana terhadap
tanggung jawab orang tua. ● Siswa
mengajutan
pertanyaan
sederhana,
misalnya apa dalil ayat Al-Qur’an atau hadis
tentang tenggung jawab orang tua. ● Siswa dapat memberikan contoh sederhana
tentang tanggung jawab orang terhadap anak. 663 Strategi Implementasi Pendekatan
M. Implikasi
Penerapan
Pendekatan
Saintifik
terhadap
Peningkatan
Keterampilan Berpikir Kritis Siswa dalam Pembelajaran Al-Qur’an Hadis
pada MAN di Provinsi Aceh Chalis, dkk 2
Membangun
keterampilan dasar
siswa (basic support)
● Penggunaan sumber/buku referensi yang
multi dan relevan dengan materi pada
pelajaran
Al-Qur’an
hadis
dapat
meningkatkan
keterampilan
dasar
siswa,
termasuk keterampilan berpikir kritis siswa
dalam proses pembelajaran Al-Qur’an hadis. ● Penggunaan sumber referensi yang relevan
berimplikasi
keterampilan
berargumentasi
siswa dalam proses pembelajaran Al-Qur’an
hadis. ● Guru memfasilitasi kegiatan belajar siswa
dengan aktivitas observasi dan menyusun
laporan
observasi
berimplikasi
pada
peningkatan keterampilan dasar siswa dalam
pembelajaran Al-Qur’an hadis pada MAN di
Aceh. 3
Menyimpulkan
(inference)
● Kemampuan membuat kesimpulan dalam
proses pembelajaran sangat penting sehingga
setiap
siswa
mampu
mengambil
atau
membuat kesimpulan dari mater belajar. ● Implementasi
pendekatan
saintifik
dalam
proses pembelajaran Al-Qur’an hadis pada
MAN
di
Aceh
berimplikasi
terhadap
peningkatan
kemampuan
siswa
dalam
membuat kesimpulan yang berdasarkan kajian
dan data yang relevan yang ditelah dan
dipelajari siswa. ● Kesimpulan yang dibuat siswa berdasarkan
data dan fakta yang diperoleh dari proses
pembelajaran
Al-Qur’an
hadis
dengan
implementasi pendekatan saintifik. ● Penggunaan sumber/buku referensi yang
multi dan relevan dengan materi pada
pelajaran
Al-Qur’an
hadis
dapat
meningkatkan
keterampilan
dasar
siswa,
termasuk keterampilan berpikir kritis siswa
dalam proses pembelajaran Al-Qur’an hadis. ● Penggunaan sumber/buku referensi yang
multi dan relevan dengan materi pada
pelajaran
Al-Qur’an
hadis
dapat
meningkatkan
keterampilan
dasar
siswa,
termasuk keterampilan berpikir kritis siswa
dalam proses pembelajaran Al-Qur’an hadis. ● Penggunaan sumber referensi yang relevan
berimplikasi
keterampilan
berargumentasi
siswa dalam proses pembelajaran Al-Qur’an
hadis. ● Guru memfasilitasi kegiatan belajar siswa
dengan aktivitas observasi dan menyusun
laporan
observasi
berimplikasi
pada
peningkatan keterampilan dasar siswa dalam
pembelajaran Al-Qur’an hadis pada MAN di
Aceh. ● Kemampuan membuat kesimpulan dalam
proses pembelajaran sangat penting sehingga
setiap
siswa
mampu
mengambil
atau
membuat kesimpulan dari mater belajar. ● Implementasi
pendekatan
saintifik
dalam
proses pembelajaran Al-Qur’an hadis pada
MAN
di
Aceh
berimplikasi
terhadap
peningkatan
kemampuan
siswa
dalam
membuat kesimpulan yang berdasarkan kajian
dan data yang relevan yang ditelah dan
dipelajari siswa. p
j
● Kesimpulan yang dibuat siswa berdasarkan
data dan fakta yang diperoleh dari proses
pembelajaran
Al-Qur’an
hadis
dengan
implementasi pendekatan saintifik. 664 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221
4
Membuat
penjelasan lebih
lanjut (advance
clarification)
● Data penelitian yang dihimpun dari berbagai
sumber data pada Madrasah Aliyah Negeri di
Aceh menunjukkan implementasi pendekatan
saintifk dalam pembelajaran Al-Qur’an hadis
berimplikasi
terhadap
peningkatan
kemampuan siswa dalam membuat penjelasan
lebih lanjut. Implikasi
Penerapan
Pendekatan
Saintifik
terhadap
Peningkatan
Keterampilan Berpikir Kritis Siswa dalam Pembelajaran Al-Qur’an Hadis
pada MAN di Provinsi Aceh ● Kemampuan membuat penjelasan lebih lanjut
tersebut menjadi indikator siswa memiliki
kemampuan berpikir kritis yang didukung
dengan penerapan pendekatan saintifik. ● Implementasi
pendekatan
saintifik
dalam
pembelajaran
Al-Qur’an
hadis
juga
berimplikasi
terhadap
peningkatan
kemampuan siswa dalam mengkonstruksi
argumen-argumen baru yang berlandaskan
pada
kajian
dan
aktivitas
belajar
yang
dilakukan siswa. 5
Strategi dan taktik
(strategy and tactic)
● Implementasi
pendekatan
saintifik
pada
Madrasah
Aliyah
di
Aceh
berimplikasi
terhadap
peningkatan
kemampuan
siswa
dalam menggunakan strategi dan taktik
belajar. ● Siswa mampu mempersiapkan beberapa
alternatif sebagai solusi dalam menyelesaikan
masalah. ● Berimplikasi
juga
terhadap
penentuan
tindakan belajar yang harus dilakukan siswa. ● Berimplikasi terhadap menciptakan interaksi
pembelajaran Al-Qur’an hadis yang efektif. ● Berimplikasi
juga
terhadap
peningkatan
keterampilan
siswa
dalam
memberikan
argumen yang tepat dan relevan dengan
materi yang dikaji. Data
tersebut
selain
memberikan
penjelasan
implikasi
positif
implementasi pendekatan saintifik dalam pembelajaran Al-Qur’an hadits pada ● Kemampuan membuat penjelasan lebih lanjut
tersebut menjadi indikator siswa memiliki
kemampuan berpikir kritis yang didukung
dengan penerapan pendekatan saintifik. g
● Implementasi
pendekatan
saintifik
dalam
pembelajaran
Al-Qur’an
hadis
juga
berimplikasi
terhadap
peningkatan
kemampuan siswa dalam mengkonstruksi
argumen-argumen baru yang berlandaskan
pada
kajian
dan
aktivitas
belajar
yang
dilakukan siswa. ● Implementasi
pendekatan
saintifik
pada
Madrasah
Aliyah
di
Aceh
berimplikasi
terhadap
peningkatan
kemampuan
siswa
dalam menggunakan strategi dan taktik
belajar. ● Siswa mampu mempersiapkan beberapa
alternatif sebagai solusi dalam menyelesaikan
masalah. ● Berimplikasi
juga
terhadap
penentuan
tindakan belajar yang harus dilakukan siswa. j
y
g
● Berimplikasi terhadap menciptakan interaksi
pembelajaran Al-Qur’an hadis yang efektif. ● Berimplikasi
juga
terhadap
peningkatan
keterampilan
siswa
dalam
memberikan
argumen yang tepat dan relevan dengan
materi yang dikaji. Data
tersebut
selain
memberikan
penjelasan
implikasi
positif
implementasi pendekatan saintifik dalam pembelajaran Al-Qur’an hadits pada
Madrasah Aliyah Negeri di Aceh terhadap peningkatan keterampilan berpikir
kritis
siswa
juga
mengisyaratkan
penerapan
pendekatan
ini
sangat
menekankan pada keaktifan siswa selama proses pembelajaran sehingga siswa
dapat mengalami, melakukan secara langsung, dan memperoleh pengetahuan
dari pengalaman belajar yang dilakukannya. Data
tersebut
selain
memberikan
penjelasan
implikasi
positif
implementasi pendekatan saintifik dalam pembelajaran Al-Qur’an hadits pada
Madrasah Aliyah Negeri di Aceh terhadap peningkatan keterampilan berpikir
kritis
siswa
juga
mengisyaratkan
penerapan
pendekatan
ini
sangat
menekankan pada keaktifan siswa selama proses pembelajaran sehingga siswa
dapat mengalami, melakukan secara langsung, dan memperoleh pengetahuan
dari pengalaman belajar yang dilakukannya. 665 Strategi Implementasi Pendekatan
M. Implikasi
Penerapan
Pendekatan
Saintifik
terhadap
Peningkatan
Keterampilan Berpikir Kritis Siswa dalam Pembelajaran Al-Qur’an Hadis
pada MAN di Provinsi Aceh Chalis, dkk Implikasi Penerapan Pendekatan Saintifik terhadap Peningkatan Sikap
Ilmiah Siswa dalam Pembelajaran Al-Qur’an Hadis pada MAN di Povinsi
Aceh Implikasi Penerapan Pendekatan Saintifik terhadap Peningkatan Sikap
Ilmiah Siswa dalam Pembelajaran Al-Qur’an Hadis pada MAN di Povinsi
Aceh Implikasi Penerapan Pendekatan Saintifik terhadap Peningkatan Sikap
Ilmiah Siswa dalam Pembelajaran Al-Qur’an Hadis pada MAN di Povinsi
Aceh Sikap ilmiah merupakan sikap yang harus dimiliki peserta didik dalam
proses pembelajaran, khususnya pembelajaran Al-Qur’an hadis, peserta didik
tidak hanya fokus pada aspek penguasaan konseptual saja. Namun juga bagian
penting yang harus ditingkatkan pada peserta didik berupa sikap ilmiah. Misalnya peserta didik dituntut untuk menunjukkan kepekaannya terhadap
lingkungan sekitarnya, rasa ingin tahu, kejujuran, keterbukaan, kolaboratif,
dan tanggung jawab. Penerapan
pendekatan
saintifik
berimplikasi
positif
terhadap
peningkatan sikap ilmiah peserta didik dalam proses pembelajaran Al-Qur’an
hadis pada Madrasah Aliyah Negeri di Provinsi Aceh. Implikasi sikap ilmiah
peserta didik tersebut lebih lanjut dapat dilihat pada gambar berikut. Gambar. Implikasi pendekatan saintifik-sikap ilmiah peserta didik Secara keseluruhan berdasarkan data penelitian tersebut bahwa
implementasi pendekatan saintifik dalam pembelajaran Al-Qur’an hadits pada
Madrasah Aliyah di Provinsi Aceh berimplikasi terhadap peningkatan sikap
ilmiah peserta didik. Oleh karena itu, guru Al-Qur’an hadis diharapkan dapat 666 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
Vol. 12 No. 3 Juli-September 2022
ISSN 2089-5127 (print) | ISSN 2460-0733 (online)
DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 mengimplementasi pendekatan ini untuk mendukung terhadap peningkatan
keterampilan siswa dalam proses pembelajaran Al-Qur’an hadis. mengimplementasi pendekatan ini untuk mendukung terhadap peningkatan
keterampilan siswa dalam proses pembelajaran Al-Qur’an hadis. PENUTUP Pendektan saintifik memiliki banyak keunggulan, diantaranya dapat
meningkatkan keterampilan berpikir kritis dan sikap ilmiah siswa sehingga
pendekatan ini sering diimplementasikan oleh guru Al-Qur’an hadis pada
Madrasah Aliyah Negeri di provinsi Aceh. Strategi implementasi pendekatan
saintifik dalam pembelajaran Al-Qur’an hadis pada MAN di provinsi Aceh
dilakukan
secara
terformat
didukung
dengan
penyusunan
Rencana
Pelaksanaan Pembelajaran (RPP) yang berbasis pada pendekatan saintifik. Secara
operasional,
strategi
pelaksanaan
pendekatan
saintifik
dalam
pembelajaran Al-Qur’an hadis mengikuti langkah-langkah pendekatan
saintifik, berupa; mengamati, menanya, mengumpulkan data, mengasosiasi,
dan mengkomunikasikan. Penerapan pendekatan saintifik dalam pembelajaran Al-Qur’an hadis
berimplikasi terhadap peningkatan keterampilan berpikir kritis peserta didik
pada Madrasah Aliyah Negeri di povinsi Aceh dengan indikator; peserta didik
mampu membuat dan mengajukan pertanyaan, membangun keterampilan
dasar peserta didik, memberi kesimpulan dari materi yang dipelajari,
memberikan penjelasan lebih lanjut, siswa menggunakan strategi dan taktik
dalam menyelesaikan masalah sesuai dengan konteks materi. Selain itu, juga
berimplikasi terhadap peningkatan siskap ilmiah siswa dengan indikator
sebagai berikut; sikap ingin tahu peserta didik dalam proses pembelajaran
hadis meningkat, sikap respek (objektif dan jujur) dalam belajar, sikap berpikir
kritis, sikap kreatif, sikap terbuka dalam kerjasama kelompok, tekun, dan siswa
menunjukkan sikap peduli antar sesama. 667 Strategi Implementasi Pendekatan
M. Chalis, dkk DAFTAR PUSTAKA Achmad, S. (2019). Pengembangan Pembelajaran Materi Qur’an Hadits
Integratif-Inklusif Di Madrasah Aliyah. INSANIA: Jurnal Pemikiran
Alternatif Kependidikan, 24 (2), 262-277. Anggito, A., & Setiawan, J. (2018). Metodologi Penelitian Kualitatif. CV Jejak. Arikunto, S. (2003). Manajemen Penelitian, Cet. VI. Jakarta: Rineka Cipta, 2003. Ciredes, A., & Ariyanto, M. P. (2014). Peningkatan Kreativitas Siswa Dalam
Pembelajaran Matematika Melalui Strategi Cooperative Tipe Teams Games
Tournament (Tgt)(PTK Pada Siswa Kelas XI OB SMK Harapan Kartasura
Tahun Ajaran 2013/2014 (Skripsi, Universitas Muhammadiyah Surakarta). Cottrell, S. (2005). Critical Thinking Skill: Developing Effective Analysis and
Argument. New York: Palcrave Macmillan. Fahrurrozi, M. (2021). Urgensi Penguatan Keterampilan Berpikir Kritis pada
Mata Pelajaran Qur’an Hadist. Jurnal Penelitian Keislaman, 17(1), 39-50. Fisher, A. (2001). Critical Thinking: An Introduction. Cambridge: Cambridge
University Press. Fitrah, A., Yantoro, Y., & Hayati, S. (2022). Strategi Guru dalam Pembelajaran
Aktif Melalui Pendekatan Saintifik dalam Mewujudkan Pembelajaran
Abad 21. Jurnal Basicedu, 6(2), 2943-2952. Hanafiah, N., & Herlina, L. (2019). Pendekatan Saintifik Untuk Meningkatkan
Mutu Pembelajaran DI MA Kabupaten Bandung. Nusantara Education
Review, 2(2), 109-116. Hidayah, N. S (2017). Pengembangan Bahan Ajar Melalui Pendekatan Saintifik
Mata Pelajaran Al Qur‟ An Hadits Kelas VII DI MTS Negeri Kiran
Sidoarjo. Tesis Universitas Islam Negeri Sunan Ampel Surabaya. Imron, M. (2016). Implementasi Metode Saintifik Pada Mata Pelajaran Al
Qur’an Hadits Di Madrasah Aliyah Negeri Lamongan. Akademika, 10 (1),
42-45. Jasin, M. (2010). Ilmu Alamiah Dasar. Ed. Rev. Jakarta: PT Raja Grafindo Persada Khakim, L., et al. (2020). Implementasi Pendekatan Saintifik Dalam
Pembelajaran Al-Qur’an Hadis Di SMP Islam Tanwirul Hija Sayung
Demak. Prosiding Konferensi Ilmiah Mahasiswa Unissula (KIMU) Klaster
Humanoira, 1530-1540. Lestari, I. L., Budhi, U., & Dwi, A. B. (2015). Meningkatkan Kemampuan
Berpikir Kritis Dan Sikap Ilmiah Siswa Melalui Perpaduan Metode
Inquiry Dan Resiprocal Teaching Pada Materi Sistem Ekskresi di Kelas
XI IPA 5 Negeri 7 Kediri Tahun Pelajaran 2014-2015. Jurnal Biologi, Sains,
Lingkungan dan Pembelajarannya, 5(8), 276-280. 668 Jurnal MUDARRISUNA: Media Kajian Pendidikan Agama Islam
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DOI: http://dx.doi.org/10.22373/jm.v12i3.15221 Machin, A. (2014). Implementasi Pendekatan Saintifik, Penanaman Karakter Dan
Konservasi Pada Pembelajaran Materi Pertumbuhan. Jurnal Pendidikan IPA
Indonesia. JPII 3 (1), 28-35. Manan, A., & Imron, M. (2020). Implementasi Metode Saintifik pada Mata
Pelajaran al-Qur’an Hadits Kelas XI IPS di Madrasah Aliyah Negeri
Lamongan. Akademika, 14(01), 49-58. Mulyasa, E. (2014). Pengembangan Dan Implementasi Kurikulum 2013. Cet. IV. Bandung: Remaja Rosdakarya. Mulyasa. E. (2007). Kurkulum Tingkat Satuan Pendidikan. Bandung. PT. DAFTAR PUSTAKA Remaja
Rosdakarya. Musfiqon. Dkk, (2015). Pendekatan Pembelajaran Saintifik. Cet. I. Nizamia
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Konsep Menggunakan Model Pembelajaran Inkuiri Terbimbing (Guided
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Building a rapid autopsy program – a step-by-step logistics guide
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Translational medicine communications
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. (2020) 5:23 (2020) 5:23 Bacon et al. Translational Medicine Communications
https://doi.org/10.1186/s41231-020-00074-x Translational Medicine
Communications Open Access Abstract Background: Rapid Autopsy Programs offer an opportunity to collect tissue from patients immediately after death,
providing critical biological material necessary to develop more effective therapies and improve patient outcomes. Here, we present a step-by-step guide to build a cancer-focused Rapid Autopsy Program, based on our own
experiences building “The Legacy Project” at the City of Hope Comprehensive Cancer Center. Methods: The linear timeline of events is separated into four phases: 1) Building the Infrastructure, 2) Recruiting
and Consenting, 3) Preparing for Death, and 4) Tissue Collection and Follow up. Important considerations and
methods for adaptation are discussed throughout the protocol. Discussion: Using these methods, we successfully collected a total of 533 specimens from 9 subjects. The average
time from death to last specimen acquisition was 6.1 h (range: 4.03–7.66 h; median: 5.71 h). A diverse team with
various areas of expertise is critical for successful program implementation. Our goal herein this protocol is to
provide a comprehensive framework and foundation for other institutions to use as a model. Keywords: Rapid autopsy program, Warm autopsy, Rapid tissue donation, Cancer research, Metastatic, Tumor
heterogeneity Building a rapid autopsy program – a step-
by-step logistics guide Eliza R. Bacon1*
, Kena Ihle1, Peter P. Lee2 and James R. Waisman1 * Correspondence: ebarragan@coh.org
1Department of Medical Oncology, City of Hope National Medical Center,
Duarte, CA 91010, USA
Full list of author information is available at the end of the article Background significant logistic and financial issues that can limit suc-
cessful development and implementation. Metastatic tumor heterogeneity presents a significant
challenge in the clinic and is believed to drive mixed and
differential responses to therapy. Better understanding of
the origins of metastatic heterogeneity and tumor evolu-
tion under the pressure of therapy is needed to improve
patient outcomes and develop more effective therapeutic
strategies. Inaccessibility to metastatic specimens for re-
search use has historically been the largest hurdle for ad-
dressing these themes. Collecting tissues from stage IV
patients shortly after death provides a promising solu-
tion. These “rapid autopsy programs” (RAPs) are grow-
ing in popularity; however, there are only roughly 20
such programs scattered around the United States. While these programs stand to revolutionize the field of
cancer research, building and maintaining RAPs presents Development of our own program began with a com-
prehensive research phase designed to ascertain the crit-
ical components of a successful program. Initially we
found ourselves overwhelmed by the lack of guidance
and resources available in the field. We reviewed numer-
ous publications from other RAPs but found that while
informative
on
a
higher
level,
these
brief
communication-styled articles did not provide the level
of detail that we so desperately needed during our pre-
planning processes. Here, we present a step-by-step guide to build a
cancer-focused RAP, based on our own experience de-
veloping and piloting “The Legacy Project”, at City of
Hope. We present the realities and challenges of build-
ing a RAP based on our experience and discuss the nu-
ances of addressing a spectrum of issues, ranging from
basic science to end-of-life themes. We highlight com-
mon challenges as well as unexpected situations that * Correspondence: ebarragan@coh.org
1Department of Medical Oncology, City of Hope National Medical Center,
Duarte, CA 91010, USA
Full list of author information is available at the end of the article Bacon et al. Translational Medicine Communications (2020) 5:23 Page 2 of 14 Page 2 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 including coordination of care in consultation with the
participant’s clinical team. Although having a team of
multiple coordinators is recommended, consistent inter-
action with the same coordinator is ideal when working
with each family. A close relationship helps to ensure
continued engagement with the RAP and families should
be given the means to contact the coordinator directly if
needed after hours. Funeral Home Pick-Up p
Release documents Death certificate information (provided by hospice) Death certificate information (provided by hospice) Background In the immediate aftermath of a par-
ticipant’s death, the coordinator should continue to
interact with the family to console them, help with
paperwork, and coordinate with the funeral home. If the
right person is indeed serving in this role, they too will
grieve the loss of the participant, alongside the family. For this reason, dealing with multiple cases at a time, or
consecutive cases back to back, can be emotionally tax-
ing. Staff should be encouraged to “recharge” as needed
between cases. should be considered carefully, and we explore the un-
predictable circumstances of death and human nature,
on both logistic and psychological levels. Lastly, we offer
detailed insights into our own solutions as well as the
many subtle, yet critical, lessons learned. Key staff and important stakeholders
Program coordinator Consenting duties and patient interactions should be re-
stricted to a small team of individuals that is highly com-
passionate and able to respectfully broach sensitive
topics. The Program Coordinator serves as a resource to
the participant and family should any urgent issues arise, Recruitment Material Softly worded introductory brochure Detailed step-by-step enrollment booklet for patients
and families Consent documents Ante-mortem Blood Collection
Blood tubes
De-identified labels
Transport container
At Death
ID bracelet
Ice packs
Body bag
Bed sheet
SOP for body preparation
Tissue Collection
Autopsy Suite
Specimens list / Collection plan
De-identified labels (plus extra blank)
Collection tubes of choice (1.5 ml, 15 ml, 50, etc.)
Media(s) of choice
Wet ice
Dry ice
Large cooler for transporting specimens
Dolly for transporting cooler
Digital Camera
Pen/markers
Tissue Ink
Blades and equipment
PPE (Personal Protective Equipment)
Tissue Cassettes
10% Formalin
Funeral Home Pick-Up
Release documents
Death certificate information (provided by hospice)
Key staff and important stakeholders
Program coordinator Ante-mortem Blood Collection Blood tubes Procurement team Depending on the institutional setting, organizational re-
sources, and local laws, programs may choose to utilize
a diener, pathologist assistant (PA), or licensed patholo-
gist for the tissue acquisition procedure. In our experi-
ence,
finding
appropriately
trained
and
educated
individuals willing to perform a “rapid” procedure was a
significant challenge. We found that pathologists and
PAs were unmotivated to participate, largely because of
the on-call and after-hours nature of the program. Con-
versely, we found that while trained autopsy technicians
and dieners were more eager to participate, their skills
were not as sophisticated, especially when it came to
lymph node systems and tissue microdissections. Depending on the program’s organizational structure
and planned number of collections, procurement teams
may or may not be employed as dedicated members of
the RAP and may be contracted on a per-case basis. However, we recommend having at least one full-time
member of the procurement team to assist with speci-
men processing, storage, and tracking. Transportation Regulations as to who may transport a decedent after
death vary from state to state (see Supplementary
Table 1). Many of the programs we surveyed stated that
finding reliable and timely transportation was a signifi-
cant
challenge
during
program
development. This
proved to be true in our own experience and we urge
new programs to consider these issues early in the devel-
opment process. Reagents & equipment Recruitment Material Recruitment Material Methods A complete overview of the RAP protocol is shown in
Fig. 1. Stakeholders Stakeholders
Critical stakeholders are shown in Table 1. Research teams At least one research team member must always be on-
call and prepared to receive specimens during and after
hours. Some programs may choose to include the Bacon et al. Translational Medicine Communications (2020) 5:23 Page 3 of 14 Bacon et al. Translational Medicine Communications research teams in the procurement process while others
may tightly restrict access to the autopsy suite. Allowing
research teams into the room allows them valuable input
regarding specimen collection and helps ensure they re-
ceive the tissue and qualitative data they need. However,
unlike medical staff, basic research personnel are likely
to have little experience with human anatomy and may
be ill-prepared to work with a human cadaver. Further-
more, working with a freshly deceased body presents
additional psychological challenges, which may trigger
new or previous trauma in untrained personnel. We ad-
vise limiting the number of untrained personnel in the
autopsy suite and providing an “orientation” to all non-
medical research staff, including desensitization methods
such as photos or videos from autopsies, as well as an
interview with an institutional psychologist to screen for
previous trauma or increased risk for PTSD. Team
members should be encouraged to discuss their emo-
tional reactions with others and seek professional coun-
seling if unsettling thoughts or memories persist. We
found that holding group “debrief” sessions after each
case, under the guidance of a trained psychologist, can
help team members process their reactions to the pro-
cedure. Most importantly, participation in collection
procedures must be voluntary and staff who decline to
assist in the autopsy suite should not face negative re-
percussions from their supervisors. ensure team availability in case of emergency, illness, or
scheduled time off. ensure team availability in case of emergency, illness, or
scheduled time off. Early-program champions In the infant stages of program development, having
passionate supporters to champion the program is crit-
ical. These “champions” can be physicians, scientists, or
other leaders within the institution. RAPs are notori-
ously difficult to start and have complex logistic require-
ments. Sustained enthusiasm during challenging periods
is important to prevent loss of momentum and can help
secure
additional
support
from
others
within
the
institution. Phase 1: building the infrastructure
Determine which laws apply Each state has its own set of laws which govern many as-
pects of a RAP. In California for example, there are min-
imal
laws
that
regulate
the
death
industry. Laws
regarding consent, transportation of a decedent, and
who may perform the tissue collection procedure vary
from state to state (Fig. 2; Supplementary Table 1; also
see Supplementary: Determine which laws apply). Considerations: “autopsy” vs. “tissue collection” Aut-
opsies and tissue collections are not subject to the same
regulatory oversight. In general, a medical autopsy must
be ordered by a physician and supervised by a licensed
pathologist or PA. Hospitals may also require additional
liability paperwork in the case of autopsies. Additionally,
consenting guidelines differ between the two procedures. Most states allow an individual to self-consent to dona-
tion of his/her body or tissues after death. This consent
is legally binding and does not require the next-of-kin to
re-consent after death. In situations where an individual
dies without providing consent, the next-of-kin may
donate the tissues, as a decedent’s remains become
property of the next-of-kin at death. In the case of
medical autopsies, most states do not allow an individual
to consent to their own autopsy. Instead, it is required
that the next-of-kin provides consent after death. This
presents itself as a logistic challenge and could poten-
tially result in delayed transportation of the body. Assess the institutional landscape and create a steering
committee p
g
A reliable staff is the most critical component of a RAP;
without seamless coordination of countless moving
parts, a program will not be successful. The 24/7 nature
of RAPs and the demanding logistics require a dedicated
and flexible team. Death often occurs outside working
hours, in the middle of the night, and on weekends. Workplace and emotional burnout can be a real concern
for these teams and competitive compensation and job
flexibility should be offered to those who frequently
work after hours and on-call. Depending on the size of
the program and the number of cases being performed
each year, multiple coordinators and procurement teams
should be utilized to minimize the on-call burden placed
on single individuals, prevent workplace burnout, and RAPs are collaborative by nature and have many stake-
holders (see Table 1). Frequent and transparent commu-
nication
and
cooperation
between
departments
is
essential. The program’s early champions will be particu-
larly important during this step, as they will drive enthu-
siasm and support among critical stakeholders. The
steering committee should also provide input on policies
relating to sharing of data and ownership of intellectual
property that is born out of the program. Depending on
the organizational structure of the RAP and the institu-
tion, clearly worded collaboration contracts may be use-
ful on projects with multiple investigators. Page 4 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications (2020) 5:23 Table 1 Summary of program stakeholders and their respective roles
Stakeholder
Interest/Responsibility
Medical oncology
Responsible for identifying patients and serving as a bridge of trust as patients are introduced to RAP staff. Many patients
prefer to be approached by their primary oncologist first. Continues to provide standard of care regardless of enrollment
into the RAP, serves as the patient’s primary medical advocate, and acts in best interest of the patient. Honest and realistic
communication with patient and family regarding prognosis and life expectancy is critical. Radiology
Reviews patients’ imaging histories throughout course of disease. Provides summary highlights of disease history in the
context of remission, progression, and relapse - by organ. Gives recommendations regarding areas of interest. Supportive care
Aid in the identification of candidate patients. Are available to meet with patients struggling to cope with end of life issues. Meeting with research staff involved in collections to ensure coping appropriately. Assess the institutional landscape and create a steering
committee Provides input on recruitment strategies and educational handouts. Aid in identifying candidate patients. Ensure enrolled patients are placed in hospice programs compatible with the RAP. Facilitate introduction and initial communication between RAP and chosen Hospice company. Aid in identifying candidate patients. Ensure enrolled patients are placed in hospice programs compatible with the RAP. Facilitate introduction and initial communication between RAP and chosen Hospice company. Aid in identifying candidate patients. Serve as a bridge of trust as patients are introduced to RAP staff. Help patient and
family process key information related to the program requirements and obligations. Aid in identifying candidate patients. Serve as a bridge of trust as patients are introduced to RAP staff. Help patient and
family process key information related to the program requirements and obligations. Facilitate discussions with Patient Advocates. Guide development of study handouts and recruitment materials. Provide scientific proposals for new projects. Perform experiments. Assist in sample collection if needed. rovide scientific proposals for new projects. Perform experiments. Assist in sample collection if needed. Aid in identifying candidate patients. Serve as a bridge of trust as patients are introduced to RAP staff. Provide Legacy team
with updated and real time information regarding patient’s health status. Aid in identifying candidate patients. Serve as a bridge of trust as patients are introduced to RAP staff. Provide Legacy tea
with updated and real time information regarding patient’s health status. Responsible for notifying RAP team IMMEDIATELY after death and preparing body for transportation. Provide support to
both patients and families dealing with end-of-life issues. Provide RAP team with updated and real time information regard-
ing patient’s health status. Strongly encouraged to provide continuous care towards last 48 h of life expectancy. Declares
death, provides details of death to funeral home, and signs final death certificate. Responsible for transporting body immediately after death, within 1 hour’s notice. Expected to be professional and
compassionate when dealing with a decedent’s family. Responsible for transporting body immediately after death, within 1 hour’s notice. Expected to be professional and
compassionate when dealing with a decedent’s family. Retrieve body from Hospital. Coordinates with RAP staff and Nursing Administration to schedule pick up. Must be fully supportive and in agreement of patient’s wishes to donate. Responsible for informing RAP or Hospice staff
sudden change in health status. Have final authority to decline donation. Assists in discharge paperwork and release of body. Fig. 1 RAP protocol overview. Assess the institutional landscape and create a steering
committee Patient and family
advocates
Serve as stakeholders in project planning meetings. Provides input on recruitment strategies and educational handouts. Case management
Aid in identifying candidate patients. Ensure enrolled patients are placed in hospice programs compatible with the RAP. Facilitate introduction and initial communication between RAP and chosen Hospice company. Social work
Aid in identifying candidate patients. Serve as a bridge of trust as patients are introduced to RAP staff. Help patient and
family process key information related to the program requirements and obligations. Community education
Facilitate discussions with Patient Advocates. Guide development of study handouts and recruitment materials. Pathology
Responsible for histological assessment of collected specimens. Basic research
Provide scientific proposals for new projects. Perform experiments. Assist in sample collection if needed. Hospitalists
Aid in identifying candidate patients. Serve as a bridge of trust as patients are introduced to RAP staff. Provide Legacy team
with updated and real time information regarding patient’s health status. Hospice
Responsible for notifying RAP team IMMEDIATELY after death and preparing body for transportation. Provide support to
both patients and families dealing with end-of-life issues. Provide RAP team with updated and real time information regard-
ing patient’s health status. Strongly encouraged to provide continuous care towards last 48 h of life expectancy. Declares
death, provides details of death to funeral home, and signs final death certificate. Transportation
Responsible for transporting body immediately after death, within 1 hour’s notice. Expected to be professional and
compassionate when dealing with a decedent’s family. Funeral homes
Retrieve body from Hospital. Coordinates with RAP staff and Nursing Administration to schedule pick up. Participants, friends, &
family
Must be fully supportive and in agreement of patient’s wishes to donate. Responsible for informing RAP or Hospice staff of
sudden change in health status. Have final authority to decline donation. Nursing administration
Assists in discharge paperwork and release of body. Research operations
Administrative and operational interests. Table 1 Summary of program stakeholders and their respective roles
Stakeholder
Interest/Responsibility Reviews patients’ imaging histories throughout course of disease. Provides summary highlights of disease history in the
context of remission, progression, and relapse - by organ. Gives recommendations regarding areas of interest. Aid in the identification of candidate patients. Are available to meet with patients struggling to cope with end of life issu
Meeting with research staff involved in collections to ensure coping appropriately. Serve as stakeholders in project planning meetings. Assess the institutional landscape and create a steering
committee A summary of the four protocol phases and their respective steps. Phase 1 involves program development and organizational
structure. Phase 2 focuses on patient education and recruiting. Phase 3 involves tracking participants, communicating with family and hospice, and preparing
for the tissue collection. Finally, Phase 4 describes what should happen at death, during the collection procedure, and the follow-up after Fig. 1 RAP protocol overview. A summary of the four protocol phases and their respective steps. Phase 1 involves program development and organizational
structure. Phase 2 focuses on patient education and recruiting. Phase 3 involves tracking participants, communicating with family and hospice, and preparing
for the tissue collection. Finally, Phase 4 describes what should happen at death, during the collection procedure, and the follow-up after Bacon et al. Translational Medicine Communications (2020) 5:23
Page 5 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 Page 5 of 14 (2020) 5:23 Bacon et al. Translational Medicine Communications Fig. 2 Determine Which Laws Apply. State and local laws dictate which regulations apply to various aspects of the program. Requirements for
consenting, transporting the decedent, and performing the procedure should be verified during Phase 1. We have compiled a state-by-state
breakdown summary, which we provide in the supplementary materials Fig. 2 Determine Which Laws Apply. State and local laws dictate which regulations apply to various aspects of the program. Requirements for
consenting, transporting the decedent, and performing the procedure should be verified during Phase 1. We have compiled a state-by-state
breakdown summary, which we provide in the supplementary materials Fig. 2 Determine Which Laws Apply. State and local laws dictate which regulations apply to various aspects of the program. Requirements for
consenting, transporting the decedent, and performing the procedure should be verified during Phase 1. We have compiled a state-by-state
breakdown summary, which we provide in the supplementary materials Consideration: piloting the program
During early
phases of development, the ability to adapt quickly is
critical. Running the program as a small scale “pilot” ini-
tially, rather than as a fully defined program, has many
benefits and may offer some protection from institu-
tional politics that may slow decision making processes. A pilot program, led by a shortlist of leadership rather
than a larger committee, will also provide more flexibil-
ity and be more adaptable should unexpected issues
arise. Assess the institutional landscape and create a steering
committee For example, the first phase of our own program
included only ten metastatic breast cancer patients. Oversight was led by a small group of key stakeholders
and the primary goal of the “pilot” was to assess feasibil-
ity and optimize logistics. Completion of the pilot then
led to the creation of an expanded program within the
institution. (CPRMC) oversight. Conversely, research uses of col-
lected specimens are outlined in separate “secondary-
use” protocols, which are regulated by the CPRMC but
may or may not be subject to IRB regulation depending
on the HSR status. Other programs however choose to
combine both tissue-collection and research-use into
one protocol to ensure research integrity of the program
itself and to simply the oversight process. Decisions re-
garding
protocol
structure
and
HSR
determination
should be made in consultation with institution leader-
ship and the program’s steering committee. Solicit feedback from your patient population to determine
the needs and unique sensitivities that should be addressed
in the program Surveying a variety of interested parties is critical to
gauge public perception and level of support for the pro-
gram. Focus groups should include current patients,
family members, and survivors. Consult the institutional review board for guidance on
oversight committees, human subjects research (HSR)
determination, and suggested protocol structure gg
p
Because tissues are collected after death, many do not
consider RAPs to be Human Subjects Research, as de-
fined by 45 CFR 46, and argue that such programs are
not subject to IRB oversight. However, if blood or clin-
ical information is collected prior to death, an argument
can be made in favor of HSR determination. Committee
oversight and HSR determination may also depend on
the structure of the program’s protocols: specifically, if
the tissue collection and subsequent research compo-
nents are tied together or housed in separate protocols. For instance, in the Legacy Program, collection of tissues
and data is housed under a tissue-repository-styled um-
brella program and is subject to full IRB oversight with
an HSR determination. No research is conducted under
this parent protocol and therefore it is not subject to
Cancer Protocol Review and Monitoring Committee Considerations: primary versus metastatic patients
We observed stark differences in opinions between our
primary cancer survivors and those with metastatic dis-
ease, especially when it came to recruitment strategies. Primary cancer survivors were more likely to be intimi-
dated by the program (and research in general) and
insisted that the conversation should only be initiated by
their primary oncologist, with whom they had a close
and trusted relationship. Our metastatic survivors how-
ever, as well as their families, held a much less sensitive
view of the program and felt that the conversation re-
garding body donation should be “normalized” in the
clinic – with the topic being introduced early on in a pa-
tient’s care and that educational brochures be freely
placed in waiting and exam rooms. Since metastatic pa-
tients were our target population, we leaned towards Bacon et al. Translational Medicine Communications (2020) 5:23 Page 6 of 14 Bacon et al. Translational Medicine Communications Resource and contact information for supportive
care, child life services, bereavement support
List of local mortuaries and funeral homes
Instructions for care taker at the time of death
Program letter in case of emergency hospitalization
at outside institution Resource and contact information for supportive
care, child life services, bereavement support
List of local mortuaries and funeral homes
Instructions for care taker at the time of death
Program letter in case of emergency hospitalization
at outside institution their input when making key decisions, while still
attempting to balance the concerns of our more sensitive
patients. Identify vendors and order reagents Vendors such as transportation, diener, and facility with
autopsy suite should be contracted well in advanced with
job expectations clearly defined. Order reagents and
equipment needed for all phases. Considerations: situations that warrant disqualification Considerations: situations that warrant disqualification
In addition to clearly defined inclusion and exclusion
criteria, we recommend outlining situations that may
disqualify an enrolled subject from participating in the
final tissue collection. These guidelines should be clearly
explained to the patient and family at the time of con-
sent to avoid confusion or disappointment should such a
situation occur. Fig. 3 Example Timeline of Events. General timeline of events per
protocol for a RAP as defined by the Legacy Project. Example
timeline showing the sequence of events in relation to life
expectancy and time of death. Depending on the circumstances of
each patient’s disease, some events may occur out of order Examples include: Subjects who receive radioactive therapy shortly
before death Subjects who require a forensic autopsy Subject those who cannot be transported to the
autopsy suite within the required timeframe Subjects who die unexpectedly at home and are not
enrolled in hospice Consult the institutional review board for guidance on
oversight committees, human subjects research (HSR)
determination, and suggested protocol structure Both primary and metastatic patients agreed
that removing the “taboo” around death and body dona-
tion would improve accrual and lessen the emotional
impact of the initial conversation. Create timeline and define sequencing of events Create timeline and define sequencing of events
Outline what events will occur and the appropriate tim-
ing. A general timeline of events per protocol as defined
by the Legacy Project is shown in Fig. 3. For example,
consider when patients will be approached for consent –
early in their disease or shortly before anticipated death? Outline inclusion and exclusion criteria for the targeted
patient population. Criteria should include “commute
time” guidelines for patients who plan to die at home. Summary of events before and after death Summary the science being done Phase 2: recruiting and consenting g
g
Through the Legacy Project we defined a series of fac-
tors that are critical to successful patient recruitment
and consent. These are outlined in Fig. 4. Consideration: the “N of 1” model Having a stream-
lined and consistent set of processes is important, how-
ever,
programs
must
also
be
flexible
enough
to
accommodate the unpredictability of death. Rather than
establishing a rigid logistics plan and applying it uni-
formly to all cases, we recommend that cases should be
considered independently, with lessons learned from
previous cases applied to the next. This “N of 1” ap-
proach allows for quick adaption and the flexibility
needed to overcome unexpected challenges, especially
during periods of program development and expansion. Determine budget and funding sources Supporting physicians in their ability to address
sensitive end-of-life themes is important as patients
will look to them for approval to participate in the
program. Physicians with longstanding relationships
with their patients may have difficulty initiating end-
of-life conversations or broaching the topic of body
donation. End-of-life conversations that are worded
too gently can do more harm than good and may
leave families unprepared for the imminent passing of
the patient. Furthermore, it can be difficult to arrange
consent conversations in a timely manner when fam-
ilies
do
not
fully
understand
the
gravity
of
the
situation. Fiscal responsibility for costs associated with clinical op-
erations, procurement reagents, tissue processing, and
downstream experiments should be considered upfront. The source and use of funds should be clearly defined
for each component of the program. Stakeholders should
have a clear understanding of which budget components
they are responsible for funding. For example, participat-
ing research labs may be responsible for all downstream
processing and experimental costs. Educate physicians and other clinical staff about the
program and how to best approach patients Meet with clinical staff multiple times to present the
program, offer tips, and answer questions related to the
program. Repeatedly provide them with resource docu-
ments, recruiting guidelines, and frequently asked ques-
tions
to
help
them
feel
comfortable
approaching
patients. Addressing the “taboo” of death and normaliz-
ing the concept of body donation can reduce the emo-
tional burden of introducing the topic. Create educational material A) Softly worded introductory brochure that provides
non-technical, high level information about the pro-
gram. This generalized brochure can be broadly dis-
tributed in waiting rooms. Readers who wish to
learn more about the program should be instructed
to speak to their physician or contact the program
coordinator. B) Detailed enrollment packet should be given to
participants and family who consent to the
program. Enrolment packet should include information such as: Program coordinator’s direct contact information Summary the science being done Summary of events before and after death Bacon et al. Translational Medicine Communications (2020) 5:23 Page 7 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications Will ante-mortem blood be collected from participants,
and if so, how often? When will imaging and clinical his-
tories be reviewed prior to death? Will participants be
expected to die at home or in the hospital? How soon
after death will tissues be collected? Who is expected to
inform the program about a participant’s death? some decisions need to be revisited and dome details re-
fined. As the program grows and new research aims are
added, some details may also need to be re-optimized. Refine and finalize operation protocol(s) As institutions move through the various steps outlined
in this phase (Phase 1), it may become apparent that Fig. 4 Critical factors to successful patient recruitment and consent within a RAPSummary of key decisions and actions that are viewed positively
or negatively by patients and their families. These guidelines were developed based on conversations with target patient populations. Fig. 4 Critical factors to successful patient recruitment and consent within a RAPSummary of key decisions and actions that are viewed positively
or negatively by patients and their families. These guidelines were developed based on conversations with target patient populations. Define the recruitment strategy using feedback from focus
group sessions and physicians Define the recruitment strategy using feedback from focus
group sessions and physicians their care or alongside an end-of-life conversation was
most effective. When death is still an abstract concept,
patients can better process sensitive themes without a
strong emotional response and are better equipped to
appreciate the practicality and utility of body donation. At the other end of the spectrum, making the decision
to donate their body to research can help patients
process the reality of imminent death by giving them the
“final word” in their fight against their disease. Con-
versely, patients who were still receiving active therapy,
and whose prognoses were uncertain, were more likely
to be intimidated by the concept and less likely to en-
gage in a conversation. Furthermore, patients who are
resistant or unable to engage in a preliminary end-of-life
conversation are more likely to be shocked or trauma-
tized by the rapid autopsy concept. Consider the advantages and disadvantage between a
broad vs. targeted recruitment. A broad strategy lends it-
self to normalizing the concept. This can be advantageous
in situations where patients or their doctors struggle to
make the transition from active therapy to comfort care at
the end of life. This is also particularly true in disease mo-
dalities where the physician and patient have long stand-
ing relationships that may span years or decades (e.g. breast cancer). The taboo surrounding conversations
about death can be overcome by initiating the conversa-
tion early in a patient’s care and introducing the RAP to
all patients, regardless of disease status. In this way, body
donation can be likened to organ donation. Normalizing
these concepts early will also lesson the pressure put on
the physician to introduce the RAP during end of life con-
versations and can limit the negative impact of the conver-
sation on the patient’s mental status. A broad strategy,
however, is likely to illicit a strong response from many
patients at once. Smaller or newer programs may become
overwhelmed if they are unable to accommodate a large
volume of study subjects. Conversely, approaching pa-
tients one by one places a greater burden on the physician
and is likely to slow recruitment. However, this strategy
may be advantageous for newer programs that are still op-
timizing processes and gaining experience. Additionally, a
targeted strategy can be easily halted if recruitment needs
to be temporarily paused for any reason. Fig. 4 Critical factors to successful patient recruitment and consent within a RAPSummary of key decisions and actions that are viewed positively
or negatively by patients and their families. These guidelines were developed based on conversations with target patient populations. Page 8 of 14 Page 8 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications C) Include family in the consenting process and clarify
expectations at the time of death. Although a participant may fully commit to donation,
it is ultimately the surviving family that must follow
through with the decedent’s wishes and allow donation
to occur. Program candidates with family who are un-
supportive or dissent to participation should be consid-
ered ineligible for consent. The timing of events that
follow at death should be clearly explained. Depending
on how quickly the body must be transported, families
should be informed if they will have limited time to
spend with the decedent after death. Lastly, hospice
partners can assist family members by reiterating what is
expected to happen at the time of death and providing
emotional and bereavement support. Define the recruitment strategy using feedback from focus
group sessions and physicians A blending of
these two strategies can also be pursued by starting with
targeted recruitment and expanding broadly as the pro-
gram grows. A) The consenting process may require multiple
conversations over time and key details may need
to be reiterated in a sensitive, patient, and
empathetic way. It is important to have candid conversations with the
participant and family about their wishes regarding fu-
neral services. Families should be encouraged to speak
with their chosen funeral home prior to death to ensure
there will not be any issues in transportation. Addition-
ally, a planned viewing may impact the tissue collection
strategy. Procedures can be conducted in a way that still
allows for open casket viewings and knowing the family’s
intension ahead of time will ensure extra care is taken:
for example, making lower chest incisions on females to
allow for lower cut clothing and carefully making cranial
incisions that can be easily hidden by the hair. Candidates and their families should be given time
and space to consider relevant information and process
their feelings about the RAP and end-of-life topics; and
they should never be pressured to make a decision. B) Not every patient and family will be receptive and it
is important to wait for cues. B) Not every patient and family will be receptive and it
is important to wait for cues. For example, past aversion to research trials are a hint
that a patient is less likely to be open to body donation. However, patients who eagerly engaged in clinical trials
and other research protocols are more likely to be inter-
ested in continuing in research efforts, even after death. We found that approaching patients either very early in D) Encourage families to make funeral home
arrangements ahead of time. A) The consenting process may require multiple
conversations over time and key details may need
to be reiterated in a sensitive, patient, and
empathetic way. E) If possible, make sensitive or visible areas optional. Participants and families should be given the oppor-
tunity to opt out of collections from visible areas, such Bacon et al. Translational Medicine Communications (2020) 5:23 Page 9 of 14 Bacon et al. Translational Medicine Communications Page 9 of 14 as the face, hands, and neck, as well as other emotionally
sensitive organs such as the brain. However, depending
on presentation of the participant’s disease, an inability
to collect certain sites may impose restrictions on the
specific research questions that can be asked. In these
cases, it may be imperative to require participants to
fully consent to the collection of any and all sites of
disease. open casket funerals after tissue collection, serious con-
sideration must be made when deciding which tissues to
target. For example, sampling certain areas of the skull
might produce visible facial deformities and would likely
cause the family to incur unnecessary reconstruction
charges from the funeral home. Collect ante-mortem blood if applicable It is realistic that not every collaborating lab will want
every specimen collected from a single participant. Add-
itionally, depending on the specific research aims, speci-
mens may need to be collected in a specific way, or with
specific matched controls. Allow research labs the
chance to review the specimen list and determine their
own samples of interest. A single time point or serial samples may be collected
while the participant is living. Confirm team member schedules and create a call tree Confirm team member schedules and create a call tree
As death becomes imminent, it is helpful to have a cal-
endar of which team members are available after-hours
and over weekends. This calendar should be updated
frequently. The RAP coordinator should verify team
member contact information and create a call tree if
appropriate. Coordinate with hospice A) Communicate the “rapid” and 24/7 nature of the
RAP. Emphasize the importance of immediate
communication. Have a pre-defined collection plan p
p
Various philosophies exist regarding how an autopsy/
tissue
collection
should
be
performed
and
which
methods are most appropriate in a given situation. Often, specific techniques are chosen based on the
personal preference of the individual performing the
procedure. Given the importance of both speed and
accuracy in a RAP setting, we suggest creating a pre-
defined collection plan using consistent methodolo-
gies. Review of participant imaging and disease history
allows for streamlined sample collection. In complex
cases, a sample by sample strategy is developed to
keep the tissue collection team on track and ensure
all relevant specimens are collected. Consideration: balancing multiple cases at once De-
pending on the size of the RAP team and how closely
participants are monitored, tracking multiple terminal
patients at once can be challenging, requiring daily com-
munications with hospice, RA program staff, and partici-
pating research teams. In addition to logistics, balancing
multiple cases at once can be emotionally challenging;
especially if a single individual is serving as the primary
coordinator. Allow research labs to review the specimen list and
prioritize tissues to be collected Collect ante-mortem blood if applicable
A single time point or serial samples may be collected
while the participant is living. Phase 3: preparing for death
Track patient health The frequency to which participants are monitored at
the end of life is proportional to the targeted post-
mortem interval (PMI), type of specimens to be col-
lected, and the intended downstream research applica-
tions. Programs that allow collections as late as 24–48 h
after death likely do not need to monitor participants
daily, or even weekly, as long as death is promptly re-
ported. However, programs that aim to collect speci-
mens within a few short hours after death, or with
research applications requiring live cells, should closely
monitor critically ill participants. Participants enrolled
into hospice are best tracked by daily communication
with the hospice team. Prepare collection reagents and labels Pre-labeled tubes for anticipated specimens can be pre-
pared prior to the procedure. Extra blank labels and con-
tainers should be packed in case more tissues are
collected than were initially expected. The procurement
team should record the appropriate information for any
additional specimens on the container label as well as
the accompanying notes. Activate teams when death occurs As soon as a death has been reported, the RAP coordin-
ator should refer to the team member schedule and call
tree (phase 3, step 2) to notify and activate teams. After
confirming transportation timeframe, the coordinator
should continue to update the procurement and re-
search teams. Review imaging data and clinical history to create a
“specimens of interest” list It is important that hospice partners initiate con-
tinuous care towards end of life (24–48 h window) so
that death can be pronounced immediately and trans-
portation
can
be
expedited. The
role
of
hospice
should be to minimize the responsibility of the family
to care for a terminal patient, keep the coordinator
updated on the participant’s heath, promptly report Determine which research labs are expected to receive
which tissues. Consideration: the need to strategize compassionately
For patients and families wishing to hold viewings or Bacon et al. Translational Medicine Communications (2020) 5:23 Page 10 of 14 Page 10 of 14 Bacon et al. Translational Medicine Communications For example, participants can be assigned a code status
upon hospice enrollment; “Yellow”, “Orange”, and “Red”
alert: death, and if applicable, prepare the body for trans-
portation. A well-trained hospice team member can
also provide invaluable emotional support to the fam-
ily that will help prepare them to say goodbye quickly
after death. Yellow alert – approximately 2 weeks life
expectancy. Updates can be delivered daily. B) Create a detailed Standard Operating Procedure
(SOP) that clearly states program requirements,
expectations from hospice, and step by step
instructions to be followed at the time of death. Orange alert – participant is declining but not
actively dying; estimated life expectancy 3–7 days. Updates can be delivered once or twice daily. p
y
Red alert – Participant is actively dying and death is
imminent within 24–48 h. Updates should be
delivered every 4–5 h or when there is a significant
change in health status. Hospice companies vary in their practices. Even
within the same company, offices may function differ-
ently, depending on their service location. This can
pose a challenge when establishing a streamlined and
uniform set of processes. Fortunately, most variances
in hospice processes can be overcome through minor
adjustments to accommodate RAP requirements. Fam-
ily should never be asked to assist in preparing the
body after death; this should be strongly emphasized
in the SOP. We have provided an example SOP as a
supplementary document (SOP: Preparation of Body
at Time of Death). C) Provide a materials kit to be used to prepare the
body for transport. The RAP coordinator should make an effort to visit the
participant while on hospice. During this time, the co-
ordinator can answer questions related to the program
and make sure that the family and participant’s needs
are being met. These visits also serve to strengthen the
relationships with the participant, family, and hospice
partner. This kit should include: body bag, identification tags,
disposable bed sheet, and ice packs. Kits should be deliv-
ered to the hospice team and not the family. Depending on the consenting process, the RAP co-
ordinator may be required to meet with the next-of-
kin immediately after death. Regardless however, ef-
forts to visit after death is reported can bring comfort
to the family and can help ensure transportation hap-
pens smoothly. Considerations: make room for family While hospice
should take charge of activities relating to keeping the
program updated regarding the participant’s health sta-
tus, informing the RAP coordinator that death as oc-
curred, and preparing the body for transportation,
occasionally, a family member may take pride in per-
forming these actions. These family members view their
involvement with the program as an act of service to
their loved one and space should be made for these
members to feel useful. Prepare the body for transportation Depending on state laws, either hospice or the transport-
ing funeral home should prepare the body for transpor-
tation. This includes removing personal articles and
clothing, cleaning the body, and placing the decedent in
the body bag. Ice packs may also be placed along the
torso and around the head and neck to help preserve tis-
sue integrity until collection. Communicate updates and timeframe to teams
As life expectancy decreases, the RAP coordinator
should notify all teams, including transportation, pro-
curement, and research, of an imminent death. An alert
system can be helpful when communicating an expected
timeline and gives teams adequate time to complete time
sensitive tasks such as alerting staff, preparing perishable
media, un-silencing phones, booking time slots on
shared laboratory equipment, and preparing ice packs if
needed. To minimize variability between cases and ensure that
transportation happens expeditiously, the responsible
party should reference the SOP for body preparation at
the time of death. Transport decedent Many states only allow funeral home directors to trans-
port decedents and a transportation permit may be Page 11 of 14 Page 11 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications location vary wildly between subjects and fat deposits
can often mimic gross lymph node structure, making
accurate identification challenging. Furthermore, while
tumor-draining and disease-involved nodes are often
enlarged or inflamed, disease free nodes may be as
small as a single grain of rice. In a surgical setting,
lymph nodes will often be marked by a pre-surgery
tracer or dye injection. Additionally, most surgical
lymph node specimens are discovered and isolated
after surgery at the grossing bench which is not an
attainable option in a RAP setting. In order to over-
come this obstacle in the autopsy room, it is neces-
sary to have someone with training, close attention to
detail, and a willingness to learn and adapt. Review of
the specimen histopathology can be used to assess ac-
curacy
of
lymph
node
collection;
however,
this
process too has its own complications. Lymph nodes
with heavy tumor burden often lose their signatory
structures and can be difficult to confirm microscop-
ically. If the node has not yet been completely over-
taken by tumor, presence of the node’s outer capsule
can aid in confirmation, in conjunction with the aut-
opsy technician’s notes, and photographs from the
collection. Small, disease-free nodes should be placed
in a cassette with smaller holes to minimize the risk
that the tissues will be lost during processing. required. In these states, a close relationship with the
participant’s chosen funeral home or mortuary should
be established prior to death to ensure transportation
happens in a timely manner. In states with limited regu-
lation, a third-party non-emergency medical transporta-
tion company is an appropriate alternative. Coordinate funeral home pick up p
p
Once the procedure is completed, the RAP coordinator
should coordinate with the funeral home to arrange re-
trieval of the remains. The RAP coordinator should also
ensure
any
required
administrative
documents
are
completed. A) Refer to the collection plan created in Phase 3, Step 5. A) Refer to the collection plan created in Phase 3, Step 5. Specimen photos and notes should be recorded as
needed. B) Consider how to account for tumor heterogeneity
and dividing tumor specimens between
experiments. Tumor heterogeneity can pose a problem when split-
ting individual specimens between labs or experiments. To account for this tumor heterogeneity when dividing
samples, we suggest dividing the specimen into 3 sec-
tions, using the middle section for histological validation
and analysis. By validating this middle section, rather
than the tissue margin, a more accurate representation
of the specimen as a whole is captured. Although not a
perfect solution, this middle section can then be refer-
enced if conflicting characteristics are observed between
experiments using distinct tumor sections. To ensure
that histology sections are oriented correctly, consider
integrating inking into your processes. By marking the
sides of each section, you can keep track of which face
of the tissue block aligns to the individual sections that
were distributed to each lab. This also ensures that tis-
sue blocks are correctly oriented when tissues are
mounted for slide sectioning. Benchmarks and expected results Of the nine candidates who were not consented, two pa-
tients immediately declined when approached by their on-
cologists, three declined after consideration with their
families, one had family approach us at the time of con-
sent stating their desire to not participate despite the pa-
tient’s interest, one patient declined after learning her
chosen funeral would not retrieve her body without a
hefty surcharge, and two interested patients passed away
suddenly before consent. The two cases in which patients
passed away suddenly occurred early in program develop-
ment. At the time, we did not have a mechanism by which
next-of kin can consent after death. In response to this,
we have modified our processes to ensure that similar sit-
uations can be accommodated should they occur again. Using this protocol, we collected a total of 533 specimens
from 9 metastatic breast cancer subjects. The average time
from death to acquisition of the last specimen was 6.1 h
(range: 4.03–7.66 h; median: 5.71 h). Total number of speci-
mens collected from each participant ranged from 38 to 75,
with an average of 60 across all patients; the number of
tumor-positive specimens collected ranged from 12 to 46,
with an average of 29; the number of non-cancer specimens
collected ranged from 25 to 45, with an average of 31. g
g
Despite initial concerns that accrual would be challen-
ging, we found that nearly all patients who were
approached expressed interest in learning more about
the program and the vast majority wanted to participate. Complicated family dynamics was the primary reason
that patients declined or were not consented, highlight-
ing the importance of the family’s perception of the pro-
gram and their relationship with the program staff. During our pilot study, a total of 19 patients were
approached for consent over a period of 13 months
(Table 2). Of these, ten (53%) were consented and nine
successfully donated tissues at the time of death. Only
one
fully
consented
patient
did
not
undergo
the y
g
At the time of tissue procurement, one third of sub-
jects exhibited clinically unidentified diseased sites in or-
gans
not
commonly
associated
with
breast
cancer
metastases including ovary, kidney, and pancreas. In two
other instances, “resolved” bone specimens (as measured
by absence of FTG uptake in PET/CT imaging) were
later determined to be > 30% tumor positive when
assessed by H&E. Follow up with family (recommended) Follow up with family (recommended)
Our patient-led focus groups stressed the importance
of continued follow up with donor families, weeks
months, and even years after death of the participant. In the immediate aftermath of death, the RAP coord-
inator should continue to keep the family updated on
status of the procedure and when the funeral home is
expected to retrieve the body. A direct phone call is
most appropriate at this time, and families should be
encouraged to reach out to the coordinator if any-
thing else is needed. Continued follow up with family
provides them with the opportunity to resolve any
outstanding clinical issues and to give feedback about
the program and logistic processes. After roughly 2
weeks, a follow-up and thank you letter can be sent
to the next-of-kin. Yearly program updates, highlight-
ing the research findings and publications made pos-
sible
by
the
decedents,
can
be
sent
if
family
is
interested. C) To ensure tumor tissues can be traced back to their
original organ, all samples should be grossed with a
“tag” of normal tissue (or low tumor burden tissue,
if normal is not available). Specimens that have been entirely replaced by tumor
can be difficult to validate, especially in terms of con-
firming specific tissue of origin. A normal tissue tag en-
sures that the specimen can be validated by pathology
and that any processing errors (such as mislabeling) can
be corrected if they occur. D) Collecting and confirming lymph nodes structures
can be challenging. Lymph nodes are historically difficult to find, both
in surgery and post mortem. Lymph node size and Page 12 of 14 Page 12 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications collection procedure due to the family’s failure to notify
our team at the time of death. Authors’ contributions JRW and PPL conceived and initiated program development. JRW funded
this work through the Waisman Innovation Fund, privately funded by
grateful patients whishing to advance research in the field of metastatic
breast cancer. ERB directed and implemented the various aspects of the
program and coordinated operations, under the supervision of JRW and PPL. KI performed all post-mortem tissue collection activities and provided expert
advice in post-mortem logistics. All authors provided significant input regard-
ing the manuscript’s scope and purpose. ERB wrote the manuscript’s primary
text and constructed the figures and tables, with contributions from KI. All authors read and approved the final manuscript. Benchmarks and expected results Table 2 Summary of patients approached for consent and reasons for screen failure
Candidate
Consent status
Tissue collected
Reason unconsented or declined
01
Not Consented
No
Patient and family interested but patient died suddenly, prior to consent. Program logistics have been modified in response. 02
Consented
Yes
03
Declined
No
Patient lived further than 1 h away and wished to die at home. Chosen funeral home would not agree to retrieve body from hospital
without significant surcharge to family. 04
Consented
Yes
05
Not Consented
No
Patient and family interested but patient died suddenly, prior to consent. Program logistics have been modified in response. 06
Consented
Yes
07
Consented
Yes
08
Declined
No
Did not want to discuss any end of life topics. 09
Not Consented
No
Patient was interested but family declined at the time of consent. 10
Declined
No
Patient interested but was already enrolled in another body donation program. 11
Consented by NOK
Yes
12
Consented by NOK
Yes
13
Declined
No
Patient was interested but family declined at the time of consent. 14
Consented by NOK
Yes
15
Consented
No
Death was not reported by family. 16
Consented
Yes
17
Declined
No
Patient did not want to discuss end of life topics. 18
Not Consented
No
Patient was interested but family declined at the time of consent. 19
Consented by NOK
Yes Did not want to discuss any end of life topics. Patient was interested but family declined at the time of consent. Patient interested but was already enrolled in another body donation program. Patient was interested but family declined at the time of consent. Bacon et al. Translational Medicine Communications (2020) 5:23 Page 13 of 14 Page 13 of 14 Page 13 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications (2020) 5:23 Bacon et al. Translational Medicine Communications Efforts can also be made to collect post-mortem tumor
tissues from patients who die from other causes than
their disease. As a preliminary assessment of tumor heterogeneity, a
subset of samples was profiled for estrogen, progester-
one, and Her2 receptors, as well as the Ki67 antigen by
immunohistochemistry. Consistent with clinical hypoth-
eses regarding disease heterogeneity, we observed vari-
able expression of the estrogen, progesterone, and Her2/
neu
receptors
across
metastatic
locations
within
patients. Big data challenges While RAPs may open the door to seemingly unlimited
experimental possibilities, a sudden over-abundance of
tissue and data can be difficult to manage. This, com-
pounded with the various omic-datasets that can be gen-
erated from a single tissue, sets the stage for a number
of “big data” challenges that must be overcome by the
research teams. Comprehensive teams with experts in
wet-lab experiments, bioinformatics, and statistics are
critical to answering the complex research questions
asked of this kind of data. Abbreviations ER: Estrogen Receptor; Her2: Human Epidermal growth factor Receptor 2;
HSR: Human Subjects Research; IRB: Institutional Review Board; NOK: Next of
Kin; PA: Pathologist’s Assistant; PMI: Post Mortem Interval; PPE: Personal
Protective Equipment; PR: Progesterone Receptor; RAP: Rapid Autopsy
Program; SOP: Standard Operating Procedures What insights are left to see at death if the “war” has
already been lost? An important question that should be ask of RAP speci-
mens is whether the disease state at the time of death is
reflective of clinically relevant disease or is largely com-
posed of artifacts of death. Further research in the RAP
setting is needed to answer this complicated question. Co-interrogation of ante-mortem tumor and blood sam-
ples paired with RAP specimens can provide insight. Conclusion We conclude that each RAP is unique and is driven by
fundamental differences in: institutional governances
and resources, state laws, IRBs, and patient populations. These differences highlight the need to tailor RAPs and
to consider the specific needs of patients, researchers,
and the overall strategic goals of the institution. Import-
antly, these findings highlight the need to use a tested
framework and protocol when planning to establish a
RAP. We hope that the framework provided herein this
text assists others in building their own successful
programs. While these preliminary findings generate more ques-
tions than answers regarding mechanisms of metastatic
progression and resistance to therapy, they highlight the
utility of rapid autopsy in a research setting. We suggest
that many unanswered clinical questions can be ad-
dressed through interrogation of post-mortem tissues
and we urge research institutions to thoughtfully con-
sider adoption of the RAP model. Supplementary Information Concerning the PMI, the time from death to completion
of the collection procedure, goals should be driven by
the specific research intents. Although DNA may remain
relatively stable in specimens collected 24–48 h after
death, RNA quality declines with increasing PMI [1] and
altered transcriptional patterns have been observed in
the hours following death [2]. Results from specimens
taken further out should be interpreted with caution. Live cell experiments, cytokine signaling, and single cell
sequencing experiments are much more sensitive to
PMI and specimens intended to be used for these appli-
cations should be collected as quickly as possible. Deci-
sions regarding PMI should be made based on feedback
from collaborating research labs. pp
y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s41231-020-00074-x. Additional file 1: Supplementary Table S1. Additional file 2. Supplementary: Determine which laws apply. Additional file 3. Preparation of Body at Time of Death. Acknowledgements The authors thank several friends and colleagues including: Nicola Welch
PhD, CMPP for editorial assistance and critical review of the manuscript; our
clinical colleagues for believing in the program and trusting us with their
patients; Emma Wolfe MPH, CHES and City of Hope’s Patient and Family
Advisory Counsel for providing input into the development of patient
education material; Ashley Baker Lee SVP, for advocating for the program
and providing expert guidance; Matt Loscalzo LCSW, SPOS Fellow and
William Dale MD, PhD for providing our patients and staff with emotional
and supportive care; the generous donors of the Waisman Innovation Fund
through which we funded this work; countless others who helped ensure
the success of our efforts. Lastly, our most sincere and enduring gratitude to
our patient donors and their families whom, in a moment of crisis, made the
decision to give back to science and help restore health to others in need. Balancing the post mortem interval (PMI) Supplementary Information
Supplementary information accompanies this paper at https://doi.org/10. 1186/s41231-020-00074-x. References 1. Fan J, et al. Quantification of nucleic acid quality in postmortem tissues
from a cancer research autopsy program. Oncotarget. 2016;7(41):66906–21. https://doi.org/10.18632/oncotarget.11836. 1. Fan J, et al. Quantification of nucleic acid quality in postmortem tissues
from a cancer research autopsy program. Oncotarget. 2016;7(41):66906–21. https://doi.org/10.18632/oncotarget.11836. 2. Ferreira PG, et al. The effects of death and post-mortem cold ischemia on
human tissue transcriptomes. Nat Commun. 2018;9:490. https://doi.org/10. 1038/s41467-017-02772-x. Funding
h d g
The development of this program was funded through the Waisman
Innovation Fund, a research fund established by the many grateful donors
and patients of Dr. James Waisman. The funding body did not participate in Page 14 of 14 Bacon et al. Translational Medicine Communications (2020) 5:23 (2020) 5:23 Bacon et al. Translational Medicine Communications the study and collection, analysis, or interpretation of data, or in writing the
manuscript. Availability of data and materials The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. Competing interests The authors declare that there are no competing interests. Ethics approval and consent to participate pp
p
p
This study was approved by City of Hope’s Institutional Review Board under
study number 17503. Participants were enrolled either by self-consent prior
to death, or had their body donated after death by next-of-kin. Author details
1D
f 1Department of Medical Oncology, City of Hope National Medical Center,
Duarte, CA 91010, USA. 2Department of Immuno-Oncology, City of Hope
National Medical Center, Duarte, CA 91010, USA. 1Department of Medical Oncology, City of Hope National Medical Center,
Duarte, CA 91010, USA. 2Department of Immuno-Oncology, City of Hope
National Medical Center, Duarte, CA 91010, USA. Received: 27 October 2020 Accepted: 29 October 2020 Received: 27 October 2020 Accepted: 29 October 2020 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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HOW DIGITAL MARKETING AND PERCEIVED PERFORMANCE ARE INFLUENCING CONSUMER BUYING BEHAVIO
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Journal of Academic Research for Humanities 3(1) Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior HJRS Link: Journal of Academic Research for Humanities (HEC-Recognized for 2022-2023)
Edition Link: Journal of Academic Research for Humanities, 3(1) January-March 2023
License: Creative Commons Attribution-Share Alike 4.0 International License
Link of the Paper: https://www.jar.bwo.org.pk/index.php/jarh/article/view/163/version/163 Corresponding & Author 1: Zehra Jabeen Khan, MBA Scholar, University of Karachi,
Email: zehrajabeenkhan@gmail.com
Dr Sarah Anjum, Assistant Professor, University of Karachi,
Email: anjum.sar@uok.edu.pk Introduction Scholars have studied the function of
digital marketing in-depth for the past three
decades. Scholars have studied the function of
digital marketing in-depth for the past three
decades. Products that are available online and that
customers may like to choose and follow up
on. Digital marketing also served as a platform
for images, videos, and customer testimonials
about their experiences using products and
services. Social media gives information about
all customers extremely clearly; therefore,
using it would transform how people explore
information about products or services. However, current research is mostly
concerned with the function and effects of
digital marketing in business-to-consumer
(B2C) settings (Kim & Moon, 2021). The recent
advancement of social platforms is one of the
most significant changes in how people
connect. This study aims to study how digital
marketing affects consumer behaviour in the
expanding online purchasing industry. We'll
also learn how the effectiveness of each
digital platform affects consumers' decisions
to shop online. For businesses to effectively compete in
the new 2.0 & 3.0 marketplaces, ICT
(Information
and
Communication
Technologies) have been incorporated into all
of these contemporary marketing techniques. The emergence of the internet and the
digital economy of business in past recent
years has forced a reconsidering of marketing
tactics and, utmost of all, the purchasing
habits of consumers by granting individuals
access to global content, the ability to
produce content, the ability to access
Spreading
infrastructure
for
consumer-
generated media, and the ability to access
viewer (Tiewul, 2020). The internet has
greatly benefited communication, sharing
knowledge,
and
encouraging
individual
creativity and invention. This technical business perspective can
help
micro,
small,
and
medium-sized
businesses Modernize, obtain the tools they need to
establish a lasting position in the market,
particularly in the 2.0 and 3.0 sectors, and
successfully identify their customers online. In
fact, by 2017, digital channels are expected to
make a claim approximately one-third of
global advertising expenditure (Stephen,
2016). The impact of digital marketing on
Pakistan's major cities was investigated in this
study. The purchasing habits of Karachi
residents are studied when they make direct
or online purchases. The segments that follow
will
go
over
the
study's
theoretical
foundation, methods, findings, discussions,
and conclusions. More than seventy per cent of the
population of Pakistan uses a digital or net
platform to access the internet and social
platform to stay in touch with close ones. Paper Information Subject Areas:
1 Economics
2 Marketing Timeline of the Paper:
Received on: 09-02-2023
Reviews Completed on: 19-03-2023
Accepted on: 25-03-2023
Online on: 31-03-2023 Key Words: Digital marketing, consumer buying
behaviour, perceived performance, digital channels,
consumer online purchase behaviour 186 | P a g e 186 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior Digital marketing is also helpful for online
customer purchases because it may be a
source for Introduction As a
result, digital marketing is the starting point
for any business looking to enter the Pakistani
market and boost online sales and leads
(Scope of Digital Marketing in Pakistan -
Digital Marketing Pakistan, n.d.). Today,
many
people
use
social
sites,
media
platforms, smartphone apps, and other digital
communication technologies regularly. For
illustration, 37% of individuals in Pakistan
currently use the internet. Paper Information p
Citation of the paper:
(APA) Khan. Zehra Jabeen and Anjum. Sarah (2023). How Digital Marketing
and
Perceived
Performance
is
Influencing
Consumer
Buying
Behavior. Journal
of
Academic
Research for Humanities, 3(1). Citation of the paper:
(APA) Khan. Zehra Jabeen and Anjum. Sarah (2023). How Digital Marketing
and
Perceived
Performance
is
Influencing
Consumer
Buying
Behavior. Journal
of
Academic
Research for Humanities, 3(1). Citation of the paper:
(APA) Khan. Zehra Jabeen and Anjum. Sarah (2023). How Digital Marketing
and
Perceived
Performance
is
Influencing
Consumer
Buying
Behavior. Journal
of
Academic
Research for Humanities, 3(1). Currently, digital marketing has become an essential factor in
campaigns to attract and retain online buyers. This study aims to
identify how different digital marketing tools impact consumer
buying behaviour. This research shows the most appropriate actions
to take while planning an online strategy and retaining users. Furthermore, the study highlights the importance of perceived
performance in shaping consumer behaviour, as consumers are
more likely to purchase if they believe that a product or service
meets their expectations. Also, this research signifies running an
effective digital marketing campaign. This research has implications
for businesses looking to improve their marketing strategies and
enhance their customer relationships in the digital age. Non-
probability sampling method was used to select the sample. Convenience sampling involves selecting participants who are easily
accessible and willing to participate in the study. The data is
gathered through online surveys from a sample of 450 respondents. To analyze the data, the study used SPSS software and Smart PLS. SPSS is a statistical software package used for data analysis. The
conclusion of this research shows factors that should be utilized
while planning a campaign with digital marketing tools. The effect on
Consumer buying behaviour for online purchases is necessary for
all SMEs, e-commerce, and online retail industries, especially their
performance. Through this research, companies can identify which
campaign is suitable for them to run in the market of Karachi. A full
detailed study is required by having different performance indicators
as mediators or moderators in the future. Literature Review On the ground of digital marketing, there
are multiple reasons to do research. This topic
is very challenging, has a wide scope and
implies unique techniques or simply it is an
extensive, exclusive, and thought-provoking
topic. Despite not knowing where or how to
start, businesses are looking for a more
defined beginning point for their digital
marketing initiatives. Social media platforms
like Facebook, Twitter, Google, and other
businesses have been effective in modifying
consumer viewpoints and attitudes in the
contemporary era, which has ultimately
changed
many
enterprises. A
sizable,
comprehensive
customer
network,
trustworthy data, and real-time customer
experience feedback were used to achieve
this. Setting a goal to increase brand awareness
abroad through the use of digital marketing
strategies necessitates the development of a
well-thought-out
strategy
that
can
successfully take advantage of new technical
developments. For businesses that use it, digital
marketing offers five significant benefits.
First, both big and small businesses can do
this.
Second, comparing the advertising space
to print and broadcast media, there are no
real restrictions.
Third, compared to express mail or even
faxes, information access and search are
fairly quick. Problem statement Ray Tomlinson, and this innovation created
the framework for enabling users to send and
receive files across various machines. The
phrase "digital marketing" was first used and
became well-known in the 1990s. Between
1994 and 1996, the number of internet users
increased from 16 to 70 million due to
consumers purchasing personal computers
and using email. This study's main objective is to determine
how digital marketing affects consumers'
purchasing
behaviours. An
increasingly
common channel for brand and consumer
involvement over the past few years has been
digital marketing. To serve customers and
advertise goods and services, marketing
experts have used a variety of media for years. The performance of those platforms is crucial
since digital marketing also serves as a route
for online purchases. With a focus on
consumer time and security, we must
examine what motivates customers to shop
online and how any digital marketing agency
may enhance its performance to attract more
users as customers on online platforms. We
can
considerably
understand
consumer
buying behaviour online by analyzing the
performance impact of each digital marketing
instrument. The broad consensus is that large
corporations, like Apple and Amazon, are
reaping all the advantages that digital
technologies can provide, whereas small or
local businesses find it difficult to gain in any
way (Makrides et al., 2020). Online surveys
which are conducted for companies and
organizations are substantially more practical
for use to collect relevant information from
certain target audiences and analyze the
outcomes based on their responses. To
choose sensibly whether to purchase a good
or use a service, potential customers usually
check for its reviews and recommendation. On either hand, companies practice pertinent
online customer feedback to understand their
customers' demands better. Research Objectives: To
measure
changes
in
consumer
purchasing patterns caused by digital
marketing technologies. To assess which digital marketing platform
is most effective at altering consumer
behaviour. To understand the behaviour of online
buyers by perceived performance. 187 | P a g e 187 | P a g e 187 Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior A transition in marketing from traditional to
digital
Fourth, people in any corner of the world
can have access to a website with no time
restrictions. The
development
of
technology
is
intrinsically related to the expansion of digital
marketing. The first email was sent in 1971 by 188 | P a g e 188 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior
Fifth, the span for doing shopping and
research is likely reduces and can be done
efficiently.
Fifth, the span for doing shopping and
research is likely reduces and can be done
efficiently.
Fifth, the span for doing shopping and
research is likely reduces and can be done
efficiently. knowledge of important brand differences. (Hawkins, 2012) Says as the level of interest a
consumer has in purchasing a good or service,
the phrase "buying engagement" might be
used. The attributes of digital marketing as it
relates to promotion as a component of the
marketing mix (4Ps). Despite the posters'
positive product experiences and dedication
to these, these effects persisted (Schlosser,
2005) there are Website, Search Engine
Optimization (SEO), Paid Search Click-based
Advertising
(PPC
advertising),
Affiliate
marketing,
and
strategic
partnerships
(affiliate marketing and strategic partnership),
online public relations (Online PR), social
networks (social networks), Email marketing
(Email marketing), Customer relationship
management
(Customer
Relationship
Management. In addition, digital marketing
has
SMM,
Mobile
marketing,
Content
marketing and management, and Marketing
analytics. The study makes an effort to
thoroughly
investigate
the
relationship
between each aspect of the digital marketing
tool and Consumer behaviour on its own. Contrarily,
several
hypotheses
have
advocated undertaking additional research to
comprehend the behaviour all aims of people
like the CBB. (Kotler P. a., 2011) Give the following
description of Assael's model of the many
customer purchase choice behaviour types: 1. Complex buying behaviour - this term
describes consumers who are highly
involved in the purchasing or buying
process and who can discern substantial
brand distinctions. 2. Dissonance-reducing buying behaviour -
describes consumers who are very
involved in the purchasing process but
who are unable to distinguish meaningful
distinctions between brands. 3. Habitual purchasing behaviour - This
relates to consumers' nonengagement in
the purchase and their incapacity to
recognize significant brand differences. 4. Variety-seeking purchasing behaviour -
describes customers' minimal purchase
engagement
and
their
capacity
to
recognize substantial variations between
the products. A transition in marketing from traditional to
digital Influence on Buyer decisions is due to
several important cultural, psychological,
social, and personal factors. Demographics,
social groupings including families and
friends, and media influence on consumer
behaviour are all examined in consumer
behaviour research. Consumer Behavior towards Online
Shopping Consumer behaviour studies how people
make purchasing or buying decisions. It makes
an effort to understand the various phases
people take before making a purchase as well
as by what method consumers select, use, and
abandon a product and a service. Consumer
behaviour is also known as how consumers'
state of mind, attitudes, and preferences are
affecting their purchasing decisions. Previous studies have shown that even a
small number of unfavourable reviews can
significantly influence consumer purchasing
behaviour and choice (Schlosser, 2005). This is
because
a
consumer
may
create
a
fundamental link with other buyers. As a
result, consumers increasingly rely on social
media networks and websites to gather
information about products before making a
decision (Rasmunder, 2011). Considering the innumerable consumer
types and their distinct purchasing behaviours
grounded on their involvement with the
purchasing
process,
their
capacity
to
recognize and awareness of how consumers
behave while making purchases necessitates 189 | P a g e 189 | P a g e How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior Journal of Academic Research for Humanities 3(1) When social media networking and
websites are changing consumer perception,
we will analyze the result of overall Digital
marketing tools over a consumer. When discussing customer behaviour in
online marketing, complicated buying should
also be taken into account in addition to
habitual purchasing. When customers look for
several different sorts of things at once, this is
called complex buying (e.g., groceries and
clothing). Habitual buying behaviour relates to
consumers' limited buying engagement and
inability to recognize important brand
differences. Or in other words Habitual
purchasing - little or no customer interaction,
regular purchases, and brand distinction
(Ramo Palalic, 2020). Customers act in this way while making
expensive, infrequently purchased purchases. Before making a high-value investment,
consumers
actively
participate
in
the
purchasing process and research. A complex
purchasing behaviour might be buying a home
or an automobile. Consumers' high purchase
engagement and capacity to recognize key
brand differences are referred to as complex
buying behaviour (Palalic et al., 2021). In
these
circumstances,
consumer
behaviour does not follow the typical belief-
attitude-behaviour chain. The consumer does
not conduct in-depth research, weigh brand
attributes, and make significant selections
over "which brand to buy." Customers or
buyers do not have strong brand loyalty. Price
and sales incentives or discounts are
frequently used by marketers of low-effort
products with few brand variations to
encourage purchasing. Consumer Behavior towards Online
Shopping In complex purchasing, the customer will
go through a learning method where they
initially form their opinion about the product,
then consider its attributes, and finally make
a learnt purchase decision (Kotler P. , 1991). When customers fully participate in the
decision-making
cycle,
complex
buying
behaviour is at stake. What customers must
do to comprehend an interesting product is
stated as “involvement”. A high level of
participation, which is characteristic among
auto or vehicle buyers, indicates that the
individual
makes
a
great
effort
to
comprehend the product. The decision's
inherent characteristics are what motivate
this conduct. It's crucial to first comprehend the idea of
habitual purchasing to comprehend how
digital marketing and customer behaviour are
related. The act of routinely buying a good or
service is known as habitual buying, and it is
frequently motivated by an emotional
connection to a brand. Over time, consumers
will buy things because they have become
emotionally attached to them. In other words,
customers are more inclined to keep buying a
brand's items if they are already familiar with
them. First
of all,
complicated
items
are
costly. High involvement stems from this. This idea can be utilized in a broad range
of ways in terms of digital marketing. For
instance,
customers
may
start
to
favour particular online brand advertisements
over others if they get used to seeing them. Promoting discounts or other incentives that
are only valid if a customer uses an online
payment method, this kind of advertising can
also be used to encourage consumers to make
purchases. Second, complicated transactions involve
powerful emotions and a strong sense of
commitment. Third, large transactions require a lot of
personal information. Consumers associate
the cost of a complex buy with the time and
effort put into making it since it is pricey. Fourth, complicated purchases don't
happen frequently. The typical consumer 190 | P a g e 190 | P a g 190 Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior views their purchases as occurring in a
predictable monthly cycle. additional assurance for the decision they are
making for the purchase is right. Fifth,
complicated
purchases
are
unfamiliar in addition to being unusual. Dissonance-reducing
purchasing
behaviour is the result of consumers' intense
involvement in the purchase process and their
inability to discern distinguishing features
between brands (Palalic et al., 2021). Consumer Behavior towards Online
Shopping purchasing Lastly, it's rare for one person to make a
complicated purchase decision. No matter
how many reviews and ratings a dealership
receives, sellers and marketers are constantly
bombarded with information. The degree of
knowledge a consumer has gained from other
people's experiences influences how easily
they can make complex purchases. The significance of cognitive dissonance. The significance of cognitive dissonance. Numerous value assessments, choices,
and evaluations are influenced by cognitive
dissonance. You can greatly enhance your
capacity to make quicker and more accurate
decisions by gaining awareness of how
opposing philosophies affect the decision-
making process. Dissonance is defined as "a disagreement
of people's beliefs, acts, or characters" in the
dictionary. In a similar vein, worry after
purchases can be understood as dissonance-
reducing buying. Individuals' propensity to seek variety in
the services or products they purchase is
known
as
variety-seeking
in
purchase
behaviour. Such variation could develop over
time, for example, when a customer picks
various restaurants throughout a series of
session occasions. Consumers are very invested in their
purchases, but they struggle to distinguish
different brands. Consumers may experience "dissonance"
if they second-guess their decision after the
fact. Products from the financial services
industry, such as insurance, and investments,
are some examples. Every brand’s marketing
strategy has been built on a thorough
understanding of customer behaviour, and for
an organization to succeed, a thorough
investigation of all its facets is now necessary. Thus, cognitive dissonance and its effect on
consumer behaviour have also been covered
in numerous important research articles or
papers (Sharma, 2014). Individuals that engage in variety-seeking
behaviour in their consumption will avoid
having their utility decline as a result of
making the same purchases or utilizing the
same products again, switching brands, types,
or objects. (Ratner, 1999). People frequently
move between options or choose numerous
options from a decision set over time (Shaddy,
2021). Variety-seeking behaviour in the
marketing field also includes varying between
marketing efforts and offerings. Even though
customers can regularly purchase their
favourite products from a specific selection
set, prior research indicated that consumers
buy a certain number of diversified products
(Ratner, 1999). An author referred to it as one of social
psychology's
most
important
theories
(Aronson 1969). Dissonance, according to
(Sweeney, 2000) both a cognitive feature, as
implied by the title cognitive dissonance, as
well as an emotional component, as
suggested by several definitions, including
Festinger's original definition. When a consumer selects a portfolio of
products, variety may also be a crucial factor. For instance, when making a decision,
Customers may have used financial services or
investments. Customers want to pick a broad
portfolio. The significance of cognitive dissonance. Such products will be selected by
consumers
based
on
their
cost
or
convenience, and they will search for 191 | P a g e 191 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior (McAlister, 1982) Categorized different
behaviours in variety seeking are derived or
direct. Derived variety-seeking conduct, or
activity not directly related to a need for
variety, was the outcome of another
incentive. Numerous
requirements,
numerous users, or numerous circumstances
led to this kind of variety-seeking. Direct
activity motivated by intrapersonal goals was
described as variety-seeking behaviour: performance. In other words, the speed is
perceived by the user of a website. After
Information technology induction in market
and marketing strategies, these performance
levels do not remain only to calculate profit
and loss margins. The theory of UTAUT was used to examine
the factors that led to the acceptance of
mobile or smartphone apps. The findings
confirmed the value of routine, hedonic
motivation, social influence, and performance
expectations (Mütterlein, 2019). Variety-seeking is motivated by a need for
novelty, change, or satiation with a product's
features. Perceived Performance first checks the
effectiveness and ineffectiveness of an
objective or approach to run on a specific
platform. Three primary elements are identified in
the research today as driving variety-seeking
behaviour. In line with the (McAlister, 1982)
direct variety-seeking theory, the first factor
describes how customers actively seek out
diversity for their reasons. To clarify what
triggers the desire for variety, we refer to this
form of motivation for variety-seeking as
satiation/stimulation. The web performance community used
Page Load Time (onLoad) and a few other
page-level navigation metrics in the past for
assessing the performance (QoE) of a website. According to (Gao et al., 2017) According to a
common industry orthodoxy, the smaller
these measurements, given the same network
conditions, content organization, and other
controllable characteristics, the better the
quality of experience of a webpage will be
(from the end-user viewpoint). The
second
factor
is
outside
circumstances, which is related to the derived
variety-seeking of (McAlister, 1982). Under these circumstances, consumers
look for diversity because of external
constraints rather than an immediate internal
demand for variation. The third incentive,
which we refer to as future preference
uncertainty, is another that does not appear
in the (McAlister, 1982) paradigm. The significance of cognitive dissonance. The above
discussion told us about so many things which
impact consumer variety-seeking perspective,
and digital online platforms can influence it
through daily updates and trending variety of
products or services. These researches were only for web
pages, but after the social era, things behaved
differently because of the expansion of Digital
platforms. Various digital marketing tools and
platforms have their performance level and
uniqueness. Load time in a webpage can
affect the website, resulting in negative
performance. These negative factors can
decrease ranking, increase bounce rate, and
ultimately the purpose of SEO and PPC
diminish. All other factors can increase and
decrease a user's or consumer’s perceived
performance. Hypothesis: In the evidence of the above literature
review following is the hypothesis which is
formed by the author: H1: Digital marketing (DM) has a positive
impact on consumer buying behaviour
(CBB) H1: Digital marketing (DM) has a positive
impact on consumer buying behaviour
(CBB) H2: Mobile Marketing (MM) has a positive
impact on Consumer Buying Behavior
(CBB). H2: Mobile Marketing (MM) has a positive
impact on Consumer Buying Behavior
(CBB). The study's primary goal is to evaluate
consumer purchasing patterns to digital
marketing when those campaigns perform
poorly with consumers who use or shop
online. The study's main goal is to gather
information and generalize findings from a
wide sample of the population. As a result, it
emphasizes
completing
a
structured
questionnaire with formal questions asked in
a precise order and structured response
options. H3: Social media marketing (SMM) has a
positive impact on Consumer buying
behaviour (CBB). H4: Pay-per-click (PPC) advertising has a
positive impact on consumer buying
behaviour (CBB) H4: Pay-per-click (PPC) advertising has a
positive impact on consumer buying
behaviour (CBB) H4: Pay-per-click (PPC) advertising has a
positive impact on consumer buying
behaviour (CBB) H5: Search Engine Optimization (SEO) has a
positive impact on consumer buying
behaviour (CBB) H6: Email marketing (EM) has a positive
impact on Consumer buying behaviour
(CBB) Whereas on our variables like Digital
marketing
and
CBB
consumer
buying
behaviour, research has already taken place,
but we are applying it to an exploratory
variable, so questions driven from both are
interlinked with descriptive and exploratory
and their correlation over each other. This
research has a proposition of scientific
knowledge and business sciences thus, the
approach of this research is Basic. Is
theoretical base and theories which are being
used in this research are UTAUT (Unified
Theory of Acceptance and Use of Technology),
TAM (technology acceptance model), and CBB
(consumer buying behaviour). H7: Consumer buying behaviour is positively
influenced by digital marketing tools. H8: If perceived performance (PP) is effective
then digital marketing (DM) tools will be
more effective. H8: If perceived performance (PP) is effective
then digital marketing (DM) tools will be
more effective. H8: If perceived performance (PP) is effective
then digital marketing (DM) tools will be
more effective. H9: Perceived performance (PP) influences
the
relationship
between
Digital
marketing (DM) and consumer buying
behaviour (CBB) H9: Perceived performance (PP) influences
the
relationship
between
Digital
marketing (DM) and consumer buying
behaviour (CBB) Perceived performance in digital marketing In
the
digital
marketing
spectrum,
performance has a different meaning and
depends upon consumer events, usage
experience, and quality spent time on that
specific platform. A subjective indicator of the
functionality,
responsiveness,
and
dependability of a website is perceived Accessibility on all platforms should be
simple for all users, and broadband speed is
important. Digital perceived performance
includes consumer experience in online 192 | P a g e 192 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior employs qualitative and quantitative for
gathering data. This is mostly because it
sparked research into the causal connections
between variables to consumer behaviour in
comparison to digital marketing and their
perceived performance in Karachi. banking, digital checkouts, and other digital
services like ordering food, getting ride-
hailing services, online shopping, and more. In this research, we are finding the facts
about consumer behaviour and digital
marketing
which
are
impacted
by
performance. With a limited sample size, the qualitative
research approach conducts an exploratory
study to better understand the issue at hand. Data is gathered using a variety of methods,
including
observations,
open-ended
questionnaires,
and
interviews. These
methods aid the researcher in collecting
replies to research questions which are based
on the respondents' reflections on their
expressions of emotions and experiences
related to the issue (O’Gorman, 2015) Methodology
Research Approach This research is a combination of
exploratory and Descriptive research, as some
variables
are
unstructured,
and
the
suggestion was so important in data
collection. Using primary data, this research 193 | P a g e 193 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior Research Design The questionnaire is created in such a way
that responders would find it engaging and
simple to understand. Additionally, only
relevant information was acquired via the
questionnaire, which only contained the
questions needed to accomplish the goals of
the study. Consisting of seven constructs mainly
focused on buyers’ behaviour towards each
online platform, their buying consistency,
their ease of shopping, and their satisfaction
with digital purchases. The idea for the third
measure, Perceived performance, was taken
from (Tran et al., 2019), which has seven
questions analyzing customer experience with
online performance, their reaction to speed
and interface while using any application,
devices they feel easy to use while purchasing
online stuff and for discussion, we asked for
their feedback that what can improve
customer experience by digital buying. The questionnaire was prepared with the
support of the research supervisor. The
language which is used in the questionnaire is
English for the targeted group of people. The
questionnaire was created in such a way that
it will be confidential, and people who fill that
out will stay anonymous. This allowed
respondents to remain anonymous and avoid
personal identification. Measures of Constructs The target audience is stratified into five
subgroups which is their area of living. The
researcher chose District East, District
West, District South, District Central and Malir
District and questionnaires were spread in
each district. The idea behind choosing these
districts is that these areas have the most
coverage of the city. The target audience is stratified into five
subgroups which is their area of living. The
researcher chose District East, District
West, District South, District Central and Malir
District and questionnaires were spread in
each district. The idea behind choosing these
districts is that these areas have the most
coverage of the city. Digital marketing measurements were
modified from (Tiago & Veríssimo, 2014)
based on nine different attributes of the
online or digital presence of people on social
media, search engine ads, emails, mobile
applications, mobile marketing and their
behaviour with online advertisement. Second,
consumer buying behaviour (CBB) was
analyzed (Verplanken & Herabadi, 2001). Sampling Technique The questionnaire followed a specific
framework and included several types of
questions. The first part of the questionnaire
is to collect demographic containing age,
gender, educational level (EL), area of living,
and in a multiple-choice format. The second
part is for the use of Digital Marketing for
online purchases in respondents’ daily
routines. The
third
part
was
to
collect Consumer
behaviour
toward
behaviour towards different online channels. Last and fourth we obtained their response on
the
Perceived
performance
of
digital
marketing which respondents are using and
what are the most sources they used for their
online purchases. According to the (worldpopulationreview,
2022) world population review, it is estimated
that the population of Karachi is 17.6 Millon. And in the report of statistics of Pakistan
(statista, 2021), it is concluded that 60% of the
population is lying between the age bracket of
15-64. It is also stated in a report published by
“date reported (Digital 2022: Pakistan, 2022)”
that around 40% of the population is lying
between 18-44. In another report of date reported (Digital
2022: Pakistan, 2022), it is stated that 36.5 per
cent of the total population is using the
internet. Therefore, according to facts and figures,
the total population size for this research is
probably 2.5 million. al., 2009). Given that the target audience for this
study is young adults, a self-administered pen-
paper intercept and online forms survey was
judged appropriate (De Vries & Carlson,
2014). Respondents were also approached
through
online
surveys
and
personal
interviews. Only those respondents were
allowed who are likely to use online channels
to buy stuff online. Results and Findings The questionnaire was spread among 600
people between the period of august 2022
and November 2022. In that period author
received 492 responses of which 458 were
selected for data analysis. The model is driven by Smart PLS 4.0. Software SPSS software and Smart PLS 4 (Ringle,
2022) were used to check confirmatory factor
analysis (CFA). The PLS-SEM model is used to
check outer loading and discriminant values of
collected responses. Data from SPSS that has
been abstracted is then imported into an
excel document to produce pie charts and bar
diagrams for better presentation. The reflecting model, which shows how
each variable is related, is drawn from Smart
PLS version 4. PP (perceived performance),
DM (Digital Marketing), and CBB (Consumer
buying behaviour). The R square of CBB is
0.639, and the R square of DM is 0.624,
showing a good relationship. Data Collection The questionnaire was used for data
collection. Every survey was conducted online
via Google Forms. The five-point Likert scale, The
stratified
Random
Sampling
technique is used for the Overall population. 194 | P a g e 194 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior loadings. The Cronbach's alpha and Construct
reliability and validity are also determined by
the same program. In this study, Cronbach's
alpha exceeds 0.70, which is beyond the
permissible range of 0.70 (Fornell & Larcker,
1981a). The acceptance rate of composite
reliability is 0.60; the three variables in this
research have Composite Reliability between
the ranges of 0.71 to 0.748, which is a good
result. Following data analysis, the average
variance value was determined (AVE) was less
than 0.50 in 2 variables, but according to
(Fornell & Larcker, 1981b) that if composite
reliability is higher than 0.60 then the value of
AVE less than 0.5 is acceptable, which
indicates that this model has accurate
reliability and validity. which is strongly agreed to strongly disagree,
includes 4 parts. The age range of the study's
target population is 18 to 40. There are
differences in Gen Y members' educational
levels and access use of digital marketing. As
a result, discrepancies between age groups
will exist depending on the greatest level of
education, gender, and occupation (Noble et
al., 2009). Data Analysis Technique: For descriptive statistics, SPSS software is
used, and to analyze inferential statistics
researchers used Smarts. For all direct
models, SPSS analytics is used. Hypothesis test
runs are performed with the same tool. Demographic Information: Construct Reliability and Validity The most crucial factors in determining if
a question is relevant are its validity and
reliability. Those questions are used in the
measurement of correlations and other
examinations of data. In this research AVE in
two variables is less than 0.5, as explained in
the methodology if Cronbach’s alpha and
composite reliability is greater than 0.6 in this
situation Average variance extracted is
acceptable at a level of 0.4. acceptable at a level of 0.4. Cron
bach'
s
alpha
Composit
e
reliability
(rho_a)
Composit
e
reliability
(rho_c)
The average
variance
extracted
(AVE)
C
B
B
0.750
0.752
0.748
0.427
D
M 0.735
0.745
0.736
0.418
P
P
0.709
0.712
0.710
0.551
Summary of results
Proposed
Paths
Pearson's
Correlation
p-
values
Hypotheses
H1: DM --->
CBB
0.412
0.00
Supported
H2: MM -->
CBB
0.215
0.00
Supported
H3: SMM -->
CBB
0.521
0.00
Supported
H4: PPC -->
CBB
0.321
0.00
Supported
H5: SEO -->
CBB
0.382
0.00
Supported In this research, the data is collected from
17% of district malir, 15% from district ease,
36% from district central, 15% from district
west, and the remaining from district south. The researcher analyzed that 48% plus
respondents are buying products from online
channels every month, and 37% are utilizing
this channel once or twice a year. Demographic Information: Demographic Information: g
p
Age of Respondent
Frequency Per cent
18-24
256
55.90%
25-30
154
33.6%
31-39
48
10.5%
Total
458
100% In SPSS linear equation model is used to
run hypothesis and correlation formation. For
analyzing data, the author uses PLS-SEM, and
for the calculation, the researcher uses a
consistent PLS algorithm technique. The measurement model was reflective,
and SmartPLS was used to examine all exterior 195 | P a g e 195 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior Gender
Frequency Per cent
Male
254
55.5%
Female
204
44.5%
Total
458
100%
Education Level
Frequency Per cent
Matric
20
4.4%
Intermediate
106
23.1%
Bachelor's
Degree
170
37.1%
Master's
Degree
144
31.4%
Professional
Certification
18
3.9%
Total
458
100%
Occupation
Frequency Per- cent
Employed
full time
232
50.7%
Self
Employed
56
12.25
A Housewife
28
6.1%
A Student
142
31%
Total
458
100%
Do you buy things over the internet? Frequency
Per
cent
Yes
408
89.1%
No
50
10.9%
Total
458
100% relationship is high with a value of 0.786. Between
Digital
marketing,
perceived
performance,
and
consumer
buying
behaviour there is a positive correlation. HTMT matrix is used for correlation for
different variables. Construct Reliability and Validity Discriminant Validity In the Discriminant validity of this paper,
the author has analyzed a strong relationship
between DM and CBB of 0.8, with PP and CBB
having a positive relation of 0.644. PP and DM 196 | P a g e 196 | P a g e Journal of Academic Research for Humanities 3(1) How Digital Marketing and Perceived Performance is Influencing Consumer Buying Behavior H6: EM -->
CBB
0.042
0.00
Unsupported
H7: CBB -->
DMT
0.488
0.00
Supported
H8: PP --> DM
0.932
0.00
Supported
H9: DM x CBB
--> PP
0.326
-
Supported
Discussion food through the online channel so those
SMEs who are newly entering this industry
have a good scope and market. For those
marketing agencies which are in the market
and working on inbound campaigns, this
research will help them to analyze the best
audience to target from specific online
mediums. This study is for a specific geographic
region and the central idea of this research is
to conclude how consumer behaviour is being
changed by different digital marketing tools
and if the perceived performance of those
tools gets better, how will this impact
consumer buying behaviour. This research can also be used from the
company’s perspective, and that will signify
how digital marketing channels are helping
their
business
and
what
performance
indicators can be improvised for more
customer buying. For the B2B market, data
should be collected in qualitative form purely
for better ideas. Karachi is one of the most populated cities
in the world, with internet users thirty-six per
cent and in this figure, around 82% of people
are those who have purchased any kind of
product or service through online stores. This
research is based on a small group of people
but if we look at overall factors, there are 17.6
million people who have the potential to buy
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Consumer Purchase Behaviour. 7. Tran, V., Zhao, S., Diop, E. B., & Song, W. (2019). Travellers’ Acceptance of Electric
Carsharing
Systems
in
Developing
Countries:
The
Case
of
China. Sustainability,
11(19). https://doi.org/10.3390/su11195348 Verplanken, B., & Herabadi, A. (2001). Individual differences in impulse buying
tendency: Feeling and no thinking. European
Journal
of
Personality,
15(1_suppl),
S71–S83. https://doi.org/10.1002/per.423 Verplanken, B., & Herabadi, A. (2001). Individual differences in impulse buying
tendency: Feeling and no thinking. European
Journal
of
Personality,
15(1_suppl),
S71–S83. https://doi.org/10.1002/per.423 199 | P a g e 199 | P a g e 199 |
|
https://openalex.org/W4361940258
|
https://figshare.com/articles/journal_contribution/Supplementary_Figure_from_Anlotinib_Induces_a_T_Cell_Inflamed_Tumor_Microenvironment_by_Facilitating_Vessel_Normalization_and_Enhances_the_Efficacy_of_PD-1_Checkpoint_Blockade_in_Neuroblastoma/22483749/1/files/39935283.pdf
|
English
| null |
Supplementary Figure from Anlotinib Induces a T Cell–Inflamed Tumor Microenvironment by Facilitating Vessel Normalization and Enhances the Efficacy of PD-1 Checkpoint Blockade in Neuroblastoma
| null | 2,023
|
cc-by
| 2,207
|
9464D 9464D 975A2
9464D
A
C
D
B B Aire
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
0
0
1
975A2
9464D
A
C
E
F
G
H
D
B
0
0
1
Immune cell migraion related genes
(GO:0050900)
Immune cell adhesion related genes
(GO:0007159)
Immune cell migraion related genes
(GO:0050900)
Immune cell adhesion related genes
(GO:0007159)
Prkcz
Aif1
Cd274
Tnf
Icos
Bmp7
Cd40lg
Cd28
Cd4
Cd209e
Sash3
Rac2
Itgb2
Ifng
Selplg
Ccl5
Tnfaip8l2
Coro1a
H2−T23
Add2
Itgal
Il2ra
Il21
Cd5
Cd6
Lck
Rasgrp1
Pdcd1lg2
Cd3e
Igf1
Il1b
Tbx21
Ptprc
Tarm1
Il18
Itgb7
Dusp22
Icam1
Btla
Tnfsf11
Prkcq
Selp
Dock8
Cd83
Stat5b
Fermt3
Cd300a
Pycard
Ptafr
Vsir
Sirpa
Zc3h12d
Madcam1
Ikzf1
Cd209e
Ass1
Il7r
Cd27
Cd274
Cd80
Adam8
Itgal
Lax1
Irf1
Zap70
H2−Ab1
H2−Aa
Lck
Spn
Tbx21
Gm9733
Dpp4
Card11
Ccr2
Prkcq
Cd244a
March7
Klf4
Stat5a
Selp
Bmp7
ll1b
Cd177
Ccr7
Il12b
Cd6
Sell
Btla
Slamf1
Cd28
Nr4a3
Igf1
Rasgrp1
Efnb1
Cebpb
Pdcd1lg2
Ccl5
Malt1
Dusp10
Cd5
Ccl2
Icos
Tarm1
Aire
Cxcl10
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Vav1
C3ar1
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2 A C
E
F D G
D C 1
G
H
D
1
Immune cell migraion related genes
(GO:0050900)
Immune cell adhesion related genes
(GO:0007159) H Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
C l2
genes
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3 Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Aire
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2
E
F
G
H
Immune cell migraion related genes
(GO:0050900)
Immune cell adhesion related genes
(GO:0007159)
Immune cell migraion related genes
(GO:0050900)
Immune cell adhesion related genes
(GO:0007159)
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Prkcz
Aif1
Cd274
Tnf
Icos
Bmp7
Cd40lg
Cd28
Cd4
Cd209e
Sash3
Rac2
Itgb2
Ifng
Selplg
Ccl5
Tnfaip8l2
Coro1a
H2−T23
Add2
Itgal
Il2ra
Il21
Cd5
Cd6
Lck
Rasgrp1
Pdcd1lg2
Cd3e
Igf1
Il1b
Tbx21
Ptprc
Tarm1
Il18
Itgb7
Dusp22
Icam1
Btla
Tnfsf11
Prkcq
Selp
Dock8
Cd83
Stat5b
Fermt3
Cd300a
Pycard
Ptafr
Vsir
Sirpa
Zc3h12d
Madcam1
Ikzf1
Ccr7
Sema4d
Il7r
Rasal3
H2−M3
Zap70
Cd27
Dpp4
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Cd209e
Ass1
Il7r
Cd27
Cd274
Cd80
Adam8
Itgal
Lax1
Irf1
Zap70
H2−Ab1
H2−Aa
Lck
Spn
Tbx21
Gm9733
Dpp4
Card11
Ccr2
Prkcq
Cd244a
March7
Klf4
Stat5a
Selp
Bmp7
ll1b
Cd177
Ccr7
Il12b
Cd6
Sell
Btla
Slamf1
Cd28
Nr4a3
Igf1
Rasgrp1
Efnb1
Cebpb
Pdcd1lg2
Ccl5
Malt1
Dusp10
Cd5
Ccl2
Icos
Tarm1
Ndfip1
Arg2
Slfn1
Icam1
Il2ra
Cd3e
Rock1
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Aire
Cxcl10
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Vav1
C3ar1
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2 HIm Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Aire
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod
Veh1
Veh2
Veh3
Anlo1
F
G
H
Immune cell migraion related genes
(GO:0050900)
related genes
59)
Immune cell migraion
(GO:00509
Immune cell adhesion related genes
(GO:0007159)
Anlo2
Anlo3
Prkcz
Aif1
Cd274
Tnf
Icos
Bmp7
Cd40lg
Cd28
Cd4
Cd209e
Sash3
Rac2
Itgb2
Ifng
Selplg
Ccl5
Tnfaip8l2
Coro1a
H2−T23
Add2
Itgal
Il2ra
Il21
Cd5
Cd6
Lck
Rasgrp1
Pdcd1lg2
Cd3e
Igf1
Il1b
Tbx21
Ptprc
Tarm1
Il18
Itgb7
Dusp22
Icam1
Btla
Tnfsf11
Prkcq
Selp
Dock8
Cd83
Stat5b
Fermt3
Cd300a
Pycard
Ptafr
Vsir
Sirpa
Zc3h12d
Madcam1
Ikzf1
Ccr7
Sema4d
Il7r
Rasal3
H2−M3
Zap70
Cd27
Dpp4
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Cd209e
Ass1
Il7r
Cd27
Cd274
Cd80
Adam8
Itgal
Lax1
Irf1
Zap70
H2−Ab1
H2−Aa
Lck
Spn
Tbx21
Gm9733
Dpp4
Card11
Ccr2
Prkcq
Cd244a
March7
Klf4
Stat5a
Selp
Bmp7
ll1b
Cd177
Ccr7
Il12b
Cd6
Sell
Btla
Slamf1
Cd28
Nr4a3
Igf1
Rasgrp1
Efnb1
Cebpb
Pdcd1lg2
Ccl5
Malt1
Dusp10
Cd5
Ccl2
Icos
Tarm1
Ndfip1
Arg2
Slfn1
Icam1
Il2ra
Cd3e
Rock1
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Aire
Cxcl10
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Vav1
C3ar1
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod
Veh1
Veh2
Veh3
Anlo1 EIm HImmune cell migraion related genes
(GO:0050900) EImmune cell adhesion related genes
(GO:0007159) GIm FIm Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Aire
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod
E
FImmune cell migraion related genes
(GO:0050900)
Immune cell adhesion related genes
(GO:0007159)
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Prkcz
Aif1
Cd274
Tnf
Icos
Bmp7
Cd40lg
Cd28
Cd4
Cd209e
Sash3
Rac2
Itgb2
Ifng
Selplg
Ccl5
Tnfaip8l2
Coro1a
H2−T23
Add2
Itgal
Il2ra
Il21
Cd5
Cd6
Lck
Rasgrp1
Pdcd1lg2
Cd3e
Igf1
Il1b
Tbx21
Ptprc
Tarm1
Il18
Itgb7
Dusp22
Icam1
Btla
Tnfsf11
Prkcq
Selp
Dock8
Cd83
Stat5b
Fermt3
Cd300a
Pycard
Ptafr
Vsir
Sirpa
Zc3h12d
Madcam1
Ikzf1
Ccr7
Sema4d
Il7r
Rasal3
H2−M3
Zap70
Cd27
Dpp4
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Aire
Cxcl10
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Vav1
C3ar1
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod Veh1
Veh2
Veh3
Anlo1
o
G
HImmune cell migraion r
(GO:0050900
Immune cell adhesion related genes
(GO:0007159)
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Cd209e
Ass1
Il7r
Cd27
Cd274
Cd80
Adam8
Itgal
Lax1
Irf1
Zap70
H2−Ab1
H2−Aa
Lck
Spn
Tbx21
Gm9733
Dpp4
Card11
Ccr2
Prkcq
Cd244a
March7
Klf4
Stat5a
Selp
Bmp7
ll1b
Cd177
Ccr7
Il12b
Cd6
Sell
Btla
Slamf1
Cd28
Nr4a3
Igf1
Rasgrp1
Efnb1
Cebpb
Pdcd1lg2
Ccl5
Malt1
Dusp10
Cd5
Ccl2
Icos
Tarm1
Ndfip1
Arg2
Slfn1
Icam1
Il2ra
Cd3e
Rock1
Veh1
Veh2
Veh3
Anlo1 A l 3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2
d genes
Anlo3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2 Veh2
Veh3
Anlo1
Anlo2
Anlo3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2
1
0
0
0
1
1
0
0
0
1
ne cell migraion related genes
(GO:0050900)
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2 GImmune cell adhesion related genes
(GO:0007159)
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Cd209e
Ass1
Il7r
Cd27
Cd274
Cd80
Adam8
Itgal
Lax1
Irf1
Zap70
H2−Ab1
H2−Aa
Lck
Spn
Tbx21
Gm9733
Dpp4
Card11
Ccr2
Prkcq
Cd244a
March7
Klf4
Stat5a
Selp
Bmp7
ll1b
Cd177
Ccr7
Il12b
Cd6
Sell
Btla
Slamf1
Cd28
Nr4a3
Igf1
Rasgrp1
Efnb1
Cebpb
Pdcd1lg2
Ccl5
Malt1
Dusp10
Cd5
Ccl2
Icos
Tarm1
Ndfip1
Arg2
Slfn1
Icam1
Il2ra
Cd3e
Rock1 Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2
0
0
1
H
0
0
1
Immune cell migraion related genes
(GO:0050900)
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Sema3g
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2 Veh1
Veh2
Veh3
Anlo
Anlo
Anlo
Aire
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod
FImmune cell migraion related genes
(GO:0050900)
Veh1
Veh2
Veh3
Anlo
Anlo
Anlo
Aire
Cxcl10
Tnf
Mcoln2
Ccl24
Bdkrb1
Adora3
Add2
Fut7
Mif
Itgb7
Gpr18
Lbp
Trpm2
Tnfsf11
Mmp9
Tnfrsf18
Cxcl16
Rac2
Ccl17
Slc8b1
Ccr7
Vav1
C3ar1
Stat5b
Cnr2
Dock8
F7
Coro1a
Cd300a
Pycard
Madcam1
Ptafr
Ptpro
Ptger4
Cxcr3
Selplg
Ccl5
Icam1
Rarres2
Slamf8
Ccl8
Itgb2
Ifng
Il1b
Aif1
Cxcl9
Tbx21
Ccl22
Myo1g
Csf3r
Jaml
Gpr35
Apod EImmune cell adhesion related genes
(GO:0007159)
Veh1
Veh2
Veh3
Anlo1
Anlo2
Anlo3
Prkcz
Aif1
Cd274
Tnf
Icos
Bmp7
Cd40lg
Cd28
Cd4
Cd209e
Sash3
Rac2
Itgb2
Ifng
Selplg
Ccl5
Tnfaip8l2
Coro1a
H2−T23
Add2
Itgal
Il2ra
Il21
Cd5
Cd6
Lck
Rasgrp1
Pdcd1lg2
Cd3e
Igf1
Il1b
Tbx21
Ptprc
Tarm1
Il18
Itgb7
Dusp22
Icam1
Btla
Tnfsf11
Prkcq
Selp
Dock8
Cd83
Stat5b
Fermt3
Cd300a
Pycard
Ptafr
Vsir
Sirpa
Zc3h12d
Madcam1
Ikzf1
Ccr7
Sema4d
Il7r
Rasal3
H2−M3
Zap70
Cd27
Dpp4 HImmune cell migraion related genes
(GO:0050900) Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Efna5
Enpp2
Sema3d
Ednra
Sema5a
Efnb1
Fgf1
Chl1
Cxcr2
Ccr2
Prkcq
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8 cq
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2
Cxcr3
Cxcl9
Nod2
Jaml
Runx3
Slamf8
Edn2
Egr2
Flrt3
Ccl7
Swap70
Trem1
Cxcl1
Ntn1
Bmp7
Ccr9
Il1b
Sell
Ccl17
Slamf1
Ccr8
Ccrl2
Ccl5
Bcl11b
Ccr7
Cxcr6
Nr4a3
Ccl2
Cxcl10
Saa3
Tubb3
Pgf
Ccr4
Cxcl2
Cxcl3
Ccl4
Gpr35
Adam8
Ccl22
Dpysl2
|
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https://europepmc.org/articles/pmc3954384?pdf=render
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English
| null |
Pelvic collection drainage by Heald anal stent
|
Annals of the Royal College of Surgeons of England
| 2,012
|
cc-by
| 694
|
TECHNIQUE The Heald anal stent (Fig 1) can be used to drain pelvic collections on
the ward after any surgery that leaves a short rectal stump. The stent
is inserted through the rectal cross-staples after the instillation of lo-
cal anaesthetic gel. After several days, once drainage is complete, the
stent is removed painlessly. stoma in low rectal anastomoses.3 We recommend this technique as
a possible method to allow free rectal drainage of a pelvic collection. We have used this technique successfully on patients with pelvic
collections (Fig 2) who have failed Foley catheter drainage. The stent
may be left for a few days until drainage is complete. (Pelvic magnetic
resonance imaging may be used to confirm this [Fig 3].) The stent can
then be removed on the ward. Pelvic collection drainage by Heald anal stent EJ Cook1, BJ Moran2, RJ Heald2, GF Nash1
1Poole General Hospital NHS Foundation Trust, UK
2Hampshire Hospitals NHS Foundation Trust, UK CORRESPONDENCE TO
Guy Nash, E: guy.nash@poole.nhs.uk BACKGROUND The use of the Heald anal stent (Basingstoke Surgical Technology Ltd,
Pinner, UK) has been described previously in the successful therapeu-
tic decompression of the rectum following a leaking ileorectal anasto-
mosis.1 The novel technique of using the Heald stent to drain a pelvic
collection following ultra low Hartmann’s operation is presented. Figure 2 Computed tomography demonstrating pelvic fluid
and gas collection incompletely drained by a rectal Foley
catheter Figure 1 The hollow silicone elastomer Heald anal stent is
flanged at both ends to prevent dislodgement in the anus. Figure 1 The hollow silicone elastomer Heald anal stent is
flanged at both ends to prevent dislodgement in the anus. Figure 3 Sagittal magnetic resonance imaging of pelvis five
days after the insertion of the Heald stent (highlighted) showing
urinary catheter in the bladder and complete drainage of the
presacral collection, now seen as air Figure 1 The hollow silicone elastomer Heald anal stent is
flanged at both ends to prevent dislodgement in the anus. Figure 3 Sagittal magnetic resonance imaging of pelvis five
days after the insertion of the Heald stent (highlighted) showing
urinary catheter in the bladder and complete drainage of the
presacral collection, now seen as air References 1. Brent A, Armstrong T, Nash GF, Heald RJ. Therapeutic use of the Heald Silastic
Anal Stent. Colorectal Dis 2007; 9: 279–280. TECHNICAL SECTION TECHNICAL SECTION Figure 2 Computed tomography demonstrating pelvic fluid
and gas collection incompletely drained by a rectal Foley
catheter Pelvic collection drainage by Heald anal stent
EJ Cook1, BJ Moran2, RJ Heald2, GF Nash1
1Poole General Hospital NHS Foundation Trust, UK
2Hampshire Hospitals NHS Foundation Trust, UK
CORRESPONDENCE TO
Guy Nash, E: guy.nash@poole.nhs.uk 1. Brent A, Armstrong T, Nash GF, Heald RJ. Therapeutic use of the Heald Silastic
Anal Stent. Colorectal Dis 2007; 9: 279–280. 2. Buie WD, MacLean AR, Attard JA et al. Neoadjuvant chemoradiation increases
the risk of pelvic sepsis after radical excision of rectal cancer. Dis Colon Rectum
2005; 48: 1,868–1,874. References
1. Brent A, Armstrong T, Nash GF, Heald RJ. Therapeutic use of the Heald Silastic
Anal Stent. Colorectal Dis 2007; 9: 279–280.
2. Buie WD, MacLean AR, Attard JA et al. Neoadjuvant chemoradiation increases
the risk of pelvic sepsis after radical excision of rectal cancer. Dis Colon Rectum
2005; 48: 1,868–1,874.
3. Amin AI, Ramalingam T, Sexton R et al. Comparison of transanal stent with
defunctioning stoma in low anterior resection for rectal cancer. Br J Surg 2003;
5: 581–582. DISCUSSION 3. Amin AI, Ramalingam T, Sexton R et al. Comparison of transanal stent with
defunctioning stoma in low anterior resection for rectal cancer. Br J Surg 2003;
5: 581–582. Pelvic sepsis is a common complication after colorectal surgery such
as Hartmann’s operation. The risk is increased following neoadjuvant
chemoradiotherapy, particularly in the presence of a suture or staple
line.2 Foley catheters may be used to decompress pelvic collections
but become blocked frequently. Being shorter and having a wider lu-
men, the Heald stent provides more effective drainage and is easy
to irrigate if necessary. It has been previously demonstrated to be an
alternative, albeit not certain, method of avoiding a defunctioning 361 Ann R Coll Surg Engl 2012; 94: 359–372
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|
English
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Surface Imaging by ABF-STEM: Lithium Ions in Diffusion Channel of LIB Electrode Materials
|
E-journal of surface science and nanotechnology
| 2,012
|
cc-by
| 4,393
|
∗This paper was presented at the 6th International Symposium on
Surface Science –Towards Nano, Bio and Green Innovation–, Tower
Hall Funabori, Tokyo, Japan, December 11-15, 2011.
†Corresponding author: lee.s.aj@m.titech.ac.jp e-Journal of Surface Science and Nanotechnology
e-J. Surf. Sci. Nanotech. Vol. 10 (2012) 454-458 e-Journal of Surface Science and Nanotechnology
e-J. Surf. Sci. Nanotech. Vol. 10 (2012) 454-458 25 August 2012
Conference - ISSS-6 - 25 August 2012 Conference - ISSS-6 - Surface Imaging by ABF-STEM: Lithium Ions in Diffusion Channel of
LIB Electrode Materials∗ Soyeon Lee†
Department of Material Science and Engineering,
Tokyo Institute of Technology, 2-12-1 Meguro-ku, Tokyo 152-8551, Japan, Yoshifumi Oshima
Research Center for Ultra HVEM, Osaka University, 7-1 Midorigaoka, Ibaraki, 567-0047, Japan. Yoshifumi Oshima
Research Center for Ultra HVEM, Osaka University, 7-1 Midorigaoka, Ibaraki, 567-0047, Japan. Hidetaka Sawada, Fumio Hosokawa, Eiji Okunishi, Toshikatsu Kaneyama, and Yukihito Kondo
JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan. Yasumasa Tanishiro and Kunio Takayanagi
Department of Physics, Tokyo Institute of Technology, 2-12-1 Meguro-ku, Tokyo 152-8551, Japan
(Received 31 January 2012; Accepted 4 July 2012; Published 25 August 2012) Yasumasa Tanishiro and Kunio Takayanagi
Department of Physics, Tokyo Institute of Technology, 2-12-1 Meguro-ku, Tokyo 152-8551, Japan
(Received 31 January 2012; Accepted 4 July 2012; Published 25 August 2012) Lithium ions in LiMn2O4 crystals have been shown observable by using an aberration corrected electron micro-
scope (R005) which has achieved a sub-50 pm resolution. Based on the annular bright-field image of the observed
image, surface imaging of a LiMn2O4 crystal is discussed through simulation. The present simulation showed that
ABF images with large convergence angles are sensitive to the top surface profile. A vacancy locating at the top
surface of the lithium or manganese atomic columns can be observable with contrast reduced by 10-25% from the
columns without vacancy. [DOI: 10.1380/ejssnt.2012.454] Keywords: Surface structure, morphology, roughness, and topography; Scanning transmission electron microscopy (STEM);
Energy technology I.
INTRODUCTION purities in the sample [16, 17]. In this report, we studied characteristics of ABF imag-
ing with a large convergence angle and the possibility of
surface imaging, imaging of vacancies at the top surface
of specimen. We show that lithium ions in a LiMn2O4
crystal are observable in ABF images by an AbC-STEM
of a 50 pm resolution (R005) [18]. The observed images
are reproducible by multi-slice simulation. For large con-
vergence angles of the incident beam, focused on the top
surface, the simulated images of LiMn2O4 surfaces show
the image contrast sensitive to the vacancy at the top
surface due to the shallow depth of focus (DOF): Lithium
ion vacancy and manganese ion vacancy, locating at the
top surface, give 20% and 25% reduction of the column
contrast, respectively. Recent development of electric vehicles has required
more powerful, stable and lighter batteries. Lithium ion
battery (LIB) is the most promising power supply because
of high energy density and stability of power [1–3]. But
the short lifetime of present LIBs is one of important sub-
jects to be improved [4]. The lifetime of LIB is known
to be determined by the interface condition between elec-
trode and electrolyte [5]. To improve the interface stabil-
ity, surface controlling [6–8] and surface profiling meth-
ods have been devised. Surface XRD has revealed the
surface structure change during battery performance [9]. AFM and SEM have given information about morphology
change [10–12]. However, direct detecting of lithium ions
had not been achieved. Recent electron microscopy studies have suggested the
possibility of direct observation of lithium ion diffusion. Lithium ions have been imaged in real space at atomic
level by annular bright field (ABF) imaging of the aberra-
tion corrected scanning transmission electron microscope
(AbC-STEM) [13, 14]. Furthermore, it has been reported
that the number of lithium ions in a diffusion channel was
countable in ABF images by quantitative STEM [15]. ISSN 1348-0391
c⃝2012 The Surface Science Society of Japan (http://www.sssj.org/ejssnt) A.
Atomic structure of a LiMn2O4 crystal A. Atomic structure of a LiMn2O4 crystal A LiMn2O4 crystal, a well-known positive electrode ma-
terial of LIB, has a spinel structure (space group: Fd¯3m,
a = 0.8245 nm [19]). Manganese ions are located in oc-
tahedral sites and lithium ions, tetrahedral sites in the
cubic close packed (ccp) array of oxygen ions. Figure 1(a)
shows the LiMn2O4 crystal viewed from the [110] direc-
tion. Each atomic column contains a single element. Man-
ganese columns (blue sphere) are positioned on the side
of rhombic unit, oxygen columns (grey sphere) are po-
sitioned at both sides of manganese columns. Lithium
columns (red one) are positioned along the long diagonal
of the rhombic unit. In this view, there are two kinds
of manganese columns, Mnα and Mnβ: Mnα column has
two manganese ions per 0.583 nm along the incident beam The modern AbC STEM give not only better spatial
resolution, but also improved depth resolution due to a
reduced depth of focus (DOF). With this advantage, an-
nular dark field imaging of STEM has been applied to
slice the sample optically to obtain 3D information of im- ISSN 1348-0391
c⃝2012 The Surface Science Society of Japan (http://www.sssj.org/ejssnt) 454 e-Journal of Surface Science and Nanotechnology Volume 10 (2012) Fig. 2
FIG. 2:
Observed ABF(annular bright-field) image of a
LiMn2O4 crystal. The images are taken in vicinity of the edge
of the sample. The convergence semi-angle of the incident
beam is 24 mrad for images (a), and 30 mrad for images (b)
The rhombic unit of a LiMn2O4 crystal viewed from the [110]
orientation is indicated by black line in the both images. Ap-
parent defocus (∆f) of ABF images are shown: +6 nm (over-
focus) to −4 nm (under-focus). The depth of focus (DOF) is
5 nm for (a), and 3 nm for (b). Fig. 1
FIG. 1: (a) Scheme of a LiMn2O4 crystal viewed from the [110]
direction. Polyhedrons indicate MnO6 units. Manganese, oxy-
gen and lithium ions are displayed by spheres of different colors
(manganese, blue; oxygen, gray; lithium, red). The rhombus
indicates the unit area of a LiMn2O4 crystal, of which the
short and long diagonal are 0.583 nm and 0.824 nm, respec-
tively. (b) Perspective view of a LiMn2O4 crystal having ion
vacancies at the top surface. The yellow circles indicate ion
vacancies of manganese (blue dashed circle) and lithium (red). Fig. 1
FIG. A.
Atomic structure of a LiMn2O4 crystal 1: (a) Scheme of a LiMn2O4 crystal viewed from the [110]
direction. Polyhedrons indicate MnO6 units. Manganese, oxy-
gen and lithium ions are displayed by spheres of different colors
(manganese, blue; oxygen, gray; lithium, red). The rhombus
indicates the unit area of a LiMn2O4 crystal, of which the
short and long diagonal are 0.583 nm and 0.824 nm, respec-
tively. (b) Perspective view of a LiMn2O4 crystal having ion
vacancies at the top surface. The yellow circles indicate ion
vacancies of manganese (blue dashed circle) and lithium (red). direction, while Mnβ column has one. In an oxygen col-
umn, two oxygen ions are positioned per 0.583 nm along
the incident beam direction and in a lithium column, one
lithium ion. The lithium columns are known as the diffu-
sion channel of ion conduction [20]; when a lithium ion dif-
fuses out from the diffusion channel, a vacancy is created
in the channel. Diffusion of lithium can induce displace
of manganese ions. The model structure of a LiMn2O4
crystal after creation of a vacancy is shown in Fig. 1(b). Each ion vacancy is positioned at the top surface: dashed
circles correspond a manganese vacancy and a lithium va-
cancy, respectively. FIG. 2:
Observed ABF(annular bright-field) image of a
LiMn2O4 crystal. The images are taken in vicinity of the edge
of the sample. The convergence semi-angle of the incident
beam is 24 mrad for images (a), and 30 mrad for images (b). The rhombic unit of a LiMn2O4 crystal viewed from the [110]
orientation is indicated by black line in the both images. Ap-
parent defocus (∆f) of ABF images are shown: +6 nm (over-
focus) to −4 nm (under-focus). The depth of focus (DOF) is
5 nm for (a), and 3 nm for (b). http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) A.
Imaging manganese vacancy at surface The calculation was performed with xHREM pro-
gram [23] based on Multi-slice method. The detector
semi-angle, β, was α/2 ≤β ≤α (α: the convergence semi-
angle of the incident beam). The thermal diffusion scat-
tering absorptive potential approximation was included
with Weikenmeier-Kohl scattering factor. The Debye-
Waller factors of manganese, oxygen, and lithium were
assumed to be 0.84, 1.11, and 1.07 ˚A2, respectively [19]. The slice thickness was 0.291 nm in the [110] direction. The cut-offscattering vector sin θB/λ was 4.5 ˚A−1, where
θB is the Bragg-angle and λ is the electron wavelength
at 300 keV (λ = 0.00197 nm). The observed images
were reproduced after convolving a Gaussian function to
the calculated one. Gaussian function with a FWHM =
65 pm reproduced the experimental images of 24 mrad,
and those of 30 mrad [24]. The Gaussian function, effec-
tive source size [24], represents the blur due to residual
aberrations, chromatic aberration, statistic noise and in-
strumental noises. ABF images were calculated for large convergence an-
gles of 50, 70, and 100 mrad. The defocus value was
zero. Figure 4 shows simulated ABF images of manganese
columns with and without one vacancy at the top surface. The upper panels of Figs. 4(a) and (b) are simulated im-
ages of the model structure illustrated in Fig. 1(b) with
100 mrad-convergence angle. In Fig. 4(a), oxygen and
Mnβ columns in the rectangled area align alternatively
from the left to the right. The second Mnβ column from
the left has one vacancy at the top surface. Although the
Mnβ columns with and without a vacancy appear dark
contrast, the intensity profile shows that one vacancy re-
duces the column contrast by 25%. The simulated image of a 5nm-thick model is shown in
Fig. 4(b). The contrast of Mnβ with a vacancy decreases
by roughly 25% from that without vacancy. For the case
of one manganese vacancy positioned at the middle of
the column or at the bottom surface, the column contrast
reduced less than 3%. Thus, one vacancy only at the top
surface reduces the column contrast regardless to the film
thickness. A smaller convergence angle of 50 mrad gave
the contrast decrease by 10%, which can also be useful for
detecting manganese vacancy at the top surface. The simulated images for α = 24, 30, 50, and 100 mrad
are summarized in Fig. 3(a). B.
DOF of ABF image: experiment Inten-
sity profiles show image contrast of oxygen and Mnβ columns
in a red rectangle of each ABF image. Solid horizontal line in
the profile is the image intensity of the columns without va-
cancy and, dashed line is the reference intensity [22]. Intensity
profile of oxygen columns is shaded. Fig. 4
FIG. 4: Simulated ABF images of one manganese vacancy at
the top surface: (a) 2 nm-thick and (b) 5 nm-thick LiMn2O4
crystal. The convergence semi-angle of the incident beam is
100 mrad. The number in bracket is the number of ions in the
Mnβ column without vacancy for a given film thickness. Inten-
sity profiles show image contrast of oxygen and Mnβ columns
in a red rectangle of each ABF image. Solid horizontal line in
the profile is the image intensity of the columns without va-
cancy and, dashed line is the reference intensity [22]. Intensity
profile of oxygen columns is shaded. FIG. 3: (a) Defocus series of simulated image of a LiMn2O4
crystal for a convergence angle (α) of 24 and 30 mrad. The
film thickness is 3 nm. The range of DOF is shown by white
region. (b) The plot of DOF as a function of the convergence
angle of the incident electron beam. Theoretical equation is
referred from Ref. [16]. B.
DOF of ABF image: experiment A LiMn2O4 crystal was observed by ABF imaging, us-
ing an AbC-STEM (R005 electron microscope) [18]. The
electron probe was accelerated at 300 kV and transmitted
the LiMn2O4 sample along the [110] direction. A series
of ABF images was obtained with a 2-nm defocus step in
order to investigate the depth of focus. The convergence
semi-angle (α) of the incident electron probe was chosen
to be 24 mrad and 30 mrad. The detector inner-outer
semi-angle, β, was 12-24 mrad (for α = 24 mrad) and
15-30 mrad (for α = 30 mrad). images become blurred and atomic columns are not distin-
guished clearly. The defocus range, in which dark contrast
of columns remains to be a half of the maximum contrast,
is defined as the depth of focus (DOF) [22]. The ABF
images obtained with the convergence angle of 30 mrad
shows similar contrast change, but gives shallower DOF. As shown in Fig. 2(b), clear images were obtained for the
defocus range of 0-2 nm. The observed DOF values for
24 and 30 mrad are plotted by triangles in Fig. 3(b) with
error bar. Figure 2(a) shows a through-focus series of ABF im-
ages observed from the [110] direction with the conver-
gence angle of 24 mrad. All atomic columns includ-
ing lithium columns appear as dark dots at over-focus
(0 ≤f ≤4 nm). Mnα columns are positioned at the
corner of the rhombic unit marked by black lines. As the
defocus goes into under-focus, column contrast changes
from dark to bright. We defined the zero defocus (0 nm)
as the defocus value where the probe is focused at the
top surface of the specimen and the plus (+) defocus as
the defocus value where the probe is focused over the top
surface [21]. Within the defocus range of 0-4 nm, ABF
images show clear contrast. Beyond that defocus range, The observed images showed firstly that the optimum
focus giving the maximum contrast is over-focus regime,
which decreased as the convergence angle increased. Sec-
ondly, the ABF images at 24 and 30 mrad showed that
the dark contrast of atomic columns was proportional to
the number of atoms aligned in the column, as reported
in the previous paper [15]. tp://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) 455 Lee, et al. Volume 10 (2012) Fig.3
FIG. C.
DOF of ABF image: simulation image can be obtained only for the probe focused at the
top surface of specimen. It imposes that the ABF image
with a large convergence angle, 50-100 mrad, is sensitive
to the top surface profile. The theoretical calculation of STEM image of a
LiMn2O4 crystal was performed under the same imag-
ing condition with the experiment. The simulated ABF
images show coincidence with the experimental images. Following the accordance between the experiments and
simulations, we calculated the ABF images of a large con-
vergence angle to find imaging conditions for detection of
a surface ion vacancy. http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) B.
DOF of ABF image: experiment 3: (a) Defocus series of simulated image of a LiMn2O4
crystal for a convergence angle (α) of 24 and 30 mrad. The
film thickness is 3 nm. The range of DOF is shown by white
region. (b) The plot of DOF as a function of the convergence
angle of the incident electron beam. Theoretical equation is
referred from Ref. [16]. C
DOF of ABF image: simulation
Fig. 4
FIG. 4: Simulated ABF images of one manganese vacancy at
the top surface: (a) 2 nm-thick and (b) 5 nm-thick LiMn2O4
crystal. The convergence semi-angle of the incident beam is
100 mrad. The number in bracket is the number of ions in the
Mnβ column without vacancy for a given film thickness. Inten-
sity profiles show image contrast of oxygen and Mnβ columns
in a red rectangle of each ABF image. Solid horizontal line in
the profile is the image intensity of the columns without va-
cancy and, dashed line is the reference intensity [22]. Intensity
profile of oxygen columns is shaded. Fig.3
FIG. 3: (a) Defocus series of simulated image of a LiMn2O4
crystal for a convergence angle (α) of 24 and 30 mrad. The
film thickness is 3 nm. The range of DOF is shown by white
region. (b) The plot of DOF as a function of the convergence
angle of the incident electron beam. Theoretical equation is
referred from Ref. [16]. Fig. 4
FIG. 4: Simulated ABF images of one manganese vacancy at
the top surface: (a) 2 nm-thick and (b) 5 nm-thick LiMn2O4
crystal. The convergence semi-angle of the incident beam is
100 mrad. The number in bracket is the number of ions in the
Mnβ column without vacancy for a given film thickness. Inten-
sity profiles show image contrast of oxygen and Mnβ columns
in a red rectangle of each ABF image. Solid horizontal line in
the profile is the image intensity of the columns without va-
cancy and, dashed line is the reference intensity [22]. Intensity
profile of oxygen columns is shaded. Fig. 4
FIG. 4: Simulated ABF images of one manganese vacancy at
the top surface: (a) 2 nm-thick and (b) 5 nm-thick LiMn2O4
crystal. The convergence semi-angle of the incident beam is
100 mrad. The number in bracket is the number of ions in the
Mnβ column without vacancy for a given film thickness. [1] J. M. Tarascon and M. Armand, Nature 414, 359 (2001).
[2] A. D. Pasquier, I. Plitz, S. Menocal, and G. Amatucci, J.
Power Sources 115, 171 (2003).
[3] E. Karden, S. Ploumen, B. Fricke, T. Miller, and K. Sny-
der. J. Power Sources 168, 2 (2007).
[4] NEDO, Battery RoadMap (Japan, 2010).
[5] J. Vetter, P. Nov´ak, M. R. Wagner, C. Veit, K.-C. M¨oller, B.
Imaging lithium vacancy at surface Figure 5 shows the simulated images of the model
with one lithium vacancy in the lithium diffusion channel
(Fig. 1(b)). The convergence angle of incident beam was
100 mrad. In the area of the red rectangle in Fig. 5(a), the
lithium column at the left side has one lithium vacancy
at the top surface. The other lithium column contains
no vacancy (four lithium ions within the film thickness
of 2 nm). The lithium column with a vacancy gives less
contrast than the other (without vacancy), as seen in the
line profile. One lithium vacancy at the top surface re-
duced the contrast by roughly 20%. Contrast decrease is
the same amount for a 5 nm-thick model (Fig. 5(b)). For
the case of one lithium vacancy positioned at middle of
the column or at the bottom surface, the column contrast
was not reduced more than 3%. Thus, a lithium vacancy
at the top surface can be observed with a decreased col-
umn contrast. As the convergence angle became small,
the contrast of the vacancy at the top surface reduced:
15% with 70 mrad which could be used to detect lithium
vacancy. A.
Imaging manganese vacancy at surface The simulated images of α =
24 and 30 mrad reproduce the experimental images, where
all atomic columns appear as dark dots at over-focuses. As the convergence angle of the probe increases, DOF of
ABF images becomes shallower. Then, well-resolved ABF http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt 456 e-Journal of Surface Science and Nanotechnology Volume 10 (2012) Fig. 5
FIG. 5:
Simulated ABF images of one lithium vacancy at
the top surface: (a) 2 nm-thick and (b) 5 nm-thick LiMn2O4
crystal. The convergence semi-angle of the incident beam is
100
d
Th
b
i
b
k t i
th
b
f i
i IV.
DISCUSSION Surface imaging by ABF imaging with the large conver-
gence angle is promising, although it needs further instru-
mental development: The convergence angle of 50 mrad
at 300 kV had already been achieved [25]. Chromatic aberration gives image blurring because of
energy spread of the cold emission source. The blurring
is represented by convolution of Gaussian function. Ac-
cording to convolution theorem, Gaussian function does
not alter the relative contrast relation (the ratio of the
vacancy contrast to the non-vacancy column contrast)
but absolute image intensity. By using monochrome-
terr [26, 27], and/or chromatic aberration corrector [28–
30], the ABF image with high contrast can be obtained. The optimum defocus of ABF image giving the max-
imum contrast is at over-focus regime, although annular
dark field (ADF) image has optimum defocus at under-
focus regime. Simultaneous ABF and ADF observation
might give much information of the surface structure. Fig. 5
FIG. 5:
Simulated ABF images of one lithium vacancy at
the top surface: (a) 2 nm-thick and (b) 5 nm-thick LiMn2O4
crystal. The convergence semi-angle of the incident beam is
100 mrad. The number in bracket is the number of ions in
the lithium column without vacancy for given film thickness. Intensity profiles show image contrast of lithium columns in a
red rectangle of each ABF image. Solid horizontal line in the
profile is the image intensity of the columns without vacancy
and, dashed line is the reference intensity. V.
CONCLUSIONS LiMn2O4 crystal was observed by ABF imaging with
an aberration corrected STEM. In ABF images, all atomic
columns including lithium columns are visible as dark dots
at over-focuses. As the convergence angle increases, the
optimum over-focus giving the maximum intensity ap-
proaches to zero-focus. The depth of focus (DOF) de-
creases, then, and ABF image becomes sensitive to the
structure of the top surface: A manganese ion vacancy
at the top surface gives the contrast reduction, 10% for
50 mrad, and 25% for 100 mrad of the convergence an-
gle. A lithium ion vacancy gives the contrast reduction of
15% for 70 mrad, and 20% for 100 mrad. This ABF imag-
ing with the large convergence angle of the incident beam
could provide the surface image of electrode materials of
lithium ion batteries. J. O. Besenhard, M. Winter, M. Wohlfahrt-Mehrens, C.
Vogler, and A. Hammouche, J. Power Sources 147, 269
(2005). [6] M. Yoshio, H. Wang, K. Fukuda, Y. Hara, and Y. Adachi,
J. Electrochem. Soc. 147, 1245 (2000). (
)
[7] Y. H. Huang, K. S. Park, and J. B. Goodenough, J. Elec-
trochem. Soc. 153 A2282 (2006). J. O. Besenhard, M. Winter, M. Wohlfahrt-Mehrens, C.
Vogler, and A. Hammouche, J. Power Sources 147, 269
(2005).
[6] M. Yoshio, H. Wang, K. Fukuda, Y. Hara, and Y. Adachi,
J. Electrochem. Soc. 147, 1245 (2000).
[7] Y. H. Huang, K. S. Park, and J. B. Goodenough, J. Elec-
trochem. Soc. 153 A2282 (2006). http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) Acknowledgments This work was supported by the Japan Science and
Technology Agency (JST) under the CREST project and
in part by a Grand-in-Aid for Scientific Research (S) from
the Ministry of Education, Culture, Sports, Science and
Technology of Japan. http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) 457 Volume 10 (2012) Lee, et al. [8] C. Li, H. P. Zhang, L. J. Fu, H. Liu, Y. P. Wu, E. Rahm,
R. Holze and H. Q. Wu, Electrochimica Acta 51, 3872
(2006). Chem. 14, 1948 (2004). Chem. 14, 1948 (2004). [20] L. A. Picciotto and M. M. Thackeray, Mater. Res. Bull. 20, 1409 (1985). (
)
[9] M. Hirayama, H. Ido, K. Kim, W. Cho, K. Tamura, J. Mizuki, and R. Kanno, J. Am. chem. Soc. 132, 15268
(2010). (
)
[21] The defocus value was determined by referring the sim-
ulated results, in which the contrast of atomic column is
reversed between ∆f = 0 and −2 nm. (
)
[10] N. Balke, S. Jesse, A. N. Morozovska, E. Eliseev, D. W. Chung, Y. Kim, L. Adamczyk, R. E. Garc´ıa, N. Dudney,
and S. V. Kalinin, Nat. Nanotech. 5, 749 (2010). [22] Dark contrast is measured as defined in Ref. [15]. [23] K. Ishizuka, Ultramicroscopy 90, 71 (2002). [
]
(
)
[24] S. Kim, Y. Oshima, H. Sawada, T. Kaneyama, Y. Kondo, M. Takeguchi, Y. Nakayama, Y. Tanishiro, and [24] S. Kim, Y. Oshima, H. Sawada, T. Kaneyama, Y. Kondo, M. Takeguchi, Y. Nakayama, Y. Tanishiro, and
K. Takayanagi, J. Electron Microsc. 60, 109 (2011). [11] M. Morcrette, P. Rozier, L. Dupont, E. Mugnier, L. San-
nier, J. Galy, and J. M. Tarascon, Nat. Mater. 2, 755
(2003). [25] H. Sawada, T. Sannomiya, F. Hosokawa, T. Nakamichi, T. Kaneyama, T. Tomita, Y. Kondo, T. Tanaka, Y. Oshima,
Y. Tanishiro, and K. Takayanagi, Ultramicroscopy 108,
1467 (2008). (
)
[12] D. Chen, S. Indris, M. Schulz, B. Gamer, and R. Monig,
J Power Sources 196 6382 (2011) J. Power Sources 196, 6382 (2011). [13] Y. Oshima, H. Sawada, F. Hosokawa, E. Okunishi, T. Kaneyama, Y. Kondo, S. Niitaka, H. Takagi, Y. Tanishiro,
and K. Takayanagi, J. Electron Microsc. 59, 457 (2010). [26] P. C. Tiemeijer,
M. Bischoff,
B. Freitag,
and
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EMC
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Microscopy
Congress 1-5, I1 I2, 53-54 (2008). [14] R. Huang, Y. H. Ikuhara, T. Mizoguchi, S. D. Findlay,
A. Kuwabara, C. A. Fisher, H. Moriwake, H. Oki, T. http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) Acknowledgments Hi-
rayama, and Y. Ikuhara, Angew. Chem. Int. Ed. 50, 3053
(2011). (
)
[27] P. C. Tiemeijer,
M. Bischoff,
B. Freitag,
and
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https://openalex.org/W3000517060
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https://pubs.rsc.org/en/content/articlepdf/2020/cp/c9cp05476f
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English
| null |
A DFT and KMC based study on the mechanism of the water gas shift reaction on the Pd(100) surface
|
Physical chemistry chemical physics/PCCP. Physical chemistry chemical physics
| 2,020
|
cc-by
| 12,584
|
a School of Chemistry, Brayford Pool, University of Lincoln, Lincoln, LN6 7TS, UK.
E-mail: achutia@lincoln.ac.uk
b UK Catalysis Hub, RCaH, Rutherford Appleton Laboratory, Didcot, OX11 OFA, UK
c Department of Chemistry, University of Manchester, UK
d Department of Chemistry, University College London, Gordon Street, London,
WC1H 0AJ, UK. E-mail: c.r.a.catlow@ucl.ac.uk
e Department of Chemical Engineering, University College London, Torrington Place,
London, WC1E 7JE, UK
f CardiffCatalysis Institute, School of Chemistry, CardiffUniversity, Cardiff,
CF10 3AT, UK
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cp05476f Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024
This article is licensed under a Creative Commons Attribution 3.0 U We present a combined density functional theory (DFT) and Kinetic Monte Carlo (KMC) study of the
water gas shift (WGS) reaction on the Pd(100) surface. We propose a mechanism comprising both the
redox and the associative pathways for the WGS within a single framework, which consists of seven core
elementary steps, which in turn involve splitting of a water molecule followed by the production of
an H-atom and an OH-species on the Pd(100) surface. In the following steps, these intermediates
then recombine with each other and with CO leading to the evolution of CO2, and H2. Seven other
elementary steps, involving the diffusion and adsorption of the surface intermediate species are also
considered for a complete description of the mechanism. The geometrical and electronic properties of
each of the reactants, products, and the transition states of the core elementary steps are presented. We
also discuss the analysis of Bader charges and spin densities for the reactants, transition states and the
products
of
these
elementary
steps. Our
study
indicates
that
the
WGS
reaction
progresses
simultaneously via the direct oxidation and the carboxyl paths on the Pd(100) surface. rsc.li/pccp associative or the carboxyl mechanism and the coupling
mechanism.3,20 In the redox mechanism, the water molecule
is dissociated into atomic H and O atoms; the dissociated
O-atom then interacts with the adsorbed CO molecule to form
CO2. In the associative mechanism, the adsorbed CO interacts
with adsorbed OH species obtained by partial dissociation of
H2O, to give a carboxyl intermediate, which then decomposes
to CO2 and an H-atom.26 Finally, in the coupling mechanism,
the evolution of CO2 takes place directly from the reaction
of CO and OH species. This study concluded that the redox
mechanism is the most favourable pathway for the WGS
reaction on the Fe3O4(111) surface. In an earlier work, Gokhale
et al. studied all the elementary steps of redox and carboxyl
mediated mechanisms for the low-temperature WGS reaction
on the Cu(111) surface, using density functional theory (DFT)
and microkinetic modelling.3 They proposed that the abstrac-
tion of H from H2O is the rate determining step, while in
another study Grabow et al. employed DFT, microkinetic mod-
elling and experimental techniques to study the WGS reaction
on the Pt(111) surface.18 They concluded that formate species act
as spectators and cannot be formed from CO and OH in a single
elementary step. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024
This article is licensed under a Creative Commons Attribution 3.0 U The CO species was identified as the most
abundant of the surface intermediates and the carboxyl group
(COOH), formed from CO and OH, subsequently combined with
OH to form CO2 and H2O. While this step was considered to be
the favoured CO2 formation path, due to its low activation energy PCCP PCCP View Article Online
View Journal | View Issue Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. PCCP This journal is ©the Owner Societies 2020 A DFT and KMC based study on the mechanism of
the water gas shift reaction on the Pd(100)
surface† Cite this: Phys.Chem.Chem.Phys.,
2020, 22, 3620 Arunabhiram Chutia,
*ab Adam Thetford,
bcd Michail Stamatakis
e and
C. Richard A. Catlow
*bdf 2. Computational details
DFT calculations In addition to pristine metallic surfaces, there are also
several reports of investigations of the WGS reaction on metals
supported on metal oxides.14,29,30 Song et al. for example,
employed DFT+U techniques to understand the mechanism
of the WGS reaction on a single Au atom versus Au cluster
supported on CeO2.14 They concluded that the carboxyl mecha-
nism has a lower energy pathway compared to the redox
mechanism. In related studies, the elementary steps involving
water splitting, and CO oxidation have also been studied. For
instance, Filhol et al. elucidated the electrochemical activation
of water over the Pd(111) surface and Peterson et al. investi-
gated low temperature CO oxidation catalysed by atomically
dispersed Pd on Al2O3.31,32 We also note that the kinetics of the
WGS reaction could be also greatly influenced by catalyst
supports. For example, earlier studies by Grenoble et al. per-
formed a detailed study on the Group VIIB, VIII and IB metals
supported on Al2O3 and showed that the rate per surface metal
atom of Pt supported on Al2O3 has an higher magnitude as
compared to Pt supported on SiO2.33 We employ the Vienna Ab initio Simulation Package (VASP)
to perform DFT calculations.38–41 We used the projector
augmented wave (PAW) method and the cut-off energy is set to
450 eV, which gave bulk energies converged to within 105 eV. We chose a convergence criterion of 0.02 eV Å1 for our structural
optimisations and the Perdew–Wang (PW91) version of the
generalized gradient approximation (GGA) to carry out total
energy calculations and perform geometry optimisations.42,43
We note that the PW91 functional has been shown to be
reliable for such studies.44,45 For all the calculations an experi-
mental lattice constant of 3.891 Å is used for bulk Pd and the
ideal Pd(100) surfaces are modelled by a 3 3 supercell with 4
atomic layers respectively from bulk Pd. Of the four layers we
have relaxed the top two atomic-layers and fixed the bottom two
layers to mimic the bulk of the system. Six equivalent layers of
vacuum between two successive metal slabs are used. To
determine the minimum energy paths for all the elementary
steps, we employed the climbing-image nudged elastic band
(CI-NEB) method.46,47 The transition state of the optimised
reaction coordinate was confirmed by calculations of the vibra-
tional frequencies. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In the following sections, we will present first the details of
our computational procedures and a description of the proposed
reaction mechanism in terms of geometric and electronic prop-
erties, which will be followed by the discussions of the results
obtained from the DFT calculations and their implementation in
the KMC analysis. We show that the water gas shift reaction
progresses simultaneously via both direct oxidation and carboxyl
pathways, which consist of seven core elementary steps. 1. Introduction The water gas shift (WGS) reaction plays a crucial role in many
industrially important processes as diverse as steam-methane
reforming for ammonia and methanol synthesis and direct
proton exchange membrane fuel cell applications.1–24 In the
last few years, the mechanism of this reaction has been studied
intensively using both theoretical and experimental techniques;
it is, however, still extensively debated.25 In previous studies,
various reaction pathways have been considered; for example, a
study by Huang et al. focused on three different pathways for
the WGS reaction on the Fe2O3(111) surface using density
functional theory with Hubbard correction (DFT+U) i.e. a redox
mechanism also known as the regenerative mechanism, the 3620 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 This journal is ©the Owner Societies 2020 View Article Online View Article Online PCCP PCCP Paper barrier, their microkinetic modelling study, shows that this
reaction is limited by the low OH coverage. They proposed that
the direct decomposition step of COOH - CO2 + H accounts
for 75–95% of the total CO2 production. In this study, we focus our study on the Pd surface as
Pd-based membranes are known to be able to isolate hydrogen
in large quantities and maintain its stability during the water
gas shift reaction. For instance, recent studies have demon-
strated that the Pd and Pd-based alloy membranes are able to
produce H2 with a high purity of 99.89%.34,35 The choice of
the Pd(100) surface, on the other hand, is largely based on the
fact that it is well characterised by a large number of recent
experimental studies using in situ methods.36 Furthermore,
Zhang et al. using periodic DFT showed that the reaction
pathways on the Pd(111) and Pd(110) surfaces display common
features despite the fact that they have different surface
symmetries.37 Recent, theoretical studies have also shown
that the surface energies of the Pd surfaces are in the order
Pd(111) o Pd(100) o Pd(110), so the Pd(111) surface is the
most stable surface followed by Pd(100) and Pd(110).28 There-
fore, we considered the Pd(100) surface to capture the WGS
reaction on the surface, which is experimentally well-known,
relatively active but sufficiently stable. Mohsenzadeh et al. recently reported a DFT based study on
the effect of three different Ni surfaces i.e., Ni(111), Ni(110) and
Ni(100) on the WGS reaction.4 They considered nine elementary
steps, which included water dissociation, CO oxidation and
the formation of hydrogen. This journal is ©the Owner Societies 2020 Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 | 3621 1. Introduction Their calculations indicated that
water dissociation, formyl formation, formate dissociation
and formation of H2 have the lowest barrier on the Ni(110)
surface, CO oxidation has the lowest barrier on the Ni(100)
surface. Their analysis showed that the rate limiting step was
CO + O - CO2. The barrier for this reaction decreased in
the order of Ni(110) 4 Ni(111) 4 Ni(100) surfaces. In another
study involving DFT, near-edge X-ray absorption fine structure
(NEXAFS) and IR spectroscopy, Senanayake et al. investigated
the role of formate, carbonate and carboxyl species as possible
intermediates in the WGS reaction on the Au(111) surface.27
They found that for formate, the bidentate is more stable
than the monodentate configuration by B0.650 eV and it is,
therefore, unlikely that formate is a key intermediate in the
WGS reaction. They concluded that carbonate species are not
present during the reaction of CO with OH on Au(111) at
90–120 K and that carbonate is much less stable on the
Au(111) surface than on oxide surfaces. However, we note that
that there are reports where the formate species have been
observed as intermediates, for example in studies on the
reverse WGS reaction by Choi et al. employing in situ FT-IR
and DFT techniques on Pd, Ni, Cu and Ag.28 3. Results and discussion Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The overall water gas shift reaction is given by the equation: CO + H2O " CO2 + H2 CO + H2O " CO2 + H2 Ead = Eadsorbate + Pd(100) (EPd(100) + Eadsorbate),
(1) To formulate the mechanism of this reaction, we first
consider the key intermediates including H, O, and OH, which
can be obtained from H2O and the reactant CO, and then
determine all the other probable intermediates in this reaction. In a recent study, Mohsenzadeh et al. have shown that HCO is
an unstable intermediate; similarly, Plauk et al. have shown
that the OOH intermediate on Pd(111) and Pd(100) surfaces will
readily dissociate to give O and OH intermediates; and as
described previously, Grabow et al. reported that the formate
species is a spectator species and cannot be formed from
CO and OH in a single elementary step.4,18,44 Therefore,
considering all these aspects, we formulated a mechanism
consisting of seven core elementary steps involving both redox
and carboxyl mechanisms as shown in Fig. 1. We propose
that the splitting of the water molecule, gives rise to an
adsorbed H-atom and an OH-species on the Pd(100) surface. In the following steps, these intermediate species combine with
CO leading to the evolution of CO2, and H2. We have also
considered the possibility that the CO2 molecule is formed via
another route involving a carboxyl species. It is worth mentioning
that the Boudouard reaction, which is 2CO " Csrf + CO2, is also
one of the probable elementary steps. However, recent studies
have shown that the rate of this reaction decreases in presence
of H2O and H2.58 Additionally, the inclusion of this elementary
step could lead to another step i.e., Csrf " Cbulk, as shown by us
in our previous studies on palladium carbides, which may
greatly affect the mechanism of the water gas shift reaction.59,60 where, Ead is the adsorption energy, Eadsorbate+Pd(100) is the
energy of the system with the adsorbate on Pd surfaces,
EPd(100) is the energy of pristine surface and Eadsorbate is the
energy of the adsorbate. The adsorption energy values include
zero-point energy corrections. 2. Computational details
DFT calculations Since a slab exposes two surfaces (top and
the bottom of the slab), placing an adsorbate on one of these
generally induces a dipole moment. To eliminate the latter, one
can place a second adsorbate symmetrically on the other It is clear that despite the wide range of theoretical and
experimental studies on the WGS reaction, there are consider-
able uncertainties regarding the key elementary steps of its
reaction mechanism. Therefore, in this study we employ density
functional theory (DFT) and Kinetic Monte Carlo (KMC) simula-
tions to understand the mechanism of this important reaction
over the Pd(100) surface. This journal is ©the Owner Societies 2020 Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 | 3621 3621 View Article Online PCCP Paper Paper on the same surface (see Table S1, ESI†).56 Pre-exponential
factors were then computed using the vibrational frequencies
from the DFT calculations. A detailed discussion on the evalua-
tion of the pre-exponential factors has been given previously by
Stamatakis and co-workers in ref. 57. surface; yet, this increases computational cost and leads to
problems when calculating transition states (two imaginary
frequencies for symmetrically placed transition state species). To avoid these issues, adsorption is allowed on only one of the
two surfaces in all of our calculations. The dipole moment for
these calculations, due to the adsorbed species is accounted
for using the methods according to the works of Makov et al. and Neugebauer et al.48,49 as implemented in VASP. A k-point
grid of 18 18 1 was used for all the calculations. The
adsorption energy of adsorbates on Pd(100) surface is then
calculated using: 3. Results and discussion The charges on various atoms
were obtained using the Bader charge analysis as implemented
by Henkelman and co-workers.50 It is also worth mentioning
that previously, Tsuzuki et al. used PW91 exchange and correla-
tion functional to evaluate nonbonding dispersive interactions,
and they found that the interaction energies are very close to
their CCSD(T) and MP2 results.51 It has been also reported that
PW91 already overestimates the binding energy; therefore,
inclusion of additional vdW term does not lead to further
improvements.52 Further to this, Ortmann et al. also showed
that the use of PW91 and PW91 + vdW results in very little
difference in lattice parameters, bulk modulus, cohesive energies,
vibrational frequencies, bond lengths, of solids and molecules.52
Therefore, in this study we have not considered the dispersive
effects separately. 3622 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 ess Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Step 2: formation of H2O and adsorption of O
(OH)srf + (OH)srf " (H2O)srf + (O)srf Step 2: formation of H2O and adsorption of O
(OH)srf + (OH)srf " (H2O)srf + (O)srf Step 2: formation of H2O and adsorption of O Kinetic Monte-Carlo simulations The KMC simulations were run using the Zacros code53,54
(http://zacros.org) with the results processed using MATLAB
(https://uk.mathworks.com/). During
the
simulations,
the
species number and process statistics were sampled every
1 ms and a snapshot of the adlayer was taken every 50 ms. We
have used H2O, CO, CO2, and the pristine Pd(100) surface as the
reference species. Stiffness scaling was used on all the diffusion
and water adsorption and desorption steps to prevent these
steps from dominating and hampering time progression past
a few milliseconds. All the steps were allowed to be reversible, so
that the simulation satisfies microscopic reversibility. It was
necessary to include 7 extra steps involving diffusion, adsorption
and desorption in the proposed mechanism considered for the
DFT calculations, in order to simulate an open system and take
into account the mobility of the adsorbates on the surface. The
pressure and temperature are kept at 1 atm and 600 K respec-
tively, which was selected so that a suitable time could be
sampled within available computational resources. The partial
pressure ratio of CO and H2O was varied, ranging from 1 : 1 to a
1000 : 1 ratio of CO and H2O.55 Lateral interactions were calcu-
lated by deriving the interaction energies using DFT on two
adjacent molecules (both for the same or for different species) Fig. 1
A mechanism consisting of seven core elementary steps involving
both redox and carboxyl mechanisms for water gas shift reaction. For
simplicity the adsorption and desorption steps are not shown. Fig. 1
A mechanism consisting of seven core elementary steps involving
both redox and carboxyl mechanisms for water gas shift reaction. For
simplicity the adsorption and desorption steps are not shown. 3622 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 This journal is ©the Owner Societies 2020 View Article Online PCCP Paper Paper In this study, to simplify the proposed mechanism we have not
included these two steps. For clarity, each of these proposed
elementary steps are summarised below (in which the subscript
‘‘srf’’ indicates a surface species): and diffusion of CO, H2O, O, H, OH, and CO2 for a complete
description of the WGS reaction, which we will discuss when
we present our KMC studies. The geometric and electronic
properties of atomic and/or molecular species involved in
reactants, products, and transition states of each of these core
elementary steps are described in the next section. Step 4: H2 evolution from (H)srf (H)srf + (H)srf " H2 Step 5: CO oxidation Step 5: CO oxidation (CO)srf + (O)srf " CO2 We find that the H-atom displays similar adsorption
energies on the four-fold hollow (4f) and the bridge (B) sites Step 6: formation of COOH from COsrf and (OH)srf
(CO)srf + (OH)srf " (COOH)srf Step 6: formation of COOH from COsrf and (OH)srf
(CO)srf + (OH)srf " (COOH)srf Fig. 2
Top and side views of the most stable structures of (a) H atom, (b)
O atom, (c) CO molecule, (d) OH species, (e) CO2 molecule, and (f) H2O
molecule. For clarity the uppermost layer is shown as ball and stick
format and for the lower subsurfaces van der Waals representation of
the atoms is adopted. Step 7: CO2 evolution (COOH)srf " (CO2)srf + (H)srf (COOH)srf " (CO2)srf + (H)srf In addition to these seven core steps, we have also
considered the adsorption and desorption of CO and H2O Table 1
The geometry and adsorption energies of various adsorbates
with zero point energy corrections on the four-fold hollow (4f), bridge (B)
and top (T) sites of Pd(100) surface. ‘‘A’’ in the table represents the atoms
closer to the Pd(100) surface Table 1
The geometry and adsorption energies of various adsorbates
with zero point energy corrections on the four-fold hollow (4f), bridge (B)
and top (T) sites of Pd(100) surface. ‘‘A’’ in the table represents the atoms
closer to the Pd(100) surface Thi j
l i ©th
O
S
i ti
2020
Species
Sites
Proximity
to Pd
Interatomic distances (Å)
Ead (eV)
Pd–A
A–B (Å)
H
4f
H
1.981
—
2.342
B
H
1.717
—
2.342
T
H
1.555
—
1.906
O
4f
O
2.149
—
1.221
B
O
1.933
—
0.890
T
O
1.825
—
0.123
CO
4f
C
2.206
1.203
1.854
B
C
2.000
1.178
1.924
T
C
1.876
1.158
1.501
OH
4f
O
2.159
0.978
3.128
B
O
1.934
0.976
3.204
T
O
1.969
0.967
2.440
CO2 (horizontal) 4f
C, O
—
1.177
0.173
B
C, O
—
1.176
0.011
T
C, O
—
1.777
0.025
H2O
4f
O
2.987
0.975
0.100
B
O
2.484
0.973
0.147
T
O
2.966
0.980
0.076 Fi
2
T
d id
i
f th
t t bl
t
t
f ( ) H
t
(b) Fig. 3.1.
Adsorption properties of the atomic and molecular
species Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (OH)srf + (OH)srf " (H2O)srf + (O)srf We first consider the adsorption energies of all the adsorbed
atomic and molecular species on the Pd(100) surface. The
adsorption energies of these species are calculated on top (T)
of a Pd atom, in between two Pd atoms i.e. the bridge site (B)
and on the four-fold hollow (4f) site. In Table 1, the calculated
distances of the atomic and/or molecular species from the
Pd(100) surface, their interatomic distance and the adsorption
energies for all these adsorbates are given. The top and side
views of all the optimised structures for the atomic and
molecular species are also shown in Fig. 2(a–f). Step 3: OH splitting (OH)srf " (O)srf + (H)srf Step 4: H2 evolution from (H)srf This journal is ©the Owner Societies 2020 Kinetic Monte-Carlo simulations Step 1: water splitting (H2O)srf " (OH)srf + (H)srf Step 4: H2 evolution from (H)srf For the
diatomic molecules (or species) such as CO, OH and H2, we
find that the adsorption at the bridge sites is of lower energy. We also note that the adsorbed OH species display an interesting
geometrical property after relaxation i.e., the H atom adopts a
slanted position with respect to the surface Pd-atom to which
O of the OH species is bound. In contrast, CO is bound via the
C-atom in a perpendicular configuration. The probable reason
for this difference can be investigated using Bader charge
analysis. The average charge on the Pd atoms on the surface is
0.034 e and 0.012 e respectively for OH/Pd(100) and CO/Pd(100)
systems. However, when we consider the Pd atoms in the
immediate neighbourhood surrounding the OH and CO species,
we see that they have a residual average negative charge i.e., 0.020
e and 0.053 e for OH/Pd(100) and CO/Pd(100) surfaces respec-
tively. On the other hand, the charge on the H-atom of OH species
is 0.595 e and 1.009 e for the O-atom in the CO species, resulting
in an electrostatic attraction between the H of OH species and the
Pd(100) surface and hence the bent structure. The CO2 molecule is
weakly adsorbed on the top site, while the H2O molecule is weakly
chemisorbed on the ‘‘bridge-top’’ site i.e., after relaxation of the H2O molecule adsorbed on the bridge site, it migrates closer to the
top site and remains tilted towards the surface. Having established the favoured sites for the adsorbed
species, we now explore their geometrical and electronic prop-
erties in more detail. Step 4: H2 evolution from (H)srf 2
Top and side views of the most stable structures of (a) H atom, (b)
O atom, (c) CO molecule, (d) OH species, (e) CO2 molecule, and (f) H2O
molecule. For clarity the uppermost layer is shown as ball and stick
format and for the lower subsurfaces van der Waals representation of
the atoms is adopted. Fig. 2
Top and side views of the most stable structures of (a) H atom, (b)
O atom, (c) CO molecule, (d) OH species, (e) CO2 molecule, and (f) H2O
molecule. For clarity the uppermost layer is shown as ball and stick
format and for the lower subsurfaces van der Waals representation of
the atoms is adopted. Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 | 3623 View Article Online PCCP Paper and is stable in both these sites. The adsorption energies of the
O-atom indicate strongest binding at the 4f site. For the
diatomic molecules (or species) such as CO, OH and H2, we
find that the adsorption at the bridge sites is of lower energy. We also note that the adsorbed OH species display an interesting
geometrical property after relaxation i.e., the H atom adopts a
slanted position with respect to the surface Pd-atom to which
O of the OH species is bound. In contrast, CO is bound via the
C-atom in a perpendicular configuration. The probable reason
for this difference can be investigated using Bader charge
analysis. The average charge on the Pd atoms on the surface is
0.034 e and 0.012 e respectively for OH/Pd(100) and CO/Pd(100)
systems. However, when we consider the Pd atoms in the
immediate neighbourhood surrounding the OH and CO species,
we see that they have a residual average negative charge i.e., 0.020
e and 0.053 e for OH/Pd(100) and CO/Pd(100) surfaces respec-
tively. On the other hand, the charge on the H-atom of OH species
is 0.595 e and 1.009 e for the O-atom in the CO species, resulting
in an electrostatic attraction between the H of OH species and the
Pd(100) surface and hence the bent structure. The CO2 molecule is
weakly adsorbed on the top site, while the H2O molecule is weakly
chemisorbed on the ‘‘bridge-top’’ site i.e., after relaxation of the and is stable in both these sites. The adsorption energies of the
O-atom indicate strongest binding at the 4f site. 3.3.
Geometry, energetics and local electronic properties of
the transition states In this section, we will describe the geometrical and local
electronic properties of the transition states with respect to This journal is ©the Owner Societies 2020
Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 | 3625
Table 2
The Pd–Pd on the surface ((Pd–Pd)srf) and in proximity ((Pd–Pd)near) of the interacting species, the distance between the nearest atom (A) of
these species with the Pd(100) surface and their Bader charges. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. On closely monitoring the Pd–Pd distances for all the reac-
tants and products, we can, however, see an exception to the
above observation in step 5 (see Table 2) i.e., in close proximity to
an adsorbed O-atom, the Pd–Pd distance is 2.778 Å, which is
similar to the average Pd–Pd distance on the surface. In this
case, the adsorbed oxygen atom, in the four-fold hollow site, is 3.2.
Geometrical and electronic properties of the reactants,
and products However, despite their similarities we can see that
in the former case the intensity of d-orbital signatures around
the Fermi energy (EF) is lower than with the pair of Pd atoms
further away from the O-adsorption site, implying weakening of
d–d interactions, which may be related to the polarisation of Pd
atoms due to presence of electronegative O-atom leading to the
increased Pd–Pd distance. Another interesting point is that,
in the former case, we can also see s and d signatures at around
7 eV (Fig. 4(a)), which are missing in the pair of Pd atoms
further away from the O-adsorption site. These additional signa-
tures are due to the interaction of the O-atom with the Pd atoms. On closely monitoring the Pd–Pd distances for all the reac-
tants and products, we can, however, see an exception to the
above observation in step 5 (see Table 2) i.e., in close proximity to
an adsorbed O-atom, the Pd–Pd distance is 2.778 Å, which is
similar to the average Pd–Pd distance on the surface. In this
case, the adsorbed oxygen atom, in the four-fold hollow site, is with each other for both pairs of Pd atoms, showing strong d–d
interactions. However, despite their similarities we can see that
in the former case the intensity of d-orbital signatures around
the Fermi energy (EF) is lower than with the pair of Pd atoms
further away from the O-adsorption site, implying weakening of
d–d interactions, which may be related to the polarisation of Pd
atoms due to presence of electronegative O-atom leading to the
increased Pd–Pd distance. Another interesting point is that,
in the former case, we can also see s and d signatures at around
7 eV (Fig. 4(a)), which are missing in the pair of Pd atoms
further away from the O-adsorption site. These additional signa-
tures are due to the interaction of the O-atom with the Pd atoms. On closely monitoring the Pd–Pd distances for all the reac-
tants and products, we can, however, see an exception to the
above observation in step 5 (see Table 2) i.e., in close proximity to
an adsorbed O-atom, the Pd–Pd distance is 2.778 Å, which is
similar to the average Pd–Pd distance on the surface. Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 | 3625 3.2.
Geometrical and electronic properties of the reactants,
and products Another interesting point
in the former case, we can also see s and d signatures a
7 eV (Fig. 4(a)), which are missing in the pair of P
further away from the O-adsorption site. These addition
tures are due to the interaction of the O-atom with the P
On closely monitoring the Pd–Pd distances for all
tants and products, we can, however, see an exceptio
above observation in step 5 (see Table 2) i.e., in close pro
an adsorbed O-atom, the Pd–Pd distance is 2.778 Å,
similar to the average Pd–Pd distance on the surface
case, the adsorbed oxygen atom, in the four-fold hollow
Table 2
The Pd–Pd on the surface ((Pd–Pd)srf) and in proximit
these species with the Pd(100) surface and their Bader charges
Step
Species on Pd
Average interatomic distances (Å)
(Pd–Pd)srf
(Pd–Pd)near
Distance
Pd close
1
H2O
2.749
2.816
O of H2O
H + OH
2.750
2.729
H
2.884
O of OH
2
OH + OH
2.729
2.885
O of OH
2.891
O of OH
O + H2O
2.754
2.763
O
2.804
O of H2O
3
OH
2.754
2.877
O of OH
O + H
2.754
2.778
H
2.760
O
4
H + H
2.751
2.785
H
2.785
H
H2
2 751
—
—
Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. shared by four Pd atoms and therefore, the effect of weakening
the d–d interactions is minimal. with each other for both pairs of Pd atoms, showing strong d–d
interactions. However, despite their similarities we can see that
in the former case the intensity of d-orbital signatures around
the Fermi energy (EF) is lower than with the pair of Pd atoms
further away from the O-adsorption site, implying weakening of
d–d interactions, which may be related to the polarisation of Pd
atoms due to presence of electronegative O-atom leading to the
increased Pd–Pd distance. Another interesting point is that,
in the former case, we can also see s and d signatures at around
7 eV (Fig. 4(a)), which are missing in the pair of Pd atoms
further away from the O-adsorption site. These additional signa-
tures are due to the interaction of the O-atom with the Pd atoms. with each other for both pairs of Pd atoms, showing strong d–d
interactions. 3.2.
Geometrical and electronic properties of the reactants,
and products The optimised geometries of all the reactants and the products
are reported in Fig. 3(a–f) and summarised in Table 2. Interestingly, the Pd–Pd distances in close proximity to the
adsorbed sites involving O-atoms are slightly longer than the
average values of 2.729 Å. For example, in Step 1, the Pd–Pd
distance is unperturbed around the adsorbed H-atom but it is
2.884 Å around the OH species (i.e., longer by B0.152 Å). This
difference can be attributed to the strong interaction between
the O-containing species bound to the neighbouring Pd atoms. To support these conclusions, we analyse the partial density of
states (PDOS) of two Pd atoms close to the O-atom (referred to as
Pd1close and Pd2close) and another pair of Pd atoms further away
from the O-adsorption site (referred to as Pd3far and Pd4far). As shown in Fig. 4, the d-orbital signatures completely overlap Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 A
This article is licensed under a Creative Commons Attribution 3.0 Unported L Fig. 3
The top view of reactants and products of (a) water splitting, (b) formation of H2O and adsorption of O, (c) OH splitting, (d) H2 evolution, (e) CO
oxidation, (f) formation of carboxyl species and (g) CO2 evolution from carboxyl species. For clarity the upper most atomic layer is shown in ball and stick
and lower sub-surfaces are shown in van der Waals representation. Fig. 3
The top view of reactants and products of (a) water splitting, (b) formation of H2O and adsorption of O, (c) OH splitting, (d) H2 evolution, (e) CO
oxidation, (f) formation of carboxyl species and (g) CO2 evolution from carboxyl species. For clarity the upper most atomic layer is shown in ball and stick
and lower sub-surfaces are shown in van der Waals representation. This journal is ©the Owner Societies 2020 3624 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 View Article Online PCCP Paper with each other for both pairs of Pd atoms, showing str
interactions. However, despite their similarities we can
in the former case the intensity of d-orbital signatures
the Fermi energy (EF) is lower than with the pair of P
further away from the O-adsorption site, implying weak
d–d interactions, which may be related to the polarisati
atoms due to presence of electronegative O-atom leadin
increased Pd–Pd distance. 3.2.
Geometrical and electronic properties of the reactants,
and products In this
case, the adsorbed oxygen atom, in the four-fold hollow site, is We further analysed the Bader charges of all the reactants and
products adsorbed on the Pd(100) surface, which indicate that the
interacting Pd (Pdint) atoms exhibit mostly positive charges ran-
ging from 0.012 e to 0.188 e, showing that due to the adsorption of
various species there is a charge transfer from the Pd surface. In
steps 1, and 4 however, we observe that Pdint has zero charge. The
analyses of charges show that O-atoms bonded to H in OH, C in
CO or CO2 and H in H2O have charges in the range of 0.966 e to
1.232 e and when they are directly adsorbed on the Pd surface
they have a charge of 0.730 e. It is interesting to note that the
H-atoms are adsorbed on the Pd surface as hydride ions. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. g
g
The fourth step, [(H)srf + (H)srf " H2], involves the evolution
of the H2 molecule due to the recombination of the adsorbed H-
atoms with an energy barrier of 0.810 eV. As shown in Table 2,
the adsorbed hydride ions are at a distance of 1.715 Å and in
the transition state of this reaction, both the H-atoms are at a
distance of approximately 2.748 Å from the nearest Pd atom
with an interatomic distance of 0.763 Å, which is close to the
H–H bond length in the H2 molecule (see Fig. 5(d)). The calcula-
tion of Bader charges indicates that in the transition state, the
H-atoms and the closest Pd atoms have approximately zero
charge (Table 3). In the spin charge density contour map, we
can, however, observe small charges on these Pd atoms (third
column in Fig. 6(e)), which may be due to weak electrostatic
interactions between the H H species and the surface. The oxidation of CO, [(CO)srf + (O)srf " (CO2)srf], takes place
in the fifth step with an energy barrier of 0.690 eV. In the
transition state (Fig. 5(e)), the Pd–O and Pd–CO distances are
slightly shorter than the Pd–O and Pd–CO distances in the
reactant (Table 2). The CO has moved from the bridge site
towards the adsorbed O-atom in the TS. The Bader charges
show that the C in CO in the TS is slightly electropositive as
compared to the CO in the reactant, which makes it more
feasible for the adsorbed O-atom to combine with the C of CO In the second step, [(OH)srf + (OH)srf " (H2O)srf + (O)srf], the
–OH species formed in the first step may recombine via an
exothermic reaction to give H2O and an adsorbed O-atom, the
energy barrier for which is found to be 0.880 eV. In the
transition state, the abstraction of one of the H-atoms from
the –OH species takes place. The distance of this H-atom from
the parent –OH species is 2.000 Å, indicating a weak interaction
via a H-bond, and its distance from the other OH species is
B0.990 Å, which is close to the normal O–H bond distance in a
water molecule. The Bader charge on the adsorbed O-atom is
0.755 e and the average charge on the interacting Pd atoms
is +0.202 e, indicating a strong interaction between them. 3.3.
Geometry, energetics and local electronic properties of
the transition states Phys., 2020, 22, 3620--3632 | 3625 This journal is ©the Owner Societies 2020 View Article Online PCCP Paper the reactants and products. The side and top views of each of
the transition states (TS) for all the steps of the proposed
mechanism are shown in Fig. 5. dissociated O-atom and the surface. In contrast, the dissociated
H-atom has a charge of +0.096 e, confirming it to be in the H+
state. The charge on the nearest Pd atom is zero. As shown in
Fig. 6(d), when we visualise the electron spin densities, we find
that the peak heights are smaller for the Pd atoms interacting
with the adsorbed O-atom (third column from the left in
Fig. 6(d)) as compared with the other Pd atoms. The Pd inter-
acting with H+ species also show smaller peak heights, which
may be related to the polarizing effects of the Pd atoms directly
interacting with the electronegative O-atom. In the first step, [H2O " (OH)srf + (H)srf], i.e. water splitting,
the energy barrier is 1.320 eV and the reaction is slightly
exothermic in nature. Previous high-resolution electron energy
loss spectroscopy (HEELS), temperature-programmed desorption
(TPD) and time-of-flight (TOF) spectroscopic studies on Pd surface
have also revealed reversible water dissociation by forming surface
hydroxyl (OHsrf) groups and H atoms adsorbed on the surface.61
In the transition state, one of the H-atoms is at a distance of
1.460 Å while the other is at a distance of 0.979 Å from the O-atom,
i.e. one of the two O–H bonds is stretched (Fig. 5(a)). At this stage,
the dissociated H-atom is B1.852 Å away from the Pd surface. The
Bader charges show that it has a charge of 0.165 e, so it is not very
strongly adsorbed to the Pd surface, unlike fully dissociated
H-atoms, which are adsorbed as hydride ions. To better under-
stand the charge distribution of the transition state, we com-
pare the spin charge densities of the interacting Pd(100) surface
with the pristine Pd(100) surface (see Fig. 5). In the pristine
Pd(100) surface, the heights of spin density peaks are uniform
and a distortion of these peak heights along with the calculated
Bader charges will indicate the nature of the interaction
between the TS and Pd(100) surface. The Pd atoms interacting
directly with the O-atom (of –OH species) have a shorter peak
height due to the electronegative effect of the O-atom. 3.3.
Geometry, energetics and local electronic properties of
the transition states The coordinates of all the files have been included in the ESI
Step
Species on Pd
Average interatomic distances (Å)
Bader charges
(Pd–Pd)srf
(Pd–Pd)near
Pd–A
Pdint
Species
Distance
Pd close to
Distance
Pd – species
1
H2O
2.749
2.816
O of H2O
2.591
O of H2O
0.001
PdH2O
1.232
O of H2O
0.635
H of H2O
H + OH
2.750
2.729
H
1.978
H
0.159
PdOH
1.052
O of OH
0.651
H of OH
2.884
O of OH
2.101
O of OH
0.045
PdH
0.086
H
2
OH + OH
2.729
2.885
O of OH
2.082
O of OH
0.185
PdOH
1.045
O of OH
2.891
O of OH
2.135
O of OH
0.635
H of H
O + H2O
2.754
2.763
O
2.154
O
0.087
PdH2O
1.266
O of H2O
2.804
O of H2O
2.613
O of H2O
0.095
PdO
0.654
H of H2O
0.760
O
3
OH
2.754
2.877
O of OH
2.104
O of OH
0.168
PdO
1.005
O of OH
0.594
H of OH
O + H
2.754
2.778
H
1.714
H
0.167
PdO
0.730
O
2.760
O
2.145
O
0.008
PdH
0.062
H
4
H + H
2.751
2.785
H
1.715
H
0.020
PdH
0.086
H
2.785
H
1.715
H
0.010
PdH
0.085
H
H2
2.751
—
—
—
—
—
—
0.027
H
0.039
H
5
CO2
2.749
—
—
—
—
—
—
2.090
C of CO2
1.068
O of CO2
CO + O
2.756
2.778
O
2.168
O
0.135
PdCO
0.731
O on Pd
2.747
C of CO
1.998
C of CO
0.188
PdO
0.976
O of CO
0.844
C of CO
6
–COOH
2.752
2.759
C of –COOH
1.994
C of –COOH
0.075
PdC
1.407
C of –COOH
1.192
OH of –COOH
1.094
O of –COOH
0.688
H of –COOH
OH + CO
2.756
2.884
O of OH
2.104
O of OH
0.191
PdOH
1.012
O of OH
0.629
H of OH
2.771
C of CO
2.006
C of CO
0.069
PdCO
0.854
C of CO
1.047
O of CO
7
H + CO2
2.747
—
CO2
—
CO2
—
—
1.970
C of CO2
1.007
O of CO2
2.780
H
1.717
H
0.012
PdH
0.087
H on Pd
This article is licensed under a Creative Com Phys. Chem. Chem. 3626 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 3.3.
Geometry, energetics and local electronic properties of
the transition states The
Bader charge of these two atoms is +0.124 e, i.e. there is charge
transfer from the surface to the –OH species. The other Pd
atoms interact weakly with the H-atom, which has a charge of
+0.016 e. There is a small distortion in the shapes of the peaks,
which may be due to the polarising effects of the Pd atom
interacting with the –OH species. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In
contrast, the charge on the Pd atom close to the water molecule
is +0.051 e revealing a weak interaction between the water
molecule and the Pd(100) surface, which is also evident in
the spin density map shown in Fig. 5c. Fig. 4
Partial density of states for (a) Pd atoms in close proximity and (b)
Pd atoms further away from the adsorbed O-atom. In the third step, [(OH)srf " (O)srf + (H)srf], the –OH obtained
from water dissociation (in the first step) can further split to give
an adsorbed O-atom and H-atom via an exothermic reaction with
an energy barrier of 0.320 eV. In the transition state, the O H
distance has increased to 1.689 Å from the normal O–H distance
of 0.978 Å. The O-atom has a charge of 0.658 e, and Pd atoms
directly interacting with it have an average charge of +0.121 e,
showing that there is a strong charge transfer effect between the Fig. 4
Partial density of states for (a) Pd atoms in close proximity and (b)
Pd atoms further away from the adsorbed O-atom. 3626 3626 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 This journal is ©the Owner Societies 2020 View Article Online Fig. 5
Top and side views of (a) TS1, (b) TS2, (c) TS3, (d) TS4, (e) TS5, (f)
TS6, and (g) TS7 where, TS represents transition states and 1–7 represents
the steps. For clarity the upper most atomic layer is shown in ball and stick
and lower sub-surfaces are shown in van der Waals representation. Paper PCCP Therefore, in addition to the above five steps, we consider two
more steps for the WGS reaction, in which the evolution of CO2
molecule may occur via the formation of a carboxyl species due
to the reaction between adsorbed COsrf with an OH species,
which is step 6 [(CO)srf + (OH)srf " (COOH)srf]. The carboxyl
species in step 6 may then decompose to give CO2 and an
adsorbed hydrogen atom in step 7 [(COOH)srf "CO2 + (H)srf]. In step 6, the OH and CO species are originally bonded to
two pairs of nearby Pd surface atoms at a distance of 2.884 Å
and 2.771 Å respectively. In the transition state (Fig. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 5(f)), the
OH and the CO species are bonded to only one surface Pd atom
with a distance of 2.078 Å and 2.331 Å respectively and the O of
OH and C of CO are at a distance of 1.669 Å. A comparison of
the charges in the reactants and the products shows that,
during the transition state, the charge on carbon changes from
+0.793 e to +1.053 e and the charge on the O-atom of the OH
species changes from 1.034 e to 1.060 e, indicating that the
OH species acts as a nucleophile to form a new C–O bond to
give a carboxyl like species. The charge spin densities show
significant changes in the peak heights directly interacting with
the CO and OH species, which agrees with the calculated
charges on these two Pd atoms (see Fig. 6(g) and Table 3). The formation of the carboxyl species is an endothermic
reaction with an energy barrier of 0.720 eV. Fig. 5
Top and side views of (a) TS1, (b) TS2, (c) TS3, (d) TS4, (e) TS5, (f)
TS6, and (g) TS7 where, TS represents transition states and 1–7 represents
the steps. For clarity the upper most atomic layer is shown in ball and stick
and lower sub-surfaces are shown in van der Waals representation. to give a CO2 molecule. Furthermore, the charges on the interacting
Pd atoms in the reactants decreased from +0.188 e to +0.110 e
indicating that the Pd–CO bond is weak. While in the reactant the
CO is adsorbed to two Pd atoms, in the transition state it is bonded
to only one, which is also clearly seen in the charge spin density
contour map for the Pd atom directly interacting with the CO
(second column third row from the top in Fig. 6(f)). Finally, in step 7, which is an exothermic reaction, the
carboxyl species decomposes to give CO2 and a hydrogen atom
(hydride ion on the surface) with an energy barrier of 0.540 eV
(Table 2). In the transition state (Fig. 5(g)), the H-atom is at a
distance of 1.465 Å, which is longer than the C–H distance of
1.109 Å in the carboxyl species indicating that the H-atom is
abstracted and has a charge of +0.208 e. The Pd atom in the
vicinity of the H-atom is almost neutral and in the vicinity of
CO2 species, slightly positive (+0.068 e). Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. i.e., interacting with the adsorbed CO2 species (Fig. 6(h)). We
also note, as shown in Table 3, that for all the transition states,
the measured Pd–Pd interatomic distances are close to those in
the pristine surface. For clarity the energy barriers for the
forward, reverse reactions and the DEreaction are summarised
in Table S2 (ESI†). For clarity, in the above reactions the surface sites in the
diffusion steps are represented by (*)srf. The CO adsorption,
H2O
adsorption,
O
diffusion
and
CO2
desorption
have
no energy barriers. The energy barriers for H diffusion, OH
diffusion and CO diffusions are calculated as 0.017 eV, 0.400 eV
and 0.090 eV respectively, which are obtained from the zero-
point corrected energy of the DFT results. The KMC simulations show that the coverage of H is
around 0.15 H atoms per site in a monolayer and as shown
in Fig. 7(a and b), the coverage of H exhibited higher fluctua-
tions than that of CO. The H surface coverage is found to
reach values as low as 0.04 and as high as 0.18 (Fig. 7a). The
KMC simulations further exhibit an average CO coverage of
0.25 molecules per site (monolayers) (see Fig. 7(b)), which does
not change significantly during any of the simulations and was
found to be almost constant after the initial adsorption of CO. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The charge spin density
maps also show that only one Pd atom has a lower peak height Previous studies have suggested that bidentate formate is a
spectator species in the WGS reaction and hence the energeti-
cally less stable carboxyl group, which is adsorbed to the
surface through the C-atom, may play a crucial role.18,62 Fig. 6
Spin densities of the exposed surface in (a) pristine Pd(100) (min = 9.45 107 e Å3, max = 4.13 105 e Å3) and during the interaction
of the transition states in (b) step 1 (min = 7.63 107 e Å3, max = 2.79 105 e Å3), (c) step 2 (min = 4.44 107 e Å3, max = 3.37 105 e Å3),
(d) step 3 (min = 5.52 108 e Å3, max = 1.80 105 e Å3), (e) step 4 (min = 1.68 107 e Å3, max = 2.74 105 e Å3), (f) step 5 (min =
3.22 105 e Å3, max = 5.89 103 e Å3), (g) step 6 (min = 7.71 108 e Å3, max = 4.69 106 e Å3) and (h) step 7 (min = 3.04 108 e Å3,
max = 1.02 105 e Å3). Fig. 6
Spin densities of the exposed surface in (a) pristine Pd(100) (min = 9.45 107 e Å3, max = 4.13 105 e Å3) and during the interaction
of the transition states in (b) step 1 (min = 7.63 107 e Å3, max = 2.79 105 e Å3), (c) step 2 (min = 4.44 107 e Å3, max = 3.37 105 e Å3),
(d) step 3 (min = 5.52 108 e Å3, max = 1.80 105 e Å3), (e) step 4 (min = 1.68 107 e Å3, max = 2.74 105 e Å3), (f) step 5 (min =
3.22 105 e Å3, max = 5.89 103 e Å3), (g) step 6 (min = 7.71 108 e Å3, max = 4.69 106 e Å3) and (h) step 7 (min = 3.04 108 e Å3,
max = 1.02 105 e Å3). Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 | 3627 This journal is ©the Owner Societies 2020 PCCP Table 3
Interatomic distances and Bader charges for the transition states. 3.4.
Kinetic Monte Carlo simulation Using the results of our DFT calculations, we are able to
simulate the WGS reaction on Pd(100) using the Kinetic Monte
Carlo (KMC) method, in order to gain deeper insight into the
kinetics of the mechanism under reaction conditions. To
simulate the reaction mechanism, in addition to the core steps
discussed in the previous sections, we consider another seven
steps relating to the adsorption, desorption and diffusion of
CO, CO2, H2O, H, and OH. The steps are: Step 8: CO adsorption This journal is ©the Owner Societies 2020
Fig. 7
(a) H and (b) CO coverage against time. CO + (*)srf " (CO)srf Step 9: H2O adsorption H2O + (*)srf " (H2O)srf Step 10: O diffusion (O)srf + (*)srf " (*)srf + (O)srf Step 11: H diffusion (H)srf + (*)srf " (H)srf + (*)srf Step 12: OH diffusion Step 12: OH diffusion (OH)srf + (*)srf " (*)srf + (OH)srf Step 13: CO diffusion Step 13: CO diffusion (CO)srf + (*)srf " (*)srf + (CO)srf Step 14: CO2 desorption (see step 7) Step 14: CO2 desorption (see step 7) Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The coordinates of all the files have been included in the ESI
TS for step
Interatomic distances
Bader charges
Pd–Pdsrf
Pd – species
Species 1
Species 2
Pdspecies 1
Pdspecies 2
1
2.764
1.846
H
0.165
H
0.585
H of OH
0.016
0.124
2.200
O of OH
1.029
O
2
2.766
2.004
O
0.755
O
1.275
O of H2O
0.202
0.051
2.617
O of H2O
0.654
H of H2O
3
2.765
1.766
H
0.096
H
0.658
O
0.009
0.234
1.977
O
4
2.753
2.831
H
0.004
H
0.000
H
0.002
2 Pd near TS
(negligible charge)
2.816
H
5
2.743
2.035
O
0.710
O
0.999
C of CO
0.185
0.110
1.947
CO
1.007
O of CO
6
2.749
2.078
C of OC–OH
1.053
C of OC–OH
1.060
O of HO–CO
0.097
0.113
2.331
O of HO–CO
1.074
O of OC–OH
0.672
H of HO–CO
7
2.755
2.057
C of COO
1.604
C of COO
0.208
H
0.068
0.005
1.858
H
1.008
O of COO Step 14: CO2 desorption (see step 7) (CO2)srf " CO2 + (*)srf Fig. 7
(a) H and (b) CO coverage against time. m. Chem. Phys., 2020, 22, 3620--3632
This journal is ©the Owne 3628 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632
This journal is ©the Owner Societies 2020 3628 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 3628 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 3628 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 View Article Online PCCP Paper Furthermore, CO forms patterns on the surface due to lateral
interactions, as shown in the snapshots in Fig. 8(a–c). Within the first 5 seconds, grain boundaries can be seen
between different adsorption patterns, which disappear to form
a single adsorption pattern after this time, suggesting an
equilibrium period of about 5 s.63 It is also worth noting that
only CO and H adsorbates are seen on the surface at any of
these snapshots meaning that these are the dominant species
and under these conditions the turnover for H2 production is
found to be low. An increase of the turnover is seen with
increasing H2O partial pressure, as shown in Fig. 9, with the
overall mechanism remaining the same over the whole range. ,
p
g
(
Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This journal is ©the Owner Societies 2020
Fig. 8
Snapshots for simulation area at (a) 0.8 s, (b) 3.2 s and (c) 5.2 s. A
simulation have 1 to 1 ratio for H2O and CO in the gas phase, under th
conditions stated in computational details section. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. When the frequencies of the simulated elementary events,
shown in Fig. 10, are examined, it is found that adsorption and
diffusion events dominate; therefore, these adsorption and
diffusion processes are limited (‘‘throttled’’) by a stiffness scaling
method.64–68 The other most common events are H2O and OH
decomposition, which are also seen to be reversible in nature. The OH molecule reacts with the CO molecule to form carboxyl
which subsequently generates CO2; while the OH molecule may
break down, thereby producing oxygen which then reacts with
CO to form CO2 via direct CO oxidation. The only other reversible
step is the –COOH formation. Step 14: CO2 desorption (see step 7) Of the remaining 5 steps i.e., 4, 5,
7 and 14 show that the reactions progress only in one direction
and step 2 shows no activity. From the above analysis it can be
summarised that WGSR mechanism involves both direct oxida-
tion and carboxyl pathways. This journal is ©the Owner Societies 2020 Conflicts of interest There are no conflicts to declare. 6 C. Ratnasamy and J. P. Wagner, Water Gas Shift Catalysis,
Catal. Rev., 2009, 51(3), 325–440. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024
This article is licensed under a Creative Commons Attribution 3.0 U Fig. 10
Event frequency per site for each of the elementary steps of the proposed reaction mechanism. References of d–d interactions due to presence of electronegative O-atoms. The calculated energy barriers demonstrate that the splitting of H2O
to give OH and H surface intermediates is the rate determining step. The energy barriers for OH and OH recombination to give H2O and
adsorbed O, and for the hydrogen evolution steps are also high. The
KMC study indicates that the H2O and OH decomposition are the
most common events i.e., the OH radical either reacts with the CO
molecule to form carboxyl which subsequently generates CO2; or the
radical may break down to oxygen which then reacts with CO to
form CO2 via direct CO oxidation. From our study we conclude that
the redox-associative mechanism of the water gas shift reaction on
Pd(100) progresses via both direct oxidation and carboxyl pathways
that occur in parallel, rather than separately, which we consider will
lead to a deeper understanding of this important chemical problem
on pristine transition metal based catalyst surfaces. 1 L. Bollmann, J. L. Ratts, A. M. Joshi, W. D. Williams, J. Pazmino,
Y. V. Joshi, J. T. Miller, A. J. Kropf, W. N. Delgass and F. H. Ribeiro, Effect of Zn Addition on the Water–Gas Shift Reaction
over Supported Palladium Catalysts, J. Catal., 2008, 257(1), 43–54. 2 T. Bunluesin, R. J. Gorte and G. W. Graham, Studies of the
Water–Gas-Shift Reaction on Ceria-Supported Pt, Pd, and
Rh: Implications for Oxygen-Storage Properties, Appl. Catal.,
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Summary and conclusions Our combined DFT and KMC study of the WGS reaction on the
Pd(100) surface has allowed us to propose a reaction mecha-
nism in which both redox and associative pathways operate
within a single framework, which consists of seven core
elementary steps. For the complete description of the WGS
chemistry, seven additional steps constituting of adsorption,
diffusion and desorption are included in the KMC simulations. Our study on the geometrical changes in each of the reactants,
products and the transition states shows that irrespective of the
type of species adsorbed on the Pd(100) surface, the average
Pd–Pd distance on the surface is always similar except when
the adsorbed species are O-atoms. The analysis of electronic
properties reveals that this exception is related to the weakening Our combined DFT and KMC study of the WGS reaction on the
Pd(100) surface has allowed us to propose a reaction mecha-
nism in which both redox and associative pathways operate
within a single framework, which consists of seven core
elementary steps. For the complete description of the WGS
chemistry, seven additional steps constituting of adsorption,
diffusion and desorption are included in the KMC simulations. Fig. 9
Plot of turnover frequency for H2 (molecules per site per s) against
the ratio of H2O/CO partial pressure (bar). Fig. 8
Snapshots for simulation area at (a) 0.8 s, (b) 3.2 s and (c) 5.2 s. All
simulation have 1 to 1 ratio for H2O and CO in the gas phase, under the
conditions stated in computational details section. Fig. 9
Plot of turnover frequency for H2 (molecules per site per s) against
the ratio of H2O/CO partial pressure (bar). Phys. Chem. Chem. Phys., 2020, 22, 3620--3632 | 3629 This journal is ©the Owner Societies 2020 View Article Online PCCP Paper Fig. 10
Event frequency per site for each of the elementary steps of the proposed reaction mechanism. PCCP
e Commons Attribution 3.0 Unported Licence. Fig. 10
Event frequency per site for each of the elementary steps of the proposed reaction mechanism. PCCP
Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:46:02 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 08 January 2020. Downloaded on 10/24/2024 5:
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Framework This journal is ©the Owner Societies 2020 3632 | Phys. Chem. Chem. Phys., 2020, 22, 3620--3632
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Evaluation of registered nurses’ interprofessional emergency care competence through the gamification of cardiopulmonary resuscitation training: a cross-sectional study
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RESEARCH Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://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. Abstract Backgrounds Cardiopulmonary resuscitation (CPR) training is generally led by instructors in a classroom; thus,
conventional teaching materials used in CPR training are often constrained by spatiotemporal factors, limiting
learners’ interest and sense of achievement in learning and preventing them from effectively applying what they
learn in practice. For greater effectiveness and more flexible application, clinical nursing education has increasingly
emphasized contextualization, individualization, and interprofessional learning. This study determined the self-
assessed emergency care competencies of nurses who received gamified emergency care training and explored the
factors associated with those competencies. Methods Quota sampling of nurses working at a certain regional hospital in central Taiwan was conducted, and a
structured questionnaire was administered to the recruited nurses. A total of 194 valid responses were collected. The
research tool was a scale measuring the participants’ emergency care competencies after they received gamified
emergency care training. The data were analyzed using descriptive and inferential statistics and multiple regression. Results Of the recruited participants, 50.52% were ≤ 30 years old; 48.45% worked in the internal medicine
department; 54.64% graduated from 2-year university technical programs; 54.12% were N2 registered nurses; 35.57%
and 21.13% had ≥ 10 and 1–3 years of work experience, respectively; and 48.45% worked in general wards. User need
(r = 0.52, p = 0.000), perceived usefulness (r = 0.54, p = 0.000), perceived ease of use (r = 0.51, p = 0.000), and usage
attitude (r = 0.41, p = 0.000) were positively correlated with emergency care competencies. Furthermore, the multiple
regression analysis revealed that perceived usefulness was the primary factor associated with the participants’
emergency care competencies. Conclusions The results of this study may serve as a reference for acute care facility authorities in designing
advanced nursing competency standards and emergency care training programs for nurses. Keywords Gamification, Game-based learning, Emergency care, Nursing competence Full list of author information is available at the end of the article © 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. BMC Medical Education BMC Medical Education Chen et al. BMC Medical Education (2023) 23:359
https://doi.org/10.1186/s12909-023-04332-y Abstract 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. Evaluation of registered nurses’
interprofessional emergency care competence
through the gamification of cardiopulmonary
resuscitation training: a cross-sectional study Tzu-Sang Chen1, Pei-Lun Hsieh2*, Chien Chien Tung3, Chao-Hsin Wu4 and Yu-Chieh Cheng5 Introduction Since the dawn of the Internet age, smart devices have
become increasingly prevalent in people’s daily lives. Many educators have adopted innovating teaching strat
egies involving the use of new technologies and flipped-
classroom models. Furthermore, GBL has been applied
in various medical education contexts [5]. Venkatesh et
al. [14] proposed the Unified Theory of Acceptance and
Use of Technology (UTAUT) model, which aims to mea
sure users’ intentions to use an information system and
their subsequent usage behaviors, summarized as the
dimensions of perceived usefulness (i.e., users’ percep
tions regarding the usefulness of learning for improving
their competence and confidence and preventing them
from making mistakes at work) and perceived ease of use
(i.e., users’ perceptions that a learning platform or system
motivates them to learn, has a clear interface, and is easy
to use). Technological advancements have transformed inter
professional health-care education. Innovative teach
ing strategies, in which newly developed technologies
are applied in clinical care nursing, have been recently
employed in health-care education [1]. Digital learn
ing—including massive open online courses, flipped
classrooms, ubiquitous learning, virtual learning envi
ronments, and, since 2017, game-based learning (GBL)—
has been applied in medical and nursing education [1–3]. In recent years, the quality of clinical medical education
has been improved through contextualization, individu
alization, gamification, and the incorporation of action
learning [4, 5]. Digital technologies have been broadly applied in
medical education curricula, and simulation-based clini
cal teaching worldwide [6, 7]. Studies have reported the
benefits of digital technology in the education of medical
students and have explored the applications of informa
tion technology in medicine, including its applications in
interactive teaching using 3D anatomical images, nursing
simulations, and objective structured clinical examina
tions [3–5, 7].i Conventional teaching models involve one-way deliv
ery of knowledge to learners. However, successful learn
ing requires learners to be motivated to learn. Insufficient
learning content undermines learners’ attention and
interest, resulting in less favorable learning outcomes. GBL combines learning content with games to inspire
learners to learn continually through practice and repeti
tion, thus enhancing their learning outcomes [9, 10, 15,
16]. Nurses are eyewitnesses of and first-aid providers dur
ing patient emergencies and are often the first profes
sionals on a health-care team to assess a patient’s need
for first aid at the scene [3, 8]. Chen et al. BMC Medical Education (2023) 23:359 Page 2 of 9 Chen et al. BMC Medical Education (2023) 23:359 Page 2 of 9 Chen et al. BMC Medical Education Research instrument
Th By providing nurses with a fun, convenient, and repeat
able memory-enhancing method not limited by space,
the gamified CPR care scenario facilitates the acquisition
of CPR knowledge and skills. The game design involves a
combination of simulated situations, game interface vari
ations, and reminder functions. The game content is pre
sented through challenge themes, time-limited answers,
and self-competition to guide players and enhance their
learning motivation and operational intent. The questionnaire was self-developed and designed
according to the goals and structure of this study. It con
sisted of three parts that respectively collected informa
tion on the nurses’ demographic backgrounds (13 items),
acceptance of the game-based emergency care training
program (23 items, revised UTAUT model [14] question
naire), and perception of emergency care competencies
(27 items). The third part involved the 27-item Nurses’
Aid Competency Scale self-developed based on the Euro
pean Research Council [11] and Hsieh et al. [12]. Details
of these three parts are presented as follows: g
Regarding the game operation process (Fig. 1), the
player enters the game website (https://tung-acls-public. web.app/) through a cell phone or computer, plays the
game as the main character, spends one day in the game
as a nurse taking care of patients, and passes the red, yel
low, and blue zones (in that order). Each of these zones
has its own challenge themes and predetermined deci
sion points. At each decision point, the user can choose
an option by tapping within 110 s; the game ends if the
player takes longer. Completing all the tasks associated
with each theme within the given time frame yields a full
score of 100. The score is revealed at the end of the game. This zone has six simulated decision points, namely ven
tricular tachycardia (VT), ventricular fibrillation, pulse
less electrical activity, asystole and other heart rhythms,
chest compression location, and defibrillation; these
points are presented randomly to train the player in iden
tifying and treating abnormal heart rhythms. The game
ends and provides feedback automatically if the player
does not pass the challenge within 15 s or chooses the
wrong path (Fig. 2). (1) Demographic information: self-reported details on
the demographic backgrounds of each participant,
including their gender, age, seniority, and experience
with using game-based/technological products. Research instrument
Th (2) Acceptance of the game-based emergency care
training program: examines the participants’
acceptance of the game-based emergency care
learning aids game software, including the user need,
perceived usefulness, perceived ease of use, and
usage attitudes toward the learning aids. Scored on
a 5-point Likert scale (“strongly agree,” “somewhat
agree,” “neither agree not disagree,” “somewhat
disagree,” and “strongly disagree”). (3) Self-assessed emergency care competencies: assesses
the participants’ clinical nursing competencies after
they completed the game-based emergency care
training. Each item was scored on a 5-point Likert
scale (“strongly agree,” “somewhat agree,” “neither
agree not disagree,” “somewhat disagree,” and
“strongly disagree”). Sample size Determination of the sample size was based on Krej
cie and Morgan’s [19] statistical tool, which provides a
sample size for a finite population at a 95% level of con
fidence. The sample size of 150 was deemed representa
tive of the estimated nursing population of approximately
1500 in the hospital, under consideration of a 5% nonre
sponse rate. Quota sampling was conducted according
to the seniority of each ward nurse. In consideration of
sample attrition, the questionnaire was administered to
200 participants recruited between January and March
2022; among these participants, 26, 40, 33, 26, and 75 had
worked for 3 months to 1 year, 1–3 years, 4–6 years, 7–9
years, and ≥ 10 years, respectively. This study adopted a cross-sectional design employing
the UTAUT [14] and GBL [10] as its theoretical basis. A
structured questionnaire was developed to assess hospi
tal nurses’ acceptance of the emergency care game soft
ware and their nursing competencies after completing
an on-the-job training course based on the gamification
software. Gamification involves using game attribute categories
outside the context of a game that can stimulate learning-
related behaviors or attitudes. The gaming software prod
uct was designed based on Landers’ gamified learning
theory [18] and by condensing the ACLS guidelines [11]. This software product has acquired a utility model patent
(Patent Number: M637730, applied on 2022/10/28). Introduction Page 3 of 9 Page 3 of 9 Chen et al. BMC Medical Education (2023) 23:359 Chen et al. BMC Medical Education (2023) 23:359 Chen et al. BMC Medical Education Introduction According to the European
Research Council, emergency care competencies are core
skills for nurses, including sufficient knowledge of first-
aid and cardiopulmonary resuscitation (CPR) techniques,
assessment of emergencies and proper administration of
first-aid procedures, maintenance of airway and recovery
position, knowledge and recognition of timely and effec
tive breathing, accurate assessment of vital signs, car
diac massage with appropriate speed and pressure, and
appropriate use of automatic external defibrillators [9]. Conventional emergency care training has been limited
by spatial and temporal factors, limiting learners’ inter
est and sense of achievement in learning and preventing
learners from applying what they learn in practice [1, 3,
4]. Recently, GBL has emerged as a potential solution
to this problem; however, further research on its effec
tiveness in improving the professional competencies of
health-care providers is needed [10, 11]. GBL has been explored in medical education research. Gallegos et al. [17] applied the 3D Gamelab gaming plat
form in a preliminary nursing education course in which
120 university nursing students participated. Each ses
sion lasted for 30–60 min and involved the use of hand
outs, videos, learning activities, and links to resources. After the students completed the learning activities,
they were awarded badges. Qualitative interviews were
conducted with the students to assess their experiences
with using the learning platform and revealed that GBL
inspired them to proactively participate in the course and
maximize their learning potential. GBL involves providing learners with a rich learning
medium and an environment that stimulates their learn
ing motivation. Games must be designed in consider
ation of elements such as the users’ knowledge, skills, and
sensory stimulation; art design; rewards, characters; and
soundtracks as well as the learners’ affective, behavioral,
cognitive, and social or cultural engagement to motivate
learners to accomplish their learning goals [8–10, 16]. This study investigated the self-assessed emergency care
competencies of nurses who received gamified emer
gency care training and explored the factors associated
with those competencies. As a matter of fact, CPR is a common clinical scenario
and a mandatory competency for medical professionals
[3, 8]. According to Taiwan’s Emergency Medical Services
Act, health-care professionals working in emergency and
intensive care units must possess advanced cardiac life
support (ACLS) certificates [3, 12, 13]. Hsieh et al. [12]
concluded that both situational simulation training and
online teaching were effective in improving the first-aid
competencies of nurses in general medical wards. Questionnaire validity and reliabilityh The participants in this study were nurses who had
worked for at least 3 months at a certain regional hospital
in central Taiwan and completed the program based on
the emergency care game developed in 2021, whose soft
ware application and website was also provided to all the
nurses (learners) in the hospital. The content validity of the questionnaire was assessed by
experts, namely two doctors, one nurse, and two nurs
ing professors. According to the content validity index
calculation method proposed by Polit and Beck [20], the
items that scored higher than 3 points were retained. Page 4 of 9 Page 4 of 9 (2023) 23:359 Chen et al. BMC Medical Education Fig. 1 Introduction to the game process, character and scenario Fig. 1 Introduction to the game process, character and scenario Data collectionh The content validity indices of the scales on the accep
tance of the game-based emergency care training pro
gram and the participants’ self-assessed emergency care
competencies were 0.7 and 1, respectively, indicating that
the content validity of both scales was satisfactory. The
Cronbach’s α values of the aforementioned scales on the
participants’ acceptance and competencies were 0.95 and
0.99, respectively, indicating that the internal consistency
of both scales was satisfactory. The participants were selected from the nurses that met
the inclusion criteria, received explanations of the goal
and procedure of this study, and signed written informed
consent documents before their data were collected. The
completed questionnaires were submitted directly to the
researchers in sealed envelopes from February 2, 2022,
to March 20, 2022. The participants were permitted to
decline answering some of the items or withdraw from
the study without consequences. Chen et al. BMC Medical Education (2023) 23:359 Page 5 of 9 (2023) 23:359 Chen et al. BMC Medical Education Fig. 2 Game answer screen and total score with feedback Fig. 2 Game answer screen and total score with feedback Fig. 2 Game answer screen and total score with feedback Data analysis BMC Medical Education participants, 90.21% and 7.22% administered CPR 1–2
times per year and 1–2 times per month, respectively
(Table 1).h
Table 1 Demographic information of the participants (N = 194)
N
%
Cumulative %
Sex
Female
190 97.94
97.94
Male
4
2.06
100.00
Age (years)
≤ 30
98 50.52
50.52
31–40
60 30.93
81.44
41–50
34 17.53
98.97
≥ 51
2
1.03
100.00
Educational level
Junior college (5-year program)
41 21.13
21.13
University (2-year technical program)
106 54.64
75.77
University (4-year technical program)
37 19.07
94.85
General university
5
2.58
97.42
Research institute
5
2.58
100.00
Doctoral program
0
0.00
100.00
Registered nurse level
N0–N1
75 38.66
38.66
N2
105 54.12
92.78
N3
14
7.22
100.00
N4
0
0.00
100.00
Seniority at the hospital (years)
< 1
27 13.92
13.92
1–3
41 21.13
35.05
4–6
28 14.43
49.48
7–9
29 14.95
64.43
≥ 10
69 35.57
100.00
Unit type
Outpatient
26 13.40
13.40
General ward
94 48.45
61.86
Operating room
27 13.92
75.78
Other
47 24.22
100.00
First-aid administration frequency
Once per day
1 0.52
0.52
1–2 times per week
4
2.06
2.58
1–2 times per month
14
7.22
9.79
1–2 times per year
175 90.21
100.00 Table 2 Participants’ (N = 194) acceptance of the game-based
emergency care training program and self-assessed emergency
care competencies Table 2 Participants’ (N = 194) acceptance of the game-based
emergency care training program and self-assessed emergency
care competencies
Dimension
Subdimension
Mean
SD
Acceptance of
the game-based
training program
(13 items)
User need
4.34
0.62
Perceived usefulness
4.30
0.66
Perceived ease of use
4.36
0.62
Usage attitude (10
items)
Cognitive
4.42
0.58
Affective
3.53
0.69
Emergency care
competencies (27
items)
Maintenance of respiratory tract and
circulation
4.30
0.70
Teamwork and exception handling
4.26
0.62
Arrhythmia assessment and
treatment
4.18
0.64
First aid assessment and initial
treatment
4.46
0.59
Nursing and emergency care
performance
4.21
0.65 Table 3 Correlation coefficients of emergency care
competencies
Variable
User
need
Perceived
usefulness
Per
ceived
ease of
use
Usage
attitude
Emergen
cy care
compe
tencies
User need
1
Perceived
usefulness
0.80***
1
Perceived
ease of use
0.76***
0.78***
1
Usage
attitude
0.58***
0.63***
0.66***
1
Emergency
care com
petencies
0.52***
0.54***
0.51***
0.41***
1
*p < 0.05; **p < 0.01; ***p < 0.001 strong, moderately strong, moderate, and weak, respec
tively (Table 3). Data analysis Results
From the 200 invitations to participate, 194 valid
responses were obtained (response rate: 97%); six ques
tionnaires were removed because they were almost
entirely incomplete. Of the participants, 97.94% were
female; 50.52% were ≤ 30 years old; 54.64% had graduated
from a 2-year technical program; 54.12% were N2 regis
tered nurse level; and 35.57% and 21.13% had provided
clinical services for ≥ 10 and 1–3 years, respectively. Regarding the types of units in which the participants
worked, 48.45%, 13.92%, 13.40%, and 24.22% worked
in general wards, operating rooms, outpatient clin
ics, and other types of units, respectively. Among the SPSS 26.0 was used to analyze the collected data. A
descriptive statistical analysis was conducted to evaluate
the distributions of the percentages, means, and standard
deviations in the data. Data were analyzed using a one-
way analysis of variance (ANOVA) with the following
independent variables: age, educational level, registered
nurse level, years of seniority at the hospital, workplace
unit type, and frequency of first-aid administration. In
addition, correlation analyses were performed to explore
the association between acceptance of the GBL training
and emergency care competencies. A p value of < 0.05
was considered statistically significant. Chen et al. BMC Medical Education (2023) 23:359 Page 6 of 9 Page 6 of 9 Chen et al. Discussion Of the participants, 97.94% were female; 50.52% were ≤ 30
years old; and 35.57% and 21.13% had worked in clinical
services for ≥ 10 and 1–3 years, respectively. These results
are nearly identical to the demographic information and
seniority distributions of acute care nurses in survey
studies conducted worldwide [8, 15, 18]. According to a
statistical report by the Taiwan Union of Nurses Asso
ciation, of the approximately 180,000 nurses registered in
Taiwan by the end of January 2021, 46.9% and 41.3% pos
sessed bachelor and junior college degrees, respectively. A high percentage of the participants in the present study
had completed higher education programs, with 54.64%
of them having graduated from a 2-year university tech
nical program. Furthermore, 90.21% and 7.22% of the
participants had administered CPR 1–2 times per year
and 1–2 times per month, respectively, while on duty. This is consistent with the findings reported by Li and
Wang [8], and Li et al. [15]. On the usage attitude scale, the participants scored
higher on the cognitive component than on the affective
component, indicating that GBL related to their emer
gency care competencies. Lin et al. [23] reported that
multimedia interactive e-book teaching, situated learn
ing, and drawing mind maps effectively increased the
accuracy of arrhythmia assessment and treatment, boost
ing medical professionals’ confidence.f i
Accordingly, GBL effectively improved the nurses’ pro
fessional competencies [12, 17, 22], consistent with the
findings of Li et al. [15] and Gallegos et al. [17]. Perceived
usefulness refers to users’ perceptions of a learning aid
as being useful for improving their self-perceived com
petence and confidence and preventing them from com
mitting mistakes at work. Perceived ease of use reflects
users’ perceptions that a learning aid motivates them to
learn, has a clear interface, and is easy to use. In this study, perceived usefulness was identified as
the factor most strongly affecting the participants’ emer
gency care competencies. GBL substantially increased
the nurses’ confidence in maintaining the respiratory
tract and circulation, teamwork, and CPR. Data analysis The participants’ emergency care com
petency scores were positively correlated with their user
need (r = 0.52, p = 0.000), perceived usefulness (r = 0.54,
p = 0.000), perceived ease of use (r = 0.51, p = 0.000), and
usage attitude (r = 0.41, p = 0.000) scores (Table 3). participants, 90.21% and 7.22% administered CPR 1–2
times per year and 1–2 times per month, respectively
(Table 1). The participants scored between 4.18 and 4.46 points
on the self-assessed emergency care competency scale. They scored the highest and lowest on “first-aid assess
ment and initial treatment” and “arrhythmia assessment
and treatment,” respectively. They scored between 3.53
and 4.42 points on the usage attitude scale, scoring the
highest and lowest on the usage attitudes components,
respectively. The participants’ average of user need, per
ceived usefulness, and perceived ease of use scores were
4.34, 4.30, and 4.36 points, respectively (Table 2). The predictive power of the predictor variables (i.e.,
user need, perceived usefulness, perceived ease of use,
and usage attitude) for the dependent variable (i.e., emer
gency care competencies) was evaluated through a multi
ple regression analysis. According to the ANOVA results,
the overall effect in the regression model reached statis
tical significance (F = 22.25, p = 0.00 < 0.05). The adjusted
coefficient of determination (0.31) indicated that the
model’s overall explanatory power was 30.6%. Although the F values of the regression model reached
statistical significance, the standardized regression coef
ficients of user need (β = 0.17, t = 1.59, p = 0.11 > 0.05),
perceived ease of use (β = 0.15, t = 1.41, p = 0.16 > 0.05), Correlations between pairs of variables for which
the r coefficient was ≥ 0.7 (p < 0.05), 0.5–0.69 (p < 0.05),
0.30–0.49 (p < 0.05), and ≤ 0.29 (p < 0.05) were considered Page 7 of 9 Page 7 of 9 Chen et al. BMC Medical Education (2023) 23:359 (2023) 23:359 Chen et al. BMC Medical Education and usage attitude (β = 0.04, t = 0.53, p = 0.60 > 0.05) did
not reach statistical significance. Perceived usefulness
(β = 0.25, t = 2.25, p = 0.03 < 0.05) significantly affected the
participants’ emergency care competencies (Table 4). study had a mean score of 4.18 in the “Arrhythmia assess
ment and treatment” subdimension, showing that they
felt secure in dealing with arrhythmia. Data analysis This positive effect
may have been yielded by the software product used, and
the sense of security was likely built or strengthened dur
ing GBL. Therefore, on-the-job training should empha
size arrhythmia assessment. Acknowledgements 7. Cheng SW, Chang KC, Chen JT. Analyzing the effectiveness of using virtual
reality in pharmacy education. Cheng Ching Med J. 2022;18:22–7. The authors are grateful to all participants who were willing to take part in the
study. 7. Cheng SW, Chang KC, Chen JT. Analyzing the effectiveness of using virtual
reality in pharmacy education. Cheng Ching Med J. 2022;18:22–7. y
y
8. Li YR, Wang YT. About first aid ability and experiences of nursing profession
als. Tzu Chi Nurs J. 2013;12:14–21. 8. Li YR, Wang YT. About first aid ability and experiences of nursing profession
als. Tzu Chi Nurs J. 2013;12:14–21. Data Availability The data presented in this study are available on request from the. 14. Venkatesh V, Morris MG, Davis GB, Davis F. D. User acceptance of information
technology: Toward a unified view. MIS Q. 2003; 425–78. Author details
1 interpretability of the results of using GBL software, an
innovative and promising educational tool was limited. interpretability of the results of using GBL software, an
innovative and promising educational tool was limited. 1Department of Nursing, Tungs’ Taichung MetroHarbor Hospital, Taichung,
Taiwan R.O.C. 1Department of Nursing, Tungs’ Taichung MetroHarbor Hospital, Taichung,
Taiwan R.O.C. 2Department of Nursing, College of Health, National Taichung University
of Science and Technology, Taichung, Taiwan R.O.C. 2Department of Nursing, College of Health, National Taichung University
of Science and Technology, Taichung, Taiwan R.O.C. Declarations 15. Li CJ, Su HC, Chen PJ, Pan WL, Hsiao CL, Hu SH, et al. The learning effective
ness of innovative game-based learning on high alert medications use
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This research was also supported by a grant from the Tungs’ Taichung
MetroHarbor Hospital [TTMHH-NUTC 110003], which was awarded to the
corresponding author. 12. Hsieh MY, Hsieh SI, Hsu LL. A study of medical center nurses’ self-assessment
in performance of advanced cardiac life support skills. New Taipei J Nurs. 2014;16:17–28. 12. Hsieh MY, Hsieh SI, Hsu LL. A study of medical center nurses’ self-assessment
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received positive feedback from the game software, such
as their perceived improvement in their emergency care
competencies and the willingness of the participants to
use the software again because of its easy-to-use inter
face and smooth game operation [4, 5, 9, 10, 16], which
boosted their job performance and confidence.h On the self-assessed emergency care competency scale,
the participants scored the highest on first-aid assess
ment and initial treatment. Accordingly, GBL proved the
participants’ abilities to assess emergency situations and
select appropriate initial treatment methods, reflecting
their critical thinking and problem-solving skills. This
finding is consistent with the findings reported by Hsu et
al. [21] and Chen and Tseng [13]. By contrast, the partici
pants obtained the lowest average scores on arrhythmia
assessment and treatment, which is consistent with the
findings of Hsieh et al. [12] in their survey on the self-
assessed emergency care competencies of nurses who
received ACLS training at a medical center. i
This study had some limitations. First, because the
participants completed the self-reported questionnaires
independently, the results indicated that their percep
tions of their own emergency care competencies and
may not have reflected their actual competencies. Sec
ond, the emergency care competency scale on the ques
tionnaire should be reconstructed, and its reliability and
validity should be reassessed. Finally, this study did not
include pretest data or a control group, and thus the According to a study conducted by Tubaishat and
Tawalbeh [22] regarding the difficulties encountered
by nursing students in arrhythmia assessment training,
nurses have the lowest confidence in and highest anxi
ety about accurately assessing arrhythmia when provid
ing emergency care. However, following the game-based
emergency care training program, the participants in our Table 4 Regression analysis of the effects of predictor variables on emergency care competencies
Model
Nonstandardized coefficients Standardized coefficients
t
Sig(t)
Adjusted
R2
F
Sig(F)
Expected β value
SD
β distribution
(Constant)
1.92
0.28
6.85
0.000
0.31
22.25***
0.000
User need
0.15
0.10
0.17
1.59
0.11
Perceived usefulness
0.21
0.09
0.25
2.25*
0.03
Perceived ease of use
0.14
0.10
0.15
1.41
0.16
Usage attitude
0.05
0.09
0.04
0.53
0.60
*p < 0.05; **p < 0.01; ***p < 0.001 Page 8 of 9 Page 8 of 9 Chen et al. BMC Medical Education (2023) 23:359 Chen et al. BMC Medical Education (2023) 23:359 Chen et al. BMC Medical Education Ethics approval and consent to participate Ethical approval for human research was obtained from the Institutional
Review Board (IRB) of Tung ' Taichung MetroHarbor Hospital, Taichung, Taiwan
(reference IRB No: 110061; approved on 17 December 2021). The participants
were selected to met the inclusion criteria, received explanations of the goal
and procedure of this study, and signed written informed consent documents
before their data were collected. All methods were conducted in accordance
with the ethical standards and regulations of the declaration of Helsinki. 16. Eseryel D, Law V, Ifenthaler D, Ge X, Miller R. An investigation of the interrela
tionships between motivation, engagement, and complex problem solving
in game-based learning. J Educ Technol Soc. 2014;17:42–53. 17. Gallegos C, Abigail J, TesarKelley C, Martz K. The use of a game-based learning
platform to engage nursing students: a descriptive, qualitative study. Nurse
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and gamification of learning. Simul gaming. 2014;45(6):752–68. 18. Landers RN. Developing a theory of gamified learning: linking serious games
and gamification of learning. Simul gaming. 2014;45(6):752–68. Authors’ contributions PLH and TSC was responsible for the study idea and design. CHW performed
the game software development. CCT performed the data collection. YCC
performed the data analysis. PLH and TSC were responsible for the drafting
of the manuscript and made critical revisions to the paper for important
intellectual content. All authors read and approved the final manuscript. 9. Perkins GD, Graesner JT, Semeraro F, Olasveengen T, Soar J, Lott C, …, Zide
man DA. European resuscitation council guidelines 2021: executive summary
Resuscitation. 2021;161:1–60. 9. Perkins GD, Graesner JT, Semeraro F, Olasveengen T, Soar J, Lott C, …, Zide
man DA. European resuscitation council guidelines 2021: executive summary
Resuscitation. 2021;161:1–60. 10. Pellas N, Mystakidis S. A systematic review of research about game-based
learning in virtual worlds. J Univers Comput Sci. 2020;26:1018–42. 10. Pellas N, Mystakidis S. A systematic review of research about game-based
learning in virtual worlds. J Univers Comput Sci. 2020;26:1018–42. 11. Plass JL, Homer BD, Kinzer CK. Foundations of game-based learning. Educ
Psychol. 2015;50:258–83. 11. Plass JL, Homer BD, Kinzer CK. Foundations of game-based learning. Educ
Psychol. 2015;50:258–83. Conclusionsh 3Jie-Rui Social Welfare Foundation Elderly Care Center, Nantou, Taiwan
R.O.C. 3Jie-Rui Social Welfare Foundation Elderly Care Center, Nantou, Taiwan
R.O.C. The usefulness of the game-based emergency care train
ing program as perceived by the participants was the
most crucial factor affecting the participants’ emergency
care competencies out of TAM in particular, aside from
other crucial factors affecting competency development. Therefore, health-care institutions should provide game-
based emergency care training software to enable nurses
to practice their skills repeatedly during and after on-
the-job training in clinical medical education, thereby
increasing their emergency care competencies in clini
cal practice settings. Emergency care training should
be retained in nursing education and routinely offered
as part of on-the-job training programs with the aid of
game-based software to improve nurses’ knowledge of
and attitudes toward CPR and, in turn, the quality of
patient care. Game-based emergency care training rein
forces nurses’ knowledge and skills, reduces their anxiety
in assessing and treating arrhythmia, and strengthens
their problem-solving abilities, thereby enhancing their
performance and professional confidence in providing
emergency care. 4Department of Emergency Medicine, Tungs’ Taichung MetroHarbor
Hospital, Taichung, Taiwan R.O.C. 4Department of Emergency Medicine, Tungs’ Taichung MetroHarbor
Hospital, Taichung, Taiwan R.O.C. 5Department of Othopedics, Tungs’ Taichung MetroHarbor Hospital,
Taichung, Taiwan R.O.C. 5Department of Othopedics, Tungs’ Taichung MetroHarbor Hospital,
Taichung, Taiwan R.O.C. Received: 7 November 2022 / Accepted: 6 May 2023 Received: 7 November 2022 / Accepted: 6 May 2023 Competing interests myths and strategies. Int J Nurs Stud. 2010;47:1451–8. The authors declare that there are no competing interests. Page 9 of 9 Page 9 of 9 Chen et al. BMC Medical Education (2023) 23:359 Chen et al. BMC Medical Education (2023) 23:359 Chen et al. BMC Medical Education 23. Lin YL, Tseng CJ, Tang MH, Kuo WY, Chang NY. Arrhythmia reading: a diverse
teaching approach to improve the ability of 2 year NPGY nurses to interpret
and manage arrhythmias. J Nurs. 2020;67:61–9. 22. Tubaishat A, Tawalbeh LI. Effect of cardiac arrhythmia simulation on
nursing students’ knowledge acquisition and retention. West J Nurs Res.
2015;37:1160–74. 21. Hsu YC, Wang HH, Liu SY, Cheng HL, Yang YM. Effect of simulated first-aid
courses combined with online teaching on the learning results of new nurs
ing staff. J Taiwan Simul Soc. 2018;5:4–14. 21. Hsu YC, Wang HH, Liu SY, Cheng HL, Yang YM. Effect of simulated first-aid
courses combined with online teaching on the learning results of new nurs
ing staff. J Taiwan Simul Soc. 2018;5:4–14.
22. Tubaishat A, Tawalbeh LI. Effect of cardiac arrhythmia simulation on
nursing students’ knowledge acquisition and retention. West J Nurs Res.
2015;37:1160–74.
23. Lin YL, Tseng CJ, Tang MH, Kuo WY, Chang NY. Arrhythmia reading: a diverse
teaching approach to improve the ability of 2 year NPGY nurses to interpret
and manage arrhythmias. J Nurs. 2020;67:61–9. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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Neonatal Imitation, Intersubjectivity, and Children With Atypical Development: Do Observations on Autism and Down Syndrome Change Our Understanding?
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HYPOTHESIS AND THEORY
published: 27 August 2021
doi: 10.3389/fpsyg.2021.701795 Neonatal Imitation, Intersubjectivity,
and Children With Atypical
Development: Do Observations on
Autism and Down Syndrome Change
Our Understanding? 1 Infant and Child Lab, Department of Behavioural Sciences and Learning, Linköping University, Linköping, Sweden,
2 Department of Behavioural Sciences and Learning, The Swedish Institute for Disability Research, Linköping University,
Linköping, Sweden Almost all studies on neonatal imitation to date seem to have focused on typically
developing children, and we thus lack information on the early imitative abilities of children
who follow atypical developmental trajectories. From both practical and theoretical
perspectives, these abilities might be relevant to study in children who develop a
neuropsychiatric diagnosis later on or in infants who later show impaired ability to
imitate. Theoretical in the sense that it will provide insight into the earliest signs of
intersubjectivity—i.e., primary intersubjectivity—and how this knowledge might influence
our understanding of children following atypical trajectories of development. Practical
in the sense that it might lead to earlier detection of certain disabilities. In the present
work, we screen the literature for empirical studies on neonatal imitation in children
with an Autism spectrum disorder (ASD) or Down syndrome (DS) as well as present
an observation of neonatal imitation in an infant that later was diagnosed with autism
and a re-interpretation of previously published data on the phenomenon in a small group
of infants with DS. Our findings suggest that the empirical observations to date are too
few to draw any definite conclusions but that the existing data suggests that neonatal
imitation can be observed both in children with ASD and in children with DS. Thus,
neonatal imitation might not represent a useful predictor of a developmental deficit. Based
on current theoretical perspectives advocating that neonatal imitation is a marker of
primary intersubjectivity, we propose tentatively that an ability to engage in purposeful
exchanges with another human being exists in these populations from birth. Edited by:
Colwyn Trevarthen,
University of Edinburgh,
United Kingdom Edited by:
Colwyn Trevarthen,
University of Edinburgh,
United Kingdom Reviewed by:
Tanu Wadhera,
Dr. B. R. Ambedkar National Institute
of Technology Jalandhar, India
Juan Carlos Gómez,
University of St Andrews,
United Kingdom Reviewed by:
Tanu Wadhera,
Dr. B. R. Ambedkar National Institute
of Technology Jalandhar, India
Juan Carlos Gómez,
University of St Andrews,
United Kingdom *Correspondence:
Mikael Heimann
mikael.heimann@liu.se *Correspondence:
Mikael Heimann
mikael.heimann@liu.se Specialty section:
This article was submitted to
Developmental Psychology,
a section of the journal
Frontiers in Psychology Received: 28 April 2021
Accepted: 30 July 2021
Published: 27 August 2021 Keywords: neonatal imitation, primary intersubjectivity, autism spectrum disorder, down syndrome, literature
search Keywords: neonatal imitation, primary intersubjectivity, autism spectrum disorder, down syndrome, literature
search INTRODUCTION, THEORY, AND MOTIVATION Imitation has been viewed as
important for children with ASD because it “supports a sense of
interpersonal connectedness and mutuality” (Sigman et al., 2004;
p. 224), capacities that people with autism often find difficult. This aspect plus the fact that imitation is an important tool for
learning through observation have made imitation training an
important part of various training and intervention programs for
children with autism (see Schreibman et al., 2015; Spjut Jansson
et al., 2020). As one central example, Rogers and Pennington
(1991) included imitation as one of the early deficits in their
theory on autism. According to them, neonatal imitation is
an early social competence that would be missing in newborn
children that later develop autism. In a subsequent theoretical
attempt, Heimann (1998) outlined two possible developmental
routes for children with autism. Building on Bråten’s (1988, 1998)
theoretical formulations that, from the beginning, the mind is
both dialogical and intersubjective, two hypothetical models of
development were formulated (see Figure 1). The observation that infants might imitate already as
newborns has been with us for a long time, and this ability
has been reported by numerous studies since the 1970’s (e.g.,
Meltzoffand Moore, 1977, 1983, 1989; Dunkeld, 1979; Maratos,
1982; Heimann and Schaller, 1985; Heimann et al., 1989;
Kugiumutzakis, 1998; Nagy et al., 2013; for a recent meta-
analysis, see Davis et al., 2021). Importantly, the existence of this
phenomenon has been argued to reflect a rudimentary capacity
for primary or innate intersubjectivity (i.e., Trevarthen, 1979,
2011a), that is, an ability to engage in intentional and purposeful
exchanges with another human being (e.g., Trevarthen and
Aitken, 2003; Rochat and Passos-Ferreira, 2009; Trevarthen,
2011a,b). Although we acknowledge the current debate around
how to understand a newborn child’s imitative-like responses
(e.g., Oostenbroek et al., 2016; Jones, 2017), this paper rests on
the assumption that those responses are best described as an
act of neonatal imitation (e.g., Simpson et al., 2014; Meltzoff
et al., 2018; Heimann and Tjus, 2019). In line with this, we
also assume that this early act of imitation reflects a potential
expression of primary intersubjectivity and that we need a
better understanding of its place in development for children
following both typical and atypical developmental pathways. INTRODUCTION, THEORY, AND MOTIVATION Heimann M and Holmer E (2021)
Neonatal Imitation, Intersubjectivity,
and Children With Atypical
Development: Do Observations on
Autism and Down Syndrome Change
Our Understanding? Front. Psychol. 12:701795. doi: 10.3389/fpsyg.2021.701795 Most published studies on imitation during the first months of life have focused on typically
developing children. It might actually be all studies for imitation of facial gestures during the
neonatal period. Researchers seem to avoid including children with any known risk factors for
non-optimal development. It follows that reports often state that the children included in studies
on neonatal imitation were born full-term and did not have any known medical complications. August 2021 | Volume 12 | Article 701795 Frontiers in Psychology | www.frontiersin.org Neonatal Imitation and Intersubjectivity Heimann and Holmer However, if imitation in the neonatal period is a real
phenomenon affecting early social interaction and development,
it becomes of uttermost importance to also investigate if children
who show developmental deviances, either specifically in the
social domain or more generally, differ from typically developing
children in this ability. Here we present: (i) a comprehensive
literature search focusing on published reports on imitation
during the first year of life in children with an Autism
Spectrum Disorder (ASD) or Down syndrome (DS); (ii) a home
video observation that in our view might be interpreted as
showing imitation during the neonatal period in a child later
diagnosed with autism; and, (iii) a re-representation of previously
published data on near-neonatal imitation in children with Down
syndrome, observations that hitherto has gone largely unnoticed
by the scientific community (Heimann et al., 1998). If neonatal imitation is one of the first signs of innate or
primary intersubjectivity as proposed by Trevarthen (1979,
2011b), Bråten (1998), Reddy (2008), and Kugiumutzakis and
Trevarthen (2015), then the question also arises to what degree
children following an atypical developmental trajectory would
show an early imitative ability. In other words: Is this capacity
of the neonate associated primarily with typical development, or
is it an ability that also can be observed among infants, following
an atypical developmental trajectory? For autism, imitation has often been highlighted as one of
the capacities that develops slowly and possibly represents a
core deficit (Sigman et al., 2004; Volkmar et al., 2005; Nadel,
2006, 2014; Rogers, 2006; Vanvuchelen et al., 2011; Gowen,
2012; Vivanti and Hamilton, 2014). INTRODUCTION, THEORY, AND MOTIVATION Our
hypothesis that newborn imitation is an example of an early social
motive that signifies an intersubjective capacity is furthermore
anchored in the works by several different theoreticians over
the years (e.g., Bråten, 1998; Reddy, 2008; Rochat and Passos-
Ferreira, 2009), but foremost on Trevarthen’s groundbreaking
ideas as exemplified in these two quotes: Heimann
(1998)
reasoned
that
neonatal
imitation,
as
a marker of primary intersubjectivity, may or may not be
observable in infants who later show signs of autism or other
developmental disabilities. In one scenario, neonatal imitation
is linked to more advanced imitation and intersubjective
abilities at later stages in development, which leads to the
expectation
that
neonatal
imitation
should
be
impaired
in infants with ASD. Thus, a diminished imitation ability
in a neonate might be a sign of an unfulfilled capacity to
partake in early intersubjective social encounters. A second
scenario noted by Heimann (1998) is that neonatal imitation
might not have a direct relationship to later imitative ability
and intersubjective development. In this scenario, neonatal
imitation might be observable in infants with autism or
other
developmental
disorders
that
are
associated
with
impairments in the social domain. This could mean that
mechanisms for primary intersubjectivity are not impaired
from birth in these populations and, instead, that atypicality
in relating to another person emerges later as other layers of
intersubjectivity develop. “Most remarkably, before a baby has competence for handling
and exploring non-living objects, he or she shows sensitive
awareness of the motive states and feelings of other persons who
offer to interact in well-timed contingency with what the infants
expresses, and the baby reacts in intricately adaptive interpersonal
ways to human expressions, often imitating, but not just by
imitating.” (Trevarthen and Aitken, 2003, p. 112). One problem in determining whether a lack of the ability
for neonatal imitation is an early marker of autism is that the
condition is not usually diagnosed before the child is several
years old and rarely before 2 years of age (Ozonoffet al., 2015;
Goldstein and Ozonoff, 2018; Zwaigenbaum et al., 2020). INTRODUCTION, THEORY, AND MOTIVATION Even if
many parents report retrospectively that they did note problems “Infant human beings imitate other humans, not just to act
like them, but to enter into a communicative and cooperative
relationship with them by some transfer of the feeling of body
action...They can, in this way, start building understandings that
may serve later to identify a particular companion in the meaning
of a shared world” (Trevarthen, 2011a, p. 124). August 2021 | Volume 12 | Article 701795 Frontiers in Psychology | www.frontiersin.org 2 Neonatal Imitation and Intersubjectivity Heimann and Holmer FIGURE 1 | Two theoretical models describing the infants state of mind with respect to intersubjectivity at birth for a child later diagnosed with autism. Explanations:
Model A describes an initially typical development, the infant is born with a capacity of primary intersubjectivity, autism emerges later. Model B describes a starting
state that is different from the beginning. A child within the autism spectrum does not have the same capacity to intersubjectivity at birth, the mind is not dialogical
from the start (Adopted from Heimann, 1998, p. 100–101. Reprinted with permission). FIGURE 1 | Two theoretical models describing the infants state of mind with respect to intersubjectivity at birth for a child later diagnosed with autism. Explanations:
Model A describes an initially typical development, the infant is born with a capacity of primary intersubjectivity, autism emerges later. Model B describes a starting
state that is different from the beginning. A child within the autism spectrum does not have the same capacity to intersubjectivity at birth, the mind is not dialogical
from the start (Adopted from Heimann, 1998, p. 100–101. Reprinted with permission). Crompton, 2021; p 650), as proposed by the dialectical mismatch
hypotheses (Bolis et al., 2017). during the first year of life, it has not been possible to pinpoint
an exact cluster of behaviors that makes it possible to reach a
definite diagnosis early in life (but see Wetherby et al., 2021
for an early identification protocol). An additional problem is
that some children with autism show a typical developmental
trajectory from birth to about 12 or 18 months of age whereafter
they start to lose abilities (Ozonoffet al., 2008). An example of
such a regressive pattern might be a child who, after being able
to point and utter his or her first words, suddenly stops both
pointing and talking. Frontiers in Psychology | www.frontiersin.org INTRODUCTION, THEORY, AND MOTIVATION FIGURE 2 | Flow diagram of the literature search. 3. In a re-use of published findings, we present a more in-depth
analysis of how five one-month-old children with DS respond
in an imitation experiment. 3. In a re-use of published findings, we present a more in-depth
analysis of how five one-month-old children with DS respond
in an imitation experiment. children younger than 4 months, was almost on par with typical
infants (Dunst reported a developmental quotient of 85), but
they were clearly below average when they reached their first
birthday (DQ = 62). This “slowing down phenomena was most
pronounced for vocal imitation” according to Dunst (1990;
p. 224). INTRODUCTION, THEORY, AND MOTIVATION For children with DS, early development differs from most
children with autism (although the two syndromes can also
overlap). DS is a chromosomal aberration usually diagnosed
at birth or shortly thereafter and almost all children with
this syndrome end up having a mild to moderate intellectual
disability (Udwin and Dennis, 1995; Di Nuovo and Buono,
2011; Ostermaier, 2019). Since the diagnosis is made early, one
would have expected that some studies on imitation at birth or
shortly thereafter in this group would have been conducted. But
this seems not to be the case. One of the few comprehensive
and longitudinal studies of the early psychological development
focusing on children with DS is the study by Dunst (1990) that
describes sensorimotor development over the first 3 years of life. Davis and Crompton (2021) highlight the growing insight that
the social difficulties associated with autism or other neurodiverse
conditions “are at least in part bidirectional” (p. 652). They also
argue for researchers to use a difference perspective, in contrast
to the more traditional deficit model. This leads to the need
for a research framework that charters socio-cognitive abilities
in detail and that avoids preconceived expectations of what to
expect or not to expect from autistic people or other neurodiverse
groups. Within the scope of this paper, this means that we must
acquire a better knowledge of the competencies of, for example,
infants with the risk of developing autism. If we ever will be
able to understand how the different social and communicative
abilities of autistic persons evolve, we must differentiate between
problems residing within the individual from problems arising
from “a mismatch of interpersonal dynamics” (Davis and The mean age of the nine children constituting the youngest
group in Dunst’s sample was 2.9 months, and they displayed
an almost typical level of imitation according to the Uzgiris-
Hunt scale (Uzgiris and Hunt, 1975). However, imitation of facial
gestures like tongue protrusion or mouth opening used in studies
of imitation in newborn children were not included. Dunst
used Piaget’s theory when chartering the early development
of children with DS and concluded that this group follows a
similar developmental trajectory as typical children, although
at a slower rate. The development of the youngest group, August 2021 | Volume 12 | Article 701795 3 Heimann and Holmer Neonatal Imitation and Intersubjectivity FIGURE 2 | Flow diagram of the literature search. STUDY 1. LITERATURE SEARCH While studies on neonatal imitation in humans to date
have almost exclusively focused on healthy infants (e.g.,
Meltzoffet al., 2018; Davis et al., 2021), our goal is to
provide
observations
relevant
for
children
developing
along
atypical
trajectories
that
might
affect
how
the
capacity
for
primary
intersubjectivity
develops. We
will
do this through three different paths presented as three
different studies: The psycINFO, PubMed, and Scopus databases were searched
for publications in English on the topic of imitation in
populations with DS or ASD younger than 2 years of age. Searches of articles were conducted by the second author (E.H.)
on December 3, 2020, in psycINFO and PubMed, and on
February 26, 2021, in Scopus (for search terms and limiters,
see Supplementary Materials). We did not set any limit for
publication date. 1. A search through scientific databases for papers on imitation
in infants at-risk for ASD and/or DS during the first year of life
with a primary focus on the neonatal period or early infancy
up to 3 months of age; A flow diagram of the search including the four phases
recommended by Liberati et al. (2009) is presented in Figure 2. The search resulted in 85 records in psycINFO, 42 in PubMed,
and 232 in Scopus. After review of titles and abstracts, 50
records were kept for full-text review. The most common
reason for exclusion was that the publication was not a study
of imitation (e.g., mimicry of medical conditions), studied a g
2. Presenting tentative observations from a brief home video
on imitation-like responses in a neonate developing along an
autism trajectory; and August 2021 | Volume 12 | Article 701795 Frontiers in Psychology | www.frontiersin.org 4 Neonatal Imitation and Intersubjectivity Heimann and Holmer can seriously influence a child’s health and further development
(Libster et al., 2012). non-human population (e.g., rodents), or included participants
that were older than the targeted populations. Full-text records
were reviewed for inclusion independently by both authors. Inclusion criteria were that the publication described an original
empirical study with at least one imitation task (e.g., experimental
procedure, observational methods, behavioral ratings), and
included participants with DS or ASD that were younger than
12 months of age. After treatment and a prolonged stay in the hospital before
the parents were allowed to take him home, Marcus seemed to
develop as expected during his first year of life. First Clinical Evaluation and Diagnosis The family contacted the health services who referred them
to a neuropsychiatric clinic that initiated an evaluation shortly
before Marcus’ second birthday. The team was made up by
a child psychiatrist, neuropsychologist, speech and hearing
therapist, and a special education teacher. Some excerpts from
the neuropsychological and medical examination provides a good
context for understanding the grounds for his diagnosis: The psychologist notes that Marcus speaks no words and does
not use gestures but is able to clearly express both joy and when
he dislikes something. He is easily frustrated, but it is relatively
easy to get him back on track. He does not initiate any interaction
and does not respond to any invitation. He uses his mother’s hand
when needing any help. The psychologist also notes that he gives
eye contact only once during the whole assessment. STUDY 1. LITERATURE SEARCH The parents
were acquainted with what to expect from a child during the
first year of life (Marcus was their third child), and they did
not note any atypical signs early on. He made adequate eye
contact according to his mother, he developed pointing as
expected, and he uttered his first word before his first birthday. However, the situation changed shortly after his first birthday. It became more and more difficult to maintain eye contact
with him, and his interest in other people decreased sharply. Parallel to this, he became less verbal, and eventually he stopped
talking. Instead, he became more focused on objects, puzzles,
and YouTube video clips. These behavioral changes began to
worry his parents and, when he was 19 months old, his mother
found a screening instrument online, the Modified Checklist for
Autism in Toddlers (MCHAT; Robins et al., 2001). She answered
the items in the checklist and received a score of 21 out of 23
with the following summary and recommendation: “This score
suggests that your toddler is at elevated risk for autism or another
developmental disorder and should be evaluated by a specialist
for early intervention services.” After full-text reviews, four articles were deemed eligible for
inclusion by both authors, and we agreed to reassess an additional
five articles due to uncertainties in study design. Four of these
five articles were included after reassessment, and both authors
agreed on excluding the fifth. The reasons for exclusion are listed
in Figure 2. The eight articles kept for this review are described
in Table 1. Studies included children with DS (n = 2), ASD (n = 4),
or children from a high-risk population for ASD (i.e., younger
sibling to a child with an ASD, n = 2). The earliest measure of
imitation that was reported was from an age of 1 month, and the
same participants were also observed at 3 and 4 months of age
(Heimann et al., 1998). Age spans in the rest of the studies were in
the range of 6–11 months. The largest study included 86 children
with a diagnosis, and the smallest was a single-case study. Two
studies reported results from an intervention, with baseline
measures of spontaneous imitation in mother-child interaction,
while the other studies used parental questionnaire (n = 1), notes
from medical records (n = 1), or experimental procedures (n =
3). STUDY 2. IMITATION IN A 3-DAY OLD
CHILD LATER DIAGNOSED AS AUTISTIC The following text tells the story of Marcus, a boy with ASD. When he was 3-days old, his parents used a smartphone to take
a video of him as parents often do, and Marcus’ mother later
provided this home video to the first author (M.H.). The very
brief home video shows the father modeling tongue protrusion
and how Marcus responds. Marcus received an autism diagnosis
before his third birthday, and his story is briefly described in
this section. The psychologist used mainly two instruments during this
initial assessment: Vineland Adaptive Behavior Scales II (Sparrow
et al., 2005), a parental questionnaire, and a developmental test,
the Griffiths Scale of Child Development I (Alin-Åkerman and
Nordberg, 1983). The results from Griffiths can be translated into
age equivalents (AE) indicating the age level that corresponds
to the responses a child gives. Marcus is 24 months old when
evaluated, and the result is an uneven profile. His scores are
close to his biological age within three of the areas included in
the Griffiths test: the gross motor, eye-hand coordination, and
performance scales (AE’s 17–22 months). In contrast, he shows
a clearly protracted development on the two scales sensitive to
language, communication, and social development (AE’s 6 ½ and
11 ½ months). The result from the Vineland parental interview
showed that Marcus’ adaptive abilities were affected. His most
severe problem area was his communicative abilities whereas his
motor abilities were judged to be at age level. STUDY 1. LITERATURE SEARCH Four of the studies had a control or comparison group, and
one of these (Keemink et al., 2021) reported that 6–9-month-old-
infants at high-risk for ASD were less likely than a control group
to spontaneously imitate facial emotion expressions; in the three
other studies, no between-group differences were detected under
the age of 12 months. Imitation ability was assessed on
experimental tasks, including object
manipulation (Meltzoff, 1988), gesture
imitation (Smith and Bryson, 2007), and
movement imitation (Bekkering et al.,
2000; Rogers et al., 2003). Infants (7 boys) diagnosed with Down
syndrome. Successful testing
performed at 1 (n = 5), 3 (n = 7), and 4
(n = 7) months of age. At 11 months of age, no statistically
significant differences were observed
between participants above the
cut-off on ADOS at 24 months
compared to those below. Evidence of imitation of tongue
protrusion but not mouth opening
was reported at 1 month of age. At 3
and 4 months of age, no evidence of
imitation was observed at the group
level, although some individuals
imitated either tongue protrusion or
mouth opening. Infants (2 boys) with Down syndrome,
aged 7, 8, and 9 months. Participants were less likely to imitate
facial emotion expressions than the
control group. At 10–12 months, there was no
difference in imitation score between
children with high and low
developmental quotient. Tongue protrusion and mouth opening
were presented to the infant by an
experimenter and responses were
video recorded. Imitation was coded
from videos, defined as a matching
response that exceeded the observed
rate of non-matching responses. Birth and Early Development Marcus was born at term (gestational age: 38 weeks). His
birthweight was within the expected range (3,030 g) and there
were no signs of asphyxia or other immediate complications. In
fact, his Apgar scores were perfect (10, 10, 10). However, the
pregnancy had not been uncomplicated. His mother had spent
some periods in the hospital due to infections and pneumonia. In spite of his perfect Apgar scores, Marcus was diagnosed with
serious complications a couple of hours after birth: early onset
GBS sepsis (Group B streptococcal septicemia), a condition that August 2021 | Volume 12 | Article 701795 Frontiers in Psychology | www.frontiersin.org 5 Neonatal Imitation and Intersubjectivity Heimann and Holmer TABLE 1 | Description of identified articles in the literature search. Study
N
Participant description
Autism spectrum disorder
Bolton et al. (2012)
86
Children diagnosed with autism
spectrum disorder at 11 years of age. Participants were part of a large sample
(N = 14,541) followed from the age of 6
months. Dawson et al. (2000)
1
A boy diagnosed with autism spectrum
disorder at 1 year of age (assessed on
the Autism Diagnostic Interview, Lord
et al., 1994). First notes/observations at
2.5 months of age. Keemink et al. (2021)
18
Infants (11 boys) with at least one older
sibling who had an autism spectrum
disorder diagnosis. Six participants
were 6 months old and 12 were 9
months old. Neimy et al. (2020)
3
Infants (one boy) aged 7, 8, and 11
months, with at least one older sibling
who had an autism spectrum disorder. Receveur et al. (2005)
18
Children (13 boys) diagnosed with
autism spectrum disorder after the age
of 4 years, and split into two groups
based on high (n = 8)/low (n = 10)
developmental quotient (as indicated by
a score above/below 50 on the
Brunet-Lezine Scales, Brunet and
Lezine, 1983). Observations started at
10–12 months. Sanefuji and
Yamamoto (2014)
21
Children (16 boys) with scores above
cut-off for autism spectrum disorder on
ADOS (Lord et al., 1989) at 24 months. Participants were part of a larger
sample of 54 high-risk children (i.e.,
younger siblings of children with an
autism spectrum disorder). First
observation at 11 months. Down syndrome
Heimann et al. (1998)
8
Infants (7 boys) diagnosed with Down
syndrome. Successful testing
performed at 1 (n = 5), 3 (n = 7), and 4
(n = 7) months of age. One child showed evidence of vocal
imitation during baseline. Birth and Early Development Bauer and Jones
(2015)
3
Infants (2 boys) with Down syndrome,
aged 7, 8, and 9 months. Frontiers in Psychology | www.frontiersin.org Imitation results Imitation assessment Imitation assessment Parental questionnaire at 6 and 11
months. Parental questionnaire at 6 and 11
months. Responses to questions referring to
imitation/play at age 6 months was
not a significant predictor of diagnosis
at age 11 years. Potentially a
predictor of later degree of autistic
trait (but overlap between
instruments). Notes from medical journal at 9 months. Notes from medical journal at 9 months. Notes suggesting that the child does
not play imitation games with parents. Participants were less likely to imitate
facial emotion expressions than the
control group. Participants and controls performed a
gaze-contingency task, looking at faces
that turned from a neutral expression to
an emotional. Spontaneous imitation in
response to emotional facial
expressions was coded. Participants and controls performed a
gaze-contingency task, looking at faces
that turned from a neutral expression to
an emotional. Spontaneous imitation in
response to emotional facial
expressions was coded. During a baseline before the
introduction of an intervention, vocal
imitation (“echoics”) was coded based
on video observation of interactions
between the mother and the child. During a baseline before the
introduction of an intervention, vocal
imitation (“echoics”) was coded based
on video observation of interactions
between the mother and the child. Participants were filmed by their parents
during the two first year of life. Imitation
was scored by using the Imitation
Disorder Evaluation scale (Malvy et al.,
1999) on video observations. Children (13 boys) diagnosed with
autism spectrum disorder after the age
of 4 years, and split into two groups
based on high (n = 8)/low (n = 10)
developmental quotient (as indicated by
a score above/below 50 on the
Brunet-Lezine Scales, Brunet and
Lezine, 1983). Observations started at
10–12 months. Participants were filmed by their parents
during the two first year of life. Imitation
was scored by using the Imitation
Disorder Evaluation scale (Malvy et al.,
1999) on video observations. Sanefuji and
Yamamoto (2014)
21 Children (16 boys) with scores above
cut-off for autism spectrum disorder on
ADOS (Lord et al., 1989) at 24 months. Participants were part of a larger
sample of 54 high-risk children (i.e.,
younger siblings of children with an
autism spectrum disorder). First
observation at 11 months. Down syndrome Down syndrome
Heimann et al. (1998) Tongue protrusion and mouth opening
were presented to the infant by an
experimenter and responses were
video recorded. Imitation was coded
from videos, defined as a matching
response that exceeded the observed
rate of non-matching responses. During a baseline before the
introduction of an intervention, an
experimenter produced vocal
utterances that the infant was
prompted to imitate (e.g., “Say,
‘mmm’.”). Imitation was assessed from
video recordings, was coded based on
video observation of interactions
between the mother and the child. Two infants showed limited evidence
of vocal imitation during baseline. Frontiers in Psychology | www.frontiersin.org August 2021 | Volume 12 | Article 701795 Frontiers in Psychology | www.frontiersin.org Neonatal Imitation and Intersubjectivity Heimann and Holmer TABLE 2 | The criteria for an autism diagnosis according to DSM-IV-TR that Marcus fulfilled according to the clinical evaluation. Abridged DSM-IV criteria for autism
Marcus’ evaluation
1. Qualitative impairment in social interactiona
a. impairment in the use of non-verbal behaviors (e.g., eye-to-eye gaze)
Yes
b. failure to develop peer relationships
Yes
c. lack of sharing enjoyments with other people
Yes
d. lack of social/emotional reciprocity
Yes
2. Qualitative impairments in communicationb
a. delay or lack of development of spoken language
Yes
b. inability to initiate conversations
No
c. idiosyncratic use of language
No
d. lack of pretend play or imitative play
Yes
3. Restricted patterns of behavior, interests, and activitiesb
a. preoccupation with restricted pattern of interest
Yes
b. inflexible adherence to routines
Yes
c. stereotyped motor mannerisms (e.g., hand flapping)
Yes
d. preoccupation with parts of objects
No TABLE 2 | The criteria for an autism diagnosis according to DSM-IV-TR that Marcus fulfilled according to the clinical evaluation. FIGURE 3 | A sequence of still photos (courtesy of the family) from the home video showing Marcus’ response. Tongue protrusion is defined as a clear forward
movement of the tongue even if the tongue was not protruded beyond the outer part of the lips (see Heimann and Tjus, 2019). FIGURE 3 | A sequence of still photos (courtesy of the family) from the home video showing Marcus’ response. Tongue protrusion is defined as a clear forward
movement of the tongue even if the tongue was not protruded beyond the outer part of the lips (see Heimann and Tjus, 2019). Down syndrome The result from the psychological evaluation is confirmed
both by the detailed analysis of his language and communicative
development conducted by the speech and language therapist
and by evaluation with the Autism Diagnostic Observation
Schedule (ADOS; Lord et al., 1989, 2000) performed by the
special education teacher. The Children’s Global Assessment
Scale (C-GAS) (Shaffer et al., 1983; Lundh et al., 2010) was
added by the child psychiatrist. Marcus received a score of 38
which indicates “major impairment in functioning in several
areas and unable to function in one of these areas” (e.g., at
home, in preschool or with peers; Shaffer et al., 1983, p. 1229). In conclusion, the child psychiatrist sees the same pattern
as his team members and concludes that Marcus fulfills the
criteria for classic autism and intellectual disability according to DSM-IV-TR (American Psychiatric Association, 2000) and
ICD-10 (World Health Organization, 1990). Specifically, the
child psychiatric evaluation established that Marcus fulfilled
all DSM-IV-TR criteria for qualitative impairment in social
interaction, two out of four criteria for qualitative impairments
in communication, and three out of the four criteria listed
for repetitive and stereotyped patterns of behavior (American
Psychiatric Association, 2000, see Table 2). Frontiers in Psychology | www.frontiersin.org aTwo criteria must be met; bAt least one criterion must be met. Background There seems to be a dearth of studies on the socio-cognitive
abilities of children with DS at birth, as evident from the literature
search described above. We could not identify a single study
investigating imitation among infants with DS during their first
2–3 weeks of life. The only study that came close was conducted
by one of the authors (M. H.) more than 20 years ago (Heimann
et al., 1998) with the goal to study facial imitation over the first 3
months in an attempt to parallel previous published observations
on typical infants. aTime averaged to whole seconds; bTP defined as a clear forward movement of the
tongue even if the tongue was not protruded beyond the outer part of the lips. The initial plan, when the study was conceived in the 1990’s,
had been to carry out the first observation when the children were
still within the neonatal period, that is, before 1 month of age. However, due to both medical and psychological reasons, this
became impossible. Children with DS often require extra medical
support and/or evaluations directly after birth. For the parents,
even if they might have known beforehand that their expecting
child had DS, the early neonatal period usually becomes a time
of adjustment when focus is on other issues than research. In the
end, we succeeded to recruit a group of eight children with DS
(see Heimann et al., 1998) born between gestational weeks 36–
39 (Mdn = 39) and, for five of them, we were able to conduct
the first observation close to the neonatal period when they on
average were 37 days old (SD = 11.0; range 25–52). The focus
here will be only on the five children (all male) observed around
1 month of age. tongue when holding Marcus, who seems both calm and attentive
although slow in his movements. The segment is only about
30 s long (see Figure 3 and Table 3), during which his father
presents six tongue protrusions and Marcus responds with three. The criteria for judging tongue protrusion in this case follows
earlier publications by accepting as a minimum criterion that
a clear forward movement of the tongue is noted although the
tongue might not be protruded beyond the back edge of the lower
lip (e.g., Meltzoffand Moore, 1983; Oostenbroek et al., 2016;
Heimann and Tjus, 2019). Home Video Suggesting a Capacity to
Imitate When Marcus was 3 days old, his mother used her smartphone
to take photos and some brief videos of him together with his
father. One of these videos show the father sticking out his August 2021 | Volume 12 | Article 701795 7 Neonatal Imitation and Intersubjectivity Heimann and Holmer TABLE 3 | A sequential overview of the home video showing Marcus responding
with tongue protrusion (TP) to his fathers’ modeling. Timelinea (s)
Father (model)
Marcus (response)b
0
Looks at Marcus
Looks slightly away
3
Presents first TP
Looks at F
6
Second TP
8
Third TP
10
Looks at Marcus
Eyes closed
12
Looks at F
13
Fourth TP
15
Looks at Marcus
Looks away
18
Fifth TP
Looks at F
22
Sixth TP
23
Looks at Marcus
TP
25
TP
26
TP
27
Smiles
Looks away
aTime averaged to whole seconds; bTP defined as a clear forward movement of the
tongue even if the tongue was not protruded beyond the outer part of the lips. TABLE 3 | A sequential overview of the home video showing Marcus responding
with tongue protrusion (TP) to his fathers’ modeling. TABLE 3 | A sequential overview of the home video showing Marcus responding
with tongue protrusion (TP) to his fathers’ modeling. Timelinea (s)
Father (model)
Marcus (response)b
0
Looks at Marcus
Looks slightly away
3
Presents first TP
Looks at F
6
Second TP
8
Third TP
10
Looks at Marcus
Eyes closed
12
Looks at F
13
Fourth TP
15
Looks at Marcus
Looks away
18
Fifth TP
Looks at F
22
Sixth TP
23
Looks at Marcus
TP
25
TP
26
TP
27
Smiles
Looks away
aTime averaged to whole seconds; bTP defined as a clear forward movement of the
tongue even if the tongue was not protruded beyond the outer part of the lips. (e.g., Vismara and Rogers, 2010; Ingersoll and Meyer, 2011; Spjut
Jansson et al., 2020). Furthermore, if we take the observation as a
true sign of neonatal imitation, then it suggests that a child with
autism (maybe all children with autism), who we know have a
different and problematic relationship with the social world as
they develop, might be no less social as newborns than “typically”
developing children. Background Marcus responses were coded by the
first author (M.H.) and independently by two other researchers. No statistical analysis was possible since there was no control
gesture presented to Marcus and no section of the video that
could be used as a possible baseline measure. Frontiers in Psychology | www.frontiersin.org GENERAL DISCUSSION We have done
this through three different venues: by searching published
reports via three different databases; by presenting a previously
unpublished observation on facial imitation in a 3-day-old infant
that later received an ASD diagnosis; and finally by a renewed
presentation of previously published observations on imitation
in five 1-month-old children with DS. Based on the observations
reported here, we tentatively propose that the little empirical
evidence that exist implies that children with ASD and DS
have a similar capacity for neonatal imitation as do typically
developing children and, thus, an innate capacity for primary
intersubjectivity. However, our most critical suggestion is that
there is a great need of studies investigating neonatal imitation
in atypical populations. Individually, all five children imitated TP during modeling. The pattern for MO was slightly different, four of the children
imitated, while the fifth child displayed a pattern of no change,
the frequency of MO stayed the same in both conditions. Putting
it differently, none of the children responded with what could be
described as a contra-imitative pattern, for instance displaying
the highest frequency of MO when TP is modeled. At least not
when only the modeling periods were analyzed. y
Searching Scopus, PubMed, and PsycInfo for papers on
neonatal imitation in atypical populations resulted in 50 papers
receiving a full-text review, of which 42 were excluded in the
end for not fulfilling our criteria (e.g., focused on diseases,
non-human populations, or included participants older than 12
months). Of the eight articles included, only two focused on
DS, and the only article that described development from birth
in a case of a boy with ASD did not comment on imitation
before the age of 9 months (Dawson et al., 2000). The one article
describing imitation around the first month of life was the one
by the first author on this paper (M.H.), described in detail
above. Thus, our literature search shows that almost no empirical
research on neonatal imitation exists in the target populations
of this paper. This is particularly surprising in the case of ASD,
since imitation is assumed to be impaired in this population
and a possible precursor of later deviant social development
(Rogers and Pennington, 1991; Vanvuchelen et al., 2011). GENERAL DISCUSSION g
We found support for imitative-like responses when the whole
period and the modeling periods were analyzed but not when
focusing only on the response periods (Heimann et al., 1998). The most convincing indication of imitation was found when
only the modeling periods were analyzed (see Figure 4). The
frequency of TP increased on average with 2.3 responses (range
1.2–3.8) when TP was modeled in comparison with the observed
frequency of TP when modeling mouth opening (Sign test; z =
2.23; p = 0.025, Wilcoxon; z = 2.02; p < 0.05). The pattern for
MO was similar, the frequency of MO increased with on average
4.0 mouth openings (range 1.6–7.4) when MO was modeled in
comparison with the number of MOs observed after modeling of
TP (Sign test; z = 2.00; p = 0.046; Wilcoxon; z = −1.75, p = 0.08). Individually, all five children imitated TP during modeling. The pattern for MO was slightly different, four of the children
imitated, while the fifth child displayed a pattern of no change,
the frequency of MO stayed the same in both conditions. Putting
it differently, none of the children responded with what could be
described as a contra-imitative pattern, for instance displaying
the highest frequency of MO when TP is modeled. At least not
when only the modeling periods were analyzed. We found support for imitative-like responses when the whole
period and the modeling periods were analyzed but not when
focusing only on the response periods (Heimann et al., 1998). The most convincing indication of imitation was found when
only the modeling periods were analyzed (see Figure 4). The
frequency of TP increased on average with 2.3 responses (range
1.2–3.8) when TP was modeled in comparison with the observed
frequency of TP when modeling mouth opening (Sign test; z =
2.23; p = 0.025, Wilcoxon; z = 2.02; p < 0.05). The pattern for
MO was similar, the frequency of MO increased with on average
4.0 mouth openings (range 1.6–7.4) when MO was modeled in
comparison with the number of MOs observed after modeling of
TP (Sign test; z = 2.00; p = 0.046; Wilcoxon; z = −1.75, p = 0.08). The aim of this endeavor has been to explore what is known
from empirical studies on the existence of imitation or imitation-
like responses near birth or during the first year of life
among children with non-typical trajectories. Results We conducted three separate analyses: (1) the overall result
across the complete 2-min period; (2) the result for the three
modeling periods; and (3) the result for the three response
periods. Based on the current knowledge at the time when the
study was conducted (in the 1990’s) that children with DS develop
“in the same sequence as that followed by normal children”
(Hodapp and Zigler, 1990; p. 10), we hypothesized that we
would find that our DS group displayed imitation similar to
what had been observed for typical children. Statistically we used
two-tailed tests (Wilcoxon and sign test). Method We acknowledge that this is anecdotal evidence that must be
interpreted with much caution (see Ozonoffet al., 2011, for a
comparison between home videos and clinical evaluations), but
it is notable from the video that Marcus shows no other facial
movements, such as mouth opening, during this brief episode. His first response is a tongue protrusion that comes more than
20 s after his father protruded his tongue for the first time. Regardless if we define Marcus’ response as imitation or not, the
video does show that a 3-day old infant later diagnosed with
autism is able to match tongue protrusion. And he does so in
a way that, in many aspects, mimics how neonatal imitation
of facial gestures has been studied and described in published
studies (e.g., Heimann and Tjus, 2019). If neonatal imitation
is linked with later imitation and social responsiveness, this
observation suggests that it is probably not a direct link–at
least not so for children with autism since imitation is a skill
that often is part of the initial training used in comprehensive
preschool behavioral training programs for children with autism All observations took place in the home of the children using
light-weight portable video equipment. The parents were often
present in the room during the observation and the sessions did
not begin until the child was judged to be awake and alert. TP
or MO were presented to the child by an experimenter and all
gestures were presented during a pre-set interval of 20 s followed
by a response time of equal length. This sequence was repeated
three times, giving each child a total observation time of 120 s (M
= 122.1 s; SD = 9.4). The order of presentation was randomized,
and the experimenter did not know beforehand which gesture
to start with. By definition a TP occurred whenever a clear
forward movement was detected, even if the tongue only passed
the posterior part of the lip (similar to the definition used for
Marcus in the previous section). MO “was defined as a clear
and visible separation of the lips that was judged to meet the
criteria of a definite change. Some children kept their mouths
open over extended periods of time which was not accepted as
a MO. Method A clear change had to take place” (Heimann et al., 1998; August 2021 | Volume 12 | Article 701795 8 Neonatal Imitation and Intersubjectivity Heimann and Holmer observation are relatively straightforward. During the periods
when the gestures were actively modeled all five children imitated
TP and four out of five MO. No child displayed a strong non-
imitative pattern of increasing the frequency of the control
gesture (e.g., TP) in comparison with the gesture being modeled
(e.g., MO). The only child not imitating showed no change, he
opened his mouth an equal number of times both when MO and
TP were modeled. The paper on which this summary is based
did “conclude that children with Down syndrome show an early
capacity for imitation similar to that usually expected for normal
infants during the first few weeks of life” (Heimann et al., 1998; p. 783). Today we would also cautiously propose that children with
DS show signs of a dialogical mind (Bråten, 1988) and a capacity
for primary intersubjectivity (Trevarthen, 2011a) already at 1
months of age. We do however not know if this ability is there
already at birth or not. p. 781). Furthermore, no concurrent forward trust of the tongue
was accepted nor was yawning. All videos were coded blindly by
two research assistants and the obtained Kappa coefficient was
0.92. Imitation was defined behaviorally: an individual child was
judged to imitate if the frequency of matching responses exceeded
the observed number of non-matching responses. Frontiers in Psychology | www.frontiersin.org GENERAL DISCUSSION The
only documented observation that we have been able to identify
is the case of Marcus described in this paper and based on that, we
do not currently see any support for the hypothesis that imitation FIGURE 4 | Imitation in infants with Down syndrome (N = 5) at 1 months of age: Mean rate increase of tongue protrusion (TP) and mouth opening (MO) after
modeling of each gesture during all periods of the experiment (6 × 20 s) as well as only during the active modeling periods (3 × 20 s). See text for more details (based
on Heimann et al., 1998, Tables 3, 4). with Down syndrome (N = 5) at 1 months of age: Mean rate increase of tongue protrusion (TP) and mouth opening (MO) after
all periods of the experiment (6 × 20 s) as well as only during the active modeling periods (3 × 20 s). See text for more details (bas FIGURE 4 | Imitation in infants with Down syndrome (N = 5) at 1 months of age: Mean rate increase of tongue protrusion (TP) and mouth opening (MO) after
modeling of each gesture during all periods of the experiment (6 × 20 s) as well as only during the active modeling periods (3 × 20 s). See text for more details (based
on Heimann et al., 1998, Tables 3, 4). is absent in the neonatal period for children on the track to
develop ASD. Clearly, more research is highly needed to test
this assumption. 2020). We therefore suggest an updated model, Model C, as
illustrated in Figure 5. This new model outlines two possible
trajectories for children later receiving an ASD diagnosis. Path
a illustrates a child developing typically over the first one to
one and a half years, whereafter a regressive pattern occurs
meaning that some social or communicative skills are lost (Parr
et al., 2011; Thompson et al., 2019). The other trajectory, path
b, shows an early deviance from typical development, notable
well before the child’s first birthday. Note that none of the
paths deviate at birth. This is not to say that genetic and
biological factors might not be different from typical children
early on–although imaging studies so far have been unsuccessful
in identifying biological indicators of autism in infants below
6 months of age (see Shen and Piven, 2017). Instead, Model
C suggests that any differences in social and communicative
skills between children with autism and non-autistic children
will not be easily detectable this early on a behavioral level. Discussion Even if this small study indicates that infants with DS seems able
to display near-neonatal imitation under some conditions it is
worth noting that the children responded a bit different to what
we had previously observed for typical infants (Heimann et al.,
1989; Heimann and Tjus, 2019). Their mean rate of responses,
especially so for TP, often exceeded what we have previously
observed for infants during the neonatal period (Heimann et al.,
1998). Furthermore, the different result for the modeling and
response periods tentatively suggests that children with DS are
helped by having stimuli in sight in order to respond. When the
modeling stops and the response period start, they lose focus and
fail to differentiate their response. It is not possible to generalize or draw any definite conclusions
from a study of only five infants. Despite this and the fact
that children with DS might be an even more heterogenous
group than typical infants, the findings from the 1-month-old August 2021 | Volume 12 | Article 701795 9 Heimann and Holmer
Neonatal Imitation and Intersubjectivity Neonatal Imitation and Intersubjectivity Heimann and Holmer Frontiers in Psychology | www.frontiersin.org Explanations of paths: Model C: (1) Path a depicts a child with autism developing along a typical trajectory over the first 12–18 months where after a halt in
development takes place and the child loses social abilities previously mastered (indicated by path a1). This child might eventually find a renewed social interest
(maybe via an intervention) and move via path a2 to develop a capacity for subject-subject relationships. (2) Path b depicts a child lagging behind in social ability
already during the first year but note that even this child has the capacity for primary intersubjectivity at birth. The child might stay in a mindset that is occupied with
subject-object relations but may also later move toward the social world (illustrated by b1). Model D: Paths c and d are almost identical to the paths in the original
model presented by Bråten (1996) for typically developing children. As in Model C, the infant has the ability to act in a complementary way and the participants step
into each other’s dialogic circle already from the start. The figure also illustrates different developmental paths for relations with people as compared to objects. The
main difference from Bråten’s original model is that instead of solid lines, the lines here are dashed in an attempt to illustrate that children with DS usually show a
slower pace of development and might not reach the expected end state. for ASD and typically developing children during the first year
of life. children in the DS sample discussed here had at least a month
of experience before they took part in the experiment. Thus, we
cannot disentangle an innate capacity from rapid learning during
their first month of life. Berger (1990, p. 137) concludes that most “infants with DS are
able to enter into reciprocal interactions with their parents soon
after birth” even if some delay can be detected for early behaviors
such as smiling, vocalizing, and eye contact. In Berger’s sample,
mutual eye contact displayed a delay of 2.5 weeks in onset. This
is also reflected in Model D (Figure 4) that illustrates two main
trajectories for how subject-subject (path c) and subject-object
(path d) relationships might develop for children with DS. Note
that these paths are similar to what we would expect for typical
children (Dunst, 1990; Bråten, 1996; Heimann, 1998). As in Model C, the infant has the ability to act in a complementary way and the participants step
into each other’s dialogic circle already from the start. The figure also illustrates different developmental paths for relations with people as compared to objects. The
main difference from Bråten’s original model is that instead of solid lines, the lines here are dashed in an attempt to illustrate that children with DS usually show a
slower pace of development and might not reach the expected end state. FIGURE 5 | Proposed new models based on the observations presented in the text. Model C represents ASD and Model D describes tentative paths for DS. Explanations of paths: Model C: (1) Path a depicts a child with autism developing along a typical trajectory over the first 12–18 months where after a halt in
development takes place and the child loses social abilities previously mastered (indicated by path a1). This child might eventually find a renewed social interest
(maybe via an intervention) and move via path a2 to develop a capacity for subject-subject relationships. (2) Path b depicts a child lagging behind in social ability
already during the first year but note that even this child has the capacity for primary intersubjectivity at birth. The child might stay in a mindset that is occupied with
subject-object relations but may also later move toward the social world (illustrated by b1). Model D: Paths c and d are almost identical to the paths in the original
model presented by Bråten (1996) for typically developing children. As in Model C, the infant has the ability to act in a complementary way and the participants step
into each other’s dialogic circle already from the start. The figure also illustrates different developmental paths for relations with people as compared to objects. The
main difference from Bråten’s original model is that instead of solid lines, the lines here are dashed in an attempt to illustrate that children with DS usually show a
slower pace of development and might not reach the expected end state. FIGURE 5 | Proposed new models based on the observations presented in the text. Model C represents ASD and Model D describes tentative paths for DS. This
proposal is in line with what we saw in the literature search, in
which few studies reported a difference between children at risk For ASD, the home video of Marcus imitating TP when only
3-days-old raises questions about the starting state of a child
developing along an autistic path. True, it is only anecdotal
evidence based on a very brief video. But even as such, the
observation challenges our knowledge of how ASD develops
over time. One might argue that Marcus is unique and that the
observation says very little about children with ASD overall. Still,
to our knowledge this is the first documented observation of
its kind. It has, for instance, direct bearing on the two models
based on Bråten’s theory (Bråten, 1988, 1998) that Heimann
(1998) outlined. Based on the video of Marcus, we suggest that
Model B should be dismissed in favor of Model A, which, by
allowing for an initial state of primary intersubjectivity, probably
is closer to the truth. However, even this model is limited since
it does not take the heterogeneity of autism into account (see
Fountain et al., 2012; Georgiades et al., 2013; Mottron and Bzdok, August 2021 | Volume 12 | Article 701795 Frontiers in Psychology | www.frontiersin.org 10 Neonatal Imitation and Intersubjectivity Heimann and Holmer FIGURE 5 | Proposed new models based on the observations presented in the text. Model C represents ASD and Model D describes tentative paths for DS. Explanations of paths: Model C: (1) Path a depicts a child with autism developing along a typical trajectory over the first 12–18 months where after a halt in
development takes place and the child loses social abilities previously mastered (indicated by path a1). This child might eventually find a renewed social interest
(maybe via an intervention) and move via path a2 to develop a capacity for subject-subject relationships. (2) Path b depicts a child lagging behind in social ability
already during the first year but note that even this child has the capacity for primary intersubjectivity at birth. The child might stay in a mindset that is occupied with
subject-object relations but may also later move toward the social world (illustrated by b1). Model D: Paths c and d are almost identical to the paths in the original
model presented by Bråten (1996) for typically developing children. Frontiers in Psychology | www.frontiersin.org CONCLUSION early parent-infant dialogues. This is not to say that autism is
caused by caregivers’ responses, only that also children on a
path to autism is affected by continuous positive or negative
social experiences. Our study highlights the lack of empirical support for the
notion that neonates with ASD or DS do not have a capacity
to imitate. Although resting on limited evidence, we believe that
our observations instead tentatively point in the direction of an
imitative capacity also for children that follows a developmental
trajectory different from neurotypical children. Thus, all
newborn children are probably ready for social encounters,
and during their first interactions with another human being,
they will use their capacity for primary intersubjectivity to
establish reciprocity. There are some limitations to consider when interpreting
the findings reported here. To start, the empirical base for
drawing any conclusion varies between our three studies. The literature search rests on a comprehensive scan across
three central and relevant databases that allow us to be more
definite about the scarcity of studies investigating imitation in
newborn and young children with ASD or DS. The empirical
support for the existence of an actual capacity to imitate for
children with ASD or DS is however much weaker, close
to non-existent. For ASD, we have a single home video
that is <1 min long and only the parents’ reassurance that
the situation was spontaneously filmed. According to the
information the parents provided they had never tried to
elicit imitation before the video was recorded. Furthermore, a
further limitation is that the video only contains documentation
of imitation of TP and no sequence when the father was
passive that could have been used as comparison. However,
the fact that no other responses than TP is produced
by Marcus during the brief video adds to the quality of
the observation. It is also striking that our literature search revealed so few
studies on neonatal or early imitation in children with ASD or
DS. For ASD, this is despite the fact that imitation in general
and neonatal imitation specifically have been theoretically in
focus for a long time. The lack of studies including infants or
neonates with DS is also surprising since this is a group of
children identified very early, often before birth. Thus, it ought to
be possible for clinicians to gather larger samples systematically
over time. CONCLUSION This would give us a more solid ground from which
to evaluate how children with DS are capable of imitation early
in life. For ASD it is more difficult to study neonatal imitation
directly, but one possibility could be to include imitation in
the neonatal period in future studies on siblings to children
with ASD. For DS, we have taken a new look at already published
observations on imitation. Although the data consists of a
small number of children (n = 5), they represent all published
observations on near neonatal imitation for this group to date,
as shown by our literature review. It should however be noted
that the DS infants responded differently to what we usually
observe for typical infants. The participating DS infants produced
a higher frequency of TP in comparison with data from studies
on typical infants (Heimann et al., 1998). We do not know if this
is a difference that is significant when it comes to the capacity
to imitate, but it should remind us that the abilities of newborn
children with DS might not be identical to typical infants. The main
difference being that children with DS usually show a much
slower pace of development and might not reach the expected
end state. From a theoretical point of view, the observations provided
suggest the possibility that both ASD and DS children are born
with a mind that has an ability for primary intersubjectivity
that makes it possible for them to enter into early dialogues as
described by, among others, Reddy (2008). In this way, their
starting state seems similar to what we expect to observe in
typical neonates. This further implies that the difficulties we see
later in development most probably are not caused by a lack of
ability for rudimentary social interaction but, instead, emerge
when the conditions for interaction changes, possibly when
typical children start to engage in secondary intersubjectivity. Another possibility is that for children with ASD, the non-
social world at some stage becomes more “attractive” than the
social. As suggested by Davis and Crompton (2021), the evolving
difficulties will, for some autistic children, also be influenced
by non-optimal bidirectional processes that repeatedly create a
mismatch between interacting partners, in this case within the According to the findings presented here and in the Heimann
et al. (1998) paper, it would, in our view, be wise to add imitation
to the list of early social behaviors that children with DS might
display. A capacity which signals that most children with DS
have the capacity to establish relationships with the quality
of primary intersubjectivity at birth or very shortly after. All August 2021 | Volume 12 | Article 701795 11 Neonatal Imitation and Intersubjectivity Heimann and Holmer ETHICS STATEMENT The studies involving human participants were reviewed
and
approved
by
The
Ethical
Review
Board,
Göteborg
University for the Down Syndrome children. For the ASD
home video, the parents gave consent and approved the
final
text. This
study
was
conducted
according
to
the
World Medical Association’s ethical principles as outlined
in the Helsinki declaration. Written informed consent to
participate in this study was provided by the participants’ legal
guardian/next of kin, including consent for the publication
of any potentially identifiable images or data included in
this article. DATA AVAILABILITY STATEMENT The datasets presented in this article are not readily available
because the Down syndrome data has been published earlier
and
the
home
video
is
not
public
material. However,
the procedure for the literature review is available in a
Supplementary Materials. Any requests regarding the Down
syndrome data or the home video should be addressed to the
corresponding author. Regarding
the
literature
search,
although
we
worked
systematically we might have overseen relevant search terms
or additional databases. Perhaps even more critical, we did not
sweep the field for gray literature (e.g., unpublished dissertations,
null findings in file drawers), and we might therefore have
failed to include relevant literature. There is reason to believe
that a publication bias might exist in the area of neonatal
imitation since there is skepticism in the field whether the
phenomenon exists (e.g., Oostenbroek et al., 2016). This, in
combination with the general issues in conducting studies on
atypical populations (e.g., small populations, large heterogeneity,
medical complications), might lead to an unwillingness to
include clinical groups like ASD or DS in studies on neonatal
imitation. Consequently, this leads to an absence of such
studies in published literature which, in our view, is very
unfortunate since it hinders theoretical advancements. A
recent meta-analysis did conclude that there is evidence to
suggest that neonatal imitation exists (Davis et al., 2021),
and therefore we believe that it is warranted to focus more
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evidence of early individual differences. Scand. J. Psychol. 30, 90–101. doi: 10.1111/j.1467-9450.1989.tb01072.x Bolton, P. F., Golding, J., Emond, A., and Steer, C. D. (2012). Autism spectrum
disorder and autistic traits in the Avon Longitudinal Study of Parents and
Children: precursors and early signs. J. Am. Acad. Child Adolescent Psychiatry
51, 249–260. doi: 10.1016/j.jaac.2011.12.009 Heimann, M., and Schaller, J. (1985). AUTHOR CONTRIBUTIONS The study was conceived by MH. Data were collected and
analyzed by MH and EH. The manuscript was written jointly by
both authors. Frontiers in Psychology | www.frontiersin.org August 2021 | Volume 12 | Article 701795 12 Neonatal Imitation and Intersubjectivity Heimann and Holmer FUNDING The original DS study was supported by grants to MH from the
Sven Jerring Foundation and Claes Groschinsky’s Foundation,
Sweden. For autism and general work on imitation MH
was partly funded by grants from The Swedish Council for
Working Life and Social Research (2008-0875) and the Swedish
Research Council (2011-1913). EH was funded by a grant from
the Swedish Research Council for Health, Working Life and
Welfare (2018-01840). Both authors were also supported by
a grant from the Marcus and Amalia Wallenberg Foundation
(MAW 2018.0084). We wish to express our gratitude to the family that provided
the home video and we also extend our thanks to all families
participating in the original study on Down Syndrome. We are
also grateful to Matt Vella for helpful editing feedback. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
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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
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Molecular profiling identifies prognostic markers of stage IA lung adenocarcinoma
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Oncotarget
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ABSTRACT We previously showed that different pathologic subtypes were associated with
different prognostic values in patients with stage IA lung adenocarcinoma (AC). We hypothesize that differential gene expression profiles of different subtypes may
be valuable factors for prognosis in stage IA lung adenocarcinoma. We performed
microarray gene expression profiling on tumor tissues micro-dissected from patients
with acinar and solid predominant subtypes of stage IA lung adenocarcinoma. These
patients had undergone a lobectomy and mediastinal lymph node dissection at the
Shanghai Chest Hospital, Shanghai, China in 2012. No patient had preoperative
treatment. We performed the Gene Set Enrichment Analysis (GSEA) analysis to look for
gene expression signatures associated with tumor subtypes. The histologic subtypes
of all patients were classified according to the 2015 WHO lung Adenocarcinoma
classification. We found that patients with the solid predominant subtype are enriched
for genes involved in RNA polymerase activity as well as inactivation of the p53
pathway. Further, we identified a list of genes that may serve as prognostic markers
for stage IA lung adenocarcinoma. Validation in the TCGA database shows that
these genes are correlated with survival, suggesting that they are novel prognostic
factors for stage IA lung adenocarcinoma. In conclusion, we have uncovered novel
prognostic factors for stage IA lung adenocarcinoma using gene expression profiling
in combination with histopathology subtyping. Molecular profiling identifies prognostic markers of stage IA
lung adenocarcinoma This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited. Molecular profiling identifies prognostic markers of stage IA
lung adenocarcinoma Jie Zhang1, Jinchen Shao1, Lei Zhu1, Ruiying Zhao1, Jie Xing1, Jun Wang2, Xiaohui
Guo3, Shichun Tu4, Baohui Han5 and Keke Yu1,6 g ,
,
,
y
g
,
g ,
g ,
Guo3, Shichun Tu4, Baohui Han5 and Keke Yu1,6
1Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pathology, Shanghai, China
2Tumor Initiation & Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
3Bioinformatics Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
4Allele Biotechnology & Pharmaceuticals, Inc., Nancy Ridge Drive, San Diego, USA
5Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pulmonary Medicine, Shanghai, China
6Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Biobank, Shanghai, China
Correspondence to: Keke Yu, email: ykkxx@shchest.org
Keywords: lung adenocarcinoma, gene expression profiling, differently expressed genes (DEGs), acinar, solid
Received: March 29, 2017 Accepted: June 16, 2017 Published: August 24, 2017
Copyright: Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited. Guo , Shichun Tu , Baohui Han and Keke Yu
1Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pathology, Shanghai, China
2Tumor Initiation & Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
3Bioinformatics Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
4Allele Biotechnology & Pharmaceuticals, Inc., Nancy Ridge Drive, San Diego, USA
5Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pulmonary Medicine, Shanghai, China
6Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Biobank, Shanghai, China
Correspondence to: Keke Yu, email: ykkxx@shchest.org
Keywords: lung adenocarcinoma, gene expression profiling, differently expressed genes (DEGs), acinar, solid
Received: March 29, 2017 Accepted: June 16, 2017 Published: August 24, 2017
Copyright: Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited. Copyright: Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited. Copyright: Zhang et al. Molecular profiling identifies prognostic markers of stage IA
lung adenocarcinoma
Jie Zhang1, Jinchen Shao1, Lei Zhu1, Ruiying Zhao1, Jie Xing1, Jun Wang2, Xiaohui
Guo3, Shichun Tu4, Baohui Han5 and Keke Yu1,6
1Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pathology, Shanghai, China
2Tumor Initiation & Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
3Bioinformatics Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
4Allele Biotechnology & Pharmaceuticals, Inc., Nancy Ridge Drive, San Diego, USA
5Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pulmonary Medicine, Shanghai, China
6Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Biobank, Shanghai, China
Correspondence to: Keke Yu, email: ykkxx@shchest.org
Keywords: lung adenocarcinoma, gene expression profiling, differently expressed genes (DEGs), acinar, solid
Received: March 29, 2017 Accepted: June 16, 2017 Published: August 24, 2017
Copyright: Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited.
www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 43), pp: 74846-74855
Research Paper Molecular profiling identifies prognostic markers of stage IA
lung adenocarcinoma
Jie Zhang1, Jinchen Shao1, Lei Zhu1, Ruiying Zhao1, Jie Xing1, Jun Wang2, Xiaohui
Guo3, Shichun Tu4, Baohui Han5 and Keke Yu1,6
1Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pathology, Shanghai, China
2Tumor Initiation & Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
3Bioinformatics Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla
4Allele Biotechnology & Pharmaceuticals, Inc., Nancy Ridge Drive, San Diego, USA
5Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Pulmonary Medicine, Shanghai, China
6Shanghai Chest Hospital, Shanghai JiaoTong University, Department of Biobank, Shanghai, China
Correspondence to: Keke Yu, email: ykkxx@shchest.org
Keywords: lung adenocarcinoma, gene expression profiling, differently expressed genes (DEGs), acinar, solid
Received: March 29, 2017 Accepted: June 16, 2017 Published: August 24, 2017
Copyright: Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited. www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 43), pp: 74846-74855
Research Paper www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget Differentially expressed genes in acinar and solid
subtype lung AC To evaluate if the identified prognostic markers
are valuable in predicting patient survival, we focused
on the top 50 differentially expressed genes between
acinar and solid subtype lung AC (Figure 4). We then
utilized OncoLnc [19], a tool developed for conveniently
exploring survival correlations with gene expression
data from 21 cancer studies performed by The Cancer
Genome Atlas (TCGA). 11 genes that are overexpressed
in acinar subtype lung AC show a positive correlation with
patient survival. Patients with higher expression of these
genes have favorable overall survival (p-value < 0.05)
(Figure 5). 5 genes that are overexpressed in solid subtype
lung AC show a negative correlation with patient survival. Patients with higher expression of these genes have worse
overall survival (p-value < 0.05) (Figure 6). To identify molecular prognostic markers for stage
IA lung AC, we decided to analyze the gene expression
profiles of lung AC from different clinicopathological
subgroups. Using tissue micro-dissection, we were able to
capture at least 500 tumor cells from each patient specimen. 4 acinar subtype lung AC, which has a better prognosis,
and 4 solid subtype lung AC, which has a worse prognosis,
were selected for comparison of gene expression profiles
using Affymetrix PrimView microarrays. Representative
images of solid and acinar subtype lung AC are presented
in Figure 1. Clinical information of the patients enrolled in
this study such as age, clinicopathological subtype, disease-
free survival (DFS) and overall survival (OS) can be found
in Table 1 and Supplementary Table 1. Using a cutoff value
of fold change > 2 and a p-value < 0.05, 521 genes were
upregulated and 534 genes were downregulated in acinar
subtype lung AC compared to solid subtype (Figure 2A). The heatmap of differentially expressed genes shows a
clear distinction between acinar and solid subtype lung AC
(Figure 2B). Together, our data suggest that acinar and solid
subtypes of lung AC have distinct gene expression
signatures. The poor prognosis of solid subtype lung
AC is potentially due to dysregulated RNA polymerase
I activity and inactivation of the p53 pathway. Candidate
prognostic marker genes from our study may be valuable
for prediction of patient survival and guidance for disease
management and treatment. INTRODUCTION stages, 5-year survival of these patients varies from 77%
to 94.4% [4]. Stage IA patients often have very different
outcomes even if undergoing the same treatment. We and
others have previously reported that the new International
Association for the Study of Lung Cancer/American
Thoracic Society/European Respiratory Society (IASLC/
ATS/ERS) lung AC classification [5] is significantly
associated with patient survival, confirming the
prognostic value of the new classification system [4, 6, 7]. Lung cancer is the leading cause of cancer related
death worldwide [1] and adenocarcinoma (AC) is the most
common histological type [2]. The TNM staging system
is often used to determine how far the cancer has spread
based on the size of the primary tumor, lymph nodes spread
and metastasis [3]. Although survival of pathologic stage
IA patients is expected to be much better than those at other www.impactjournals.com/oncotarget Oncotarget 74846 With the recent advances of genome-wide sequencing
and gene expression profiling, we have gained a deeper
understanding of cancers on a molecular level. Multiple
studies involving gene expression profiling of lung cancers
suggest that gene expression signatures in addition to the
IASLC/ATS/ERS system can be used to improve the
prediction of patient survival [8–11]. In this study, we
focus on identifying molecular prognostic markers that
can predict the survival of stage IA lung adenocarcinoma
patients, with the intention of finding reliable prognostic
markers for better management and treatment of stage IA
lung adenocarcinoma. is involved in RNA polymerase I transcriptional activity
is among the top gene sets enriched in solid subtype lung
AC (Figure 3). Dysregulated RNA polymerase I activity is
commonly associated with upregulated cell growth such
as cancer [15, 16], indicating that poor prognosis of solid
subtype lung AC may be due to aberrant activation of RNA
polymerase I activity. Interestingly, p53 target genes were
downregulated in solid subtype lung AC (Figure 3). The
p53 pathway has been shown to regulate RNA polymerase
I activity by repressing rRNA genes [17, 18], suggesting
that dysregulated RNA polymerase I activity may be due to
inactivation of the p53 pathway in solid subtype lung AC. DISCUSSION Aberrations in key pathways are common in lung
AC [12]. We thus used Gene Set Enrichment Analysis
(GSEA) [13, 14] to identify key pathways and gene sets
that are altered in acinar and solid subtypes of lung AC. 1174 gene sets were upregulated in acinar subtype, among
which 9 gene sets were significant at false discovery
rate (FDR) < 0.25 and 40 gene sets were significantly
enriched at a nominal p-value < 0.01. 2691 gene sets were
upregulated in solid subtype, among which 84 gene sets
were significantly enriched at FDR < 0.25 and 114 gene sets
were significantly enriched at a nominal p-value < 0.01. The
top 9 enriched gene sets based on their normalized enriched
score (NES) are shown for acinar subtype and solid subtype
in Tables 2 and 3, respectively. Notably, the gene set that Stage IA lung adenocarcinoma is a very heterogeneous
disease. Previous studies have shown a wide range of 5-year
survival and outcome from treatments for stage IA lung
AC patients [20–22]. The new multidisciplinary IASLC/
ATS/ERS lung AC classification system has improved
the prediction of patient survival [4, 6, 7]. With the recent
advances in genomics, gene expression profiling has been
shown to predict the survival of patients with lung cancer
accurately [23–26]. Moreover, molecular profiling may
help predict patient response to treatments [27]. Several
recent studies have identified gene expression signatures
in non-small cell lung cancers, including lung AC, that
predict patient survival. In our study, we focused on stage
IA lung AC and specifically compared the gene expression www.impactjournals.com/oncotarget Oncotarget 74847 Table 1: Clinical information of pathological stage IA lung AC patients
Patient ID
Histological
Subtype
Gender
Age
DFS (Months)
OS (Months)
12-6305
Acinar
Male
43
46
46
12-3960
Acinar
Female
55
50
50
12-406
Acinar
Female
61
54
54
12-5316
Acinar
Female
52
48
48
12-6242
Solid
Male
59
10
40
12-3765
Solid
Male
62
34
50
12-1427
Solid
Female
59
53
53
12-6734
Solid
Female
60
45
45
DFS, disease free survival; OS, overall survival. Table 1: Clinical information of pathological stage IA lung AC patients DFS, disease free survival; OS, overall survival. Figure 1: Representative images (100x) of solid and acinar subtypes of Lung AC. Images in the top row show HE staining of
solid (left) and acinar (right) AC. Images in the 2nd row show solid (left) and acinar (right) lung tumor sections before laser microdissection
(LMD). DISCUSSION Images in the bottom two rows show sections with tumor tissues removed (3rd row) and tumor fractions that were dissected out
(4th row). Figure 1: Representative images (100x) of solid and acinar subtypes of Lung AC. Images in the top row show HE staining of
solid (left) and acinar (right) AC. Images in the 2nd row show solid (left) and acinar (right) lung tumor sections before laser microdissection
(LMD). Images in the bottom two rows show sections with tumor tissues removed (3rd row) and tumor fractions that were dissected out
(4th row). www.impactjournals.com/oncotarget Oncotarget 74848 Table 2: Top 9 gene sets enriched in acinar subtype lung AC
NAME
SIZE
ES
NES
NOM p-val
FDR q-val
REACTOME_CREB_
PHOSPHORYLATION_THROUGH_THE_
ACTIVATION_OF_RAS
27
0.59
1.95
0
0.07
LEIN_ASTROCYTE_MARKERS
42
0.52
1.81
0
0.26
REACTOME_INWARDLY_
RECTIFYING_K_CHANNELS
30
0.57
1.81
0
0.19
REACTOME_POST_NMDA_RECEPTOR_
ACTIVATION_EVENTS
33
0.48
1.77
0
0.23
REACTOME_GABA_B_RECEPTOR_
ACTIVATION
37
0.49
1.73
0
0.29
BILANGES_SERUM_SENSITIVE_VIA_
TSC1
23
0.53
1.73
0
0.25
TANG_SENESCENCE_TP53_TARGETS_
UP
33
0.54
1.73
0
0.22
REACTOME_INHIBITION_OF_INSULIN_
SECRETION_BY_ADRENALINE_
NORADRENALINE
24
0.55
1.73
0
0.20
REACTOME_RAS_ACTIVATION_UOPN_
CA2_INFUX_THROUGH_NMDA_
RECEPTOR
17
0.62
1.71
0
0.22
ES, enrichment sore; NES, normalized enrichment score; NOM p-value, nominal p value; FDR q-val, false discovery rate q
value. Table 3: Top 9 gene sets enriched in solid subtype lung AC
NAME
SIZE
ES
NES
NOM p-val
FDR q-val
REACTOME_TELOMERE_
MAINTENANCE
75
-0.59
-1.95
0
0.16
REACTOME_RNA_POL_I_
TRANSCRIPTION
82
-0.62
-1.93
0
0.13
REACTOME_RNA_POL_I_RNA_
POL_III_AND_MITOCHONDRIAL_
TRANSCRIPTION
114
-0.55
-1.92
0
0.11
REACTOME_PACKAGING_OF_
TELOMERE_ENDS
48
-0.66
-1.89
0
0.13
DELPUECH_FOXO3_TARGETS_DN
40
-0.57
-1.88
0
0.11
KEGG_FRUCTOSE_AND_MANNOSE_
METABOLISM
34
-0.51
-1.86
0
0.13
REACTOME_MEIOTIC_
RECOMBINATION
81
-0.59
-1.85
0
0.12
REACTOME_RNA_POL_I_PROMOTER_
OPENING
58
-0.66
-1.82
0
0.14
GALINDO_IMMUNE_RESPONSE_TO_
ENTEROTOXIN
82
-0.49
-1.81
0
0.15 Table 3: Top 9 gene sets enriched in solid subtype lung AC www.impactjournals.com/oncotarget Oncotarget www.impactjournals.com/oncotarget 74849 Figure 2: Microarray gene expression profiling of acinar and solid subtype of Lung AC. (A) Volcano plot shows differentially
expressed genes comparing acinar and solid subtype of Lung AC. Red dots highlight genes overexpressed in acinar subtype (fold change
> 2, p value < 0.05). Green dots highlight genes underexpressed in acinar subtype (fold change > 2, p value < 0.05). (B) Heatmap shows
differentially expressed genes in acinar subtype samples (12-406, 12-6305, 12-3960, 12-5316) and solid subtype samples (12-3765, 12-
1427, 12-6734, 12-6242). Figure 2: Microarray gene expression profiling of acinar and solid subtype of Lung AC. (A) Volcano plot shows differentially
expressed genes comparing acinar and solid subtype of Lung AC. Red dots highlight genes overexpressed in acinar subtype (fold change
> 2, p value < 0.05). Green dots highlight genes underexpressed in acinar subtype (fold change > 2, p value < 0.05). (B) Heatmap shows
differentially expressed genes in acinar subtype samples (12-406, 12-6305, 12-3960, 12-5316) and solid subtype samples (12-3765, 12-
1427, 12-6734, 12-6242). Figure 3: Cancer related signaling pathways are altered in different subtypes of lung AC. Gene Set Enrichment Analysis
(GSEA) was used to identify dysregulated signaling pathways in acinar and solid subtype of lung AC samples. Among the top dysregulated
gene sets, we found enriched RNA polymerase I transcriptional activity, a common feature in human cancers, in solid subtype lung AC. Interestingly, the p53 pathway, which often regulates RNA polymerase I activity, is down-regulated in solid subtype lung AC, indicating
that a worse prognosis in solid subtype lung AC is potentially due to inactivation of p53 target genes, which leads to dysregulated RNA
polymerase I transcription. Figure 3: Cancer related signaling pathways are altered in different subtypes of lung AC. Gene Set Enrichment Analysis
(GSEA) was used to identify dysregulated signaling pathways in acinar and solid subtype of lung AC samples. Among the top dysregulated
gene sets, we found enriched RNA polymerase I transcriptional activity, a common feature in human cancers, in solid subtype lung AC. Interestingly, the p53 pathway, which often regulates RNA polymerase I activity, is down-regulated in solid subtype lung AC, indicating
that a worse prognosis in solid subtype lung AC is potentially due to inactivation of p53 target genes, which leads to dysregulated RNA
polymerase I transcription. www.impactjournals.com/oncotarget Oncotarget 74850 igure 4: Heatmap shows top 50 differentially expressed genes between acinar and solid subtypes of lung AC. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Red
dicates overexpression and blue indicates underexpression. Heatmap was generated in GSEA. Figure 4: Heatmap shows top 50 differentially expressed genes between acinar and solid subtypes of lung AC. Red
indicates overexpression and blue indicates underexpression. Heatmap was generated in GSEA. www.impactjournals.com/oncotarget Oncotarget 74851 Figure 5: Kaplan-Meier survival curves using TCGA data validate the prognostic value of genes overexpressed in
acinar subtype lung AC. Figure 5: Kaplan-Meier survival curves using TCGA data validate the prognostic value of genes overexpressed in
acinar subtype lung AC. Figure 6: Kaplan-Meier survival curves using TCGA data validate the prognostic value of genes overexpressed in solid
subtype lung AC. Figure 6: Kaplan-Meier survival curves using TCGA data validate the prognostic value of genes overexpressed in solid
subtype lung AC. www.impactjournals.com/oncotarget Oncotarget 74852 approved this study. None of the subjects had received
radiation therapy or chemotherapy prior to surgery. Smoking history was obtained from the patients via
interview. Regular follow-up evaluations after surgery
for all patients included physical examination, blood
examination, computed tomography scan of the chest and
abdomen, brain magnetic resonance imaging, and bone
scanning. Postoperative follow-up involved examination
every 3 months for 2 years, every 6 months for years 3-5,
and every 12 months from year 5. The median follow-
up time for the entire group was 70 months (range, 10-
150 months). All cases were confirmed by retrospective
pathological analysis of paraffin-embedded sections. signatures of specimens from 8 acinar and solid subtype
lung AC patients. We identified genes that are positively
correlated with survival (CLDN2, PGM5, USP44, CYP4B1,
PLA2G1B, ZDHHC15, CYP4Z1, RASGRF1, ZNF391,
LRRC36, TMEM178A) and negatively correlated with
survival (C5orf34, MRPL42, GREM1, RRM2, HELLS). In addition, we identified gene sets and pathways that are
differentially activated in acinar and solid subtype lung AC
that may help predict response to treatments.i signatures of specimens from 8 acinar and solid subtype
lung AC patients. We identified genes that are positively
correlated with survival (CLDN2, PGM5, USP44, CYP4B1,
PLA2G1B, ZDHHC15, CYP4Z1, RASGRF1, ZNF391,
LRRC36, TMEM178A) and negatively correlated with
survival (C5orf34, MRPL42, GREM1, RRM2, HELLS). In addition, we identified gene sets and pathways that are
differentially activated in acinar and solid subtype lung AC
that may help predict response to treatments. y
p p
p
The identified genes that predict survival of stage
IA lung AC patients may reveal important information for
developing subtype specific therapies. Survival correlation OncoLnc (www.oncolnc.org) is a web-based database
for exploring correlations between gene expression and
cancer patient survival. It contains survival data for 8,647
patients from 21 cancer studies performed by The Cancer
Genome Atlas (TCGA), along with RNA-SEQ expression for
mRNAs and miRNAs from TCGA [19]. OncoLnc was used
to link TCGA survival data to microarray gene expression
data [19]. A 33% upper percentile and 33% lower percentile
of expression was used to generate a Kaplan-Meier survival
curve for individual genes of interest. Microarray At least 500 tumor cells were micro-dissected from
each patient specimen. All samples were submitted to
the WuXi Genome Center for microarray analysis. RNA
extraction was performed followed by QC. Samples
were then processed for microarray expression analysis
using PrimeView Human Gene Expression Array
(Thermo Fisher Scientific). Data QC was processed using
Affymetrix Expression Console software (version 1.3.1). Microarray data was processed using R with the limma
package. RMA normalization was applied. www.impactjournals.com/oncotarget CLDN2 encodes
for Claudin-2, which belongs to major integral membrane
proteins localized in tight junctions. CLDN2 was previously
shown to be a good prognostic factor in non-small cell lung
cancer [28]. PGM5 is a phosphoglucomutase involved in
interconversion of glucose-1-phosphate and glucose-6-
phosphage [29]. PGM5 has been found to be down-regulated
in colorectal cancers [30]. USP44 is a deubiquitinase that
functions as a key regulator of mitotic spindle checkpoint
and loss of Usp44 in mice resulted in spontaneous tumor
development, especially in lung [31]. USP44 is also
frequently downregulated in human lung cancers and its
decreased expression is associated with a poor prognosis
[31]. CYP4B1 and CYP4Z1 are members of the cytochrome
P450 monooxygenase involved in drug metabolism. It has
been reported that the expression of CYP4B1 is reduced
in neoplastic lung tissues compared with normal lung [32]. Mutations of the CYP4Z1 gene have been associated with
progression of NSCLC [33]. GREM1 is a bone morphogenic
protein (BMP) antagonist. It is overexpressed in lung AC and
forced expression increases proliferation in normal lung cells
[34]. RRM2 is ribonucleotide reductase regulatory subunit
M2, which catalyzes the formation of deoxyribonucleotides
from ribonucleotides. Multiple studies have suggested that
high expression of RRM2 correlates with poor prognosis in
NSCLC patients [35, 36]. GSEA analysis GSEA is a computational methodology to identify
classes of genes that are overexpressed in a large set of genes
between two biological states, such as disease phenotypes
[13, 14]. This analysis was done using GenePattern (https://
genepattern.broadinstitute.org/). The Molecular Signatures
Database (MSigDB) C2 collection, consisting of canonical
pathways and experimental signatures curated from
publications, was used for the analysis. In conclusion, our gene expression analysis on
different subtypes of stage IA lung AC have revealed valuable
prognostic factors that may help improve the prediction
of patient survival. Furthermore, it may provide important
implications for identifying targeted therapies in a subtype
specific manner. Due to the relatively small sample size in
this study, follow-up studies with a larger cohort of patient
specimens will strengthen our discovery and hopefully help
further improve the clinical outcomes of patients. www.impactjournals.com/oncotarget Statistical analysis We used data from the records of 8 patients with
pathological stage IA AC, who had undergone a lobectomy
and mediastinal lymph node dissection at Shanghai Chest
Hospital, Shanghai, China in 2012. Written consent was
obtained from all participants and our ethics committee For microarray analysis, the significance of
differential gene expression is determined by a cut-
off of p-value < 0.05 and fold change > 2. Statistics
used in GSEA analysis was previously described [13]. www.impactjournals.com/oncotarget Oncotarget 74853 american thoracic society/european respiratory society
international multidisciplinary classification of lung
adenocarcinoma. J Thorac Oncol. 2011; 6:244-285. Significance of enriched gene sets is determined by a
cut-off of FDR < 0.25 and nominal p-value < 0.01. For
OncoLnc, multivariate Cox regressions were used to
conduct survival analysis [19] and a p-value < 0.05 is
considered significant. 6. Yoshizawa A, Motoi N, Riely GJ, Sima CS, Gerald
WL, Kris MG, Park BJ, Rusch VW, Travis WD. Impact
of proposed IASLC/ATS/ERS classification of lung
adenocarcinoma: prognostic subgroups and implications for
further revision of staging based on analysis of 514 stage I
cases. Mod Pathol. 2011; 24:653-664. FUNDING This work was supported by the Major and Key
Foundation of Shanghai Chest Hospital (grant no. 2014YZDC10200). 11. Chen HY, Yu SL, Chen CH, Chang GC, Chen CY, Yuan A,
Cheng CL, Wang CH, Terng HJ, Kao SF, Chan WK, Li HN,
Liu CC, et al. A five-gene signature and clinical outcome in
non-small-cell lung cancer. N Engl J Med. 2007; 356:11-20. Abbreviations AC: adenocarcinoma; GSEA: gene set enrichment
analysis; DFS: disease free survival; OS: overall survival;
TCGA: the cancer genome atlas; NES: normalized
enriched
score;
IASLC/ATS/ERS:
International
Association for the Study of Lung Cancer/American
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W, Su B. Two-gene signature improves the discriminatory
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the PainDETECT Questionnaire Hiroaki Abe1,
Masahiko Sumitani2*,
Yoshitaka Matsubayashi3,
Rikuhei Tsuchida1,
Yasushi Oshima3,
Katsushi Takeshita4 and
Yoshitsugu Yamada1 Abstract Int J Anesth
Pain Med. Vol. 3 No. 1:3 Citation: Abe H, Sumitani M, Matsubayashi
Y, Tsuchida R, Oshima Y, et al. (2017)
Validation of Pain Severity Assessment using
the PainDETECT Questionnaire. Int J Anesth
Pain Med. Vol. 3 No. 1:3 Abstract Background: The PainDETECT Questionnaire (PD-Q) is a screening tool for
Neuropathic Pain (NeP). A cut-off value of ≥ 13 indicates the possibility of NeP
components. The PD-Q score seems to reliably distinguish the severity of NeP;
however, it has not yet been validated whether changes of the PD-Q score can
follow changes of pain severity assessed using an 11-point Numerical Rating Scale
(NRS). 1 Department of Anesthesiology and Pain
Relief Center, The University of Tokyo
Hospital, Japani 2 Department of Pain and Palliative
Medicine, The University of Tokyo
Hospital, Japan Methods: Sixty patients diagnosed with NeP answered the PD-Q, NRS, and
Neuropathic Pain Symptom Inventory (NPSI). We analyzed correlations among
them by using the Pearson correlation test. Another 49 patients with NeP
answered the PD-Q and NRS twice, with the second survey being conducted 8
weeks after the first survey. Correlations were analyzed between the %decrease
of PD-Q and NRS scores. In both experiments, the participants were divided into
two groups according to the PD-Q score (cut-off value of 13). For these groups, we
also analyzed the correlations. 3 Department of Orthopaedic Surgery and
Spinal Surgery, The University of Tokyo
Hospital, Japan 4 Department of Orthopaedic Surgery,
Jichi Medical University, Japan Results: The PD-Q showed fair to moderate correlation with the NRS and NPSI. The PD-Q could linearly track changes of the NRS. Analyses of subsets revealed
that patients with PD-Q scores ≥ 13 showed similar correlations, whereas those
with PD-Q scores <13 did not. Corresponding author:
Masahiko Sumitani, MD, PhD Conclusion: Despite the limited number of patients included, our findings suggest
that the PD-Q can be suitable for assessing and tracking pain severity in patients
with NeP at least in common clinical settings. Sufficient attention should be paid
when using the PD-Q as an outcome measure for patients with a low PD-Q score. Department of Pain and Palliative Medicine,
The University of Tokyo Hospital, Hongo 7-3-
1, Bunkyo, Tokyo 113-0033, Japan. Keywords: Neuropathic pain; painDETECT; Screening; Severity of pain; Validation Tel: +81-3-3815-5411 Received: August 20, 2017; Accepted: September 09, 2017; Published: September
16, 2017 Citation: Abe H, Sumitani M, Matsubayashi
Y, Tsuchida R, Oshima Y, et al. (2017)
Validation of Pain Severity Assessment using
the PainDETECT Questionnaire. Int J Anesth
Pain Med. Vol. 3 No. 1:3 Citation: Abe H, Sumitani M, Matsubayashi
Y, Tsuchida R, Oshima Y, et al. (2017)
Validation of Pain Severity Assessment using
the PainDETECT Questionnaire. © Under License of Creative Commons Attribution 3.0 License | This article is available in: http://anaesthesia-painmedicine.i Experiment 1 Subjects: The study protocol was approved by the institutional
review board of the University of Tokyo Hospital and adhered to
the guidelines of the Helsinki Declaration. To determine criterion-related validity, we calculated the
Pearson correlation coefficient between the PD-Q score and
three-type NRS score, the NPSI score, the SF-MPQ score, PCS
of SF-36, and MCS of SF-36. The following ranges are generally
accepted rankings for coefficients: 1.0-0.81 (excellent); 0.80-0.61
(very good); 0.60-0.41 (good); 0.40-0.21 (fair); and 0.2-0.0 (poor)
[24]. Demographic data of the two patient groups were analyzed
using the Mann-Whitney test and Chi-square test as appropriate. All statistical analyses were performed using IBM SPSS Statistics
for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA). Sixty patients referred to the outpatient clinic of the Department
of Anesthesiology and Pain Relief Center at The University of
Tokyo Hospital was eligible for participation in this study. The
inclusion criteria were as follows: (i) diagnosed with NeP by
their attending pain specialist as per guidelines established by
the IASP Neuropathic Pain Special-Interest-Group [16]; (ii) mean
pain intensity in the past month (recorded at inclusion) of ≥
3 on an 11-point NRS (0 = no pain; 10 = worst possible pain);
(iii) pain duration of ≥ 3 months; and (iv) age ≥ 20 years. The
exclusion criteria were comorbid psychiatric disorders such
as schizophrenia, personality disorders, and other psychotic
disorders as defined by the ICD-10. Patients were enrolled after
they provided informed consent. They were divided into two
groups according to the PD-Q score; patients with a low PD-Q
score (<13) indicating nociceptive pain and those with a high
PD-Q score (≥ 13) indicating possible and likely NeP components. International Journal of Anesthesiology & Pain Medicine
ISSN 2471-982X Some studies have reported the PD-Q
score and used it to identify patients with NeP components at
baseline [8-11]. Other studies have used the PD-Q to assess the
response of NeP components to therapy [12,13]. These studies
showed that the PD-Q score significantly improved at the end
of the treatment period. The PD-Q seems to reliably distinguish
the severity of pain in patients with NeP [14]; however, it has
not yet been validated whether the changes of the PD-Q score
can follow changes of pain severity assessed using the NRS in
both the high and low PD-Q score subsets. In fact, one of the
largest longitudinal observational studies on a drug treatment
demonstrated significant improvement in NRS scores but not in
the PD-Q scores [15]. In the present study, we examined whether
the PD-Q score reflected pain intensity and could be used to track
the course of pain symptoms over time in both patients with NeP
components (i.e., PD-Q score ≥ 13) and without NeP components
(i.e., PD-Q score <13). Short-Form Health Survey (SF-36) for assessing their health-
related quality of life (QOL), and the Neuropathic Pain Symptoms
Inventory (NPSI) Japanese version for specifically assessing the
severity of NeP [17-23]. The PD-Q identifies and rates seven
pathological pain qualities: burning, tingling or prickling sensations,
tactile and thermal allodynia, electric shock-like sensations,
numbness, and pressure-evoked pain sensation. Moreover, the
PD-Q evaluates the presence or absence of gradation of pain, pain
course pattern, and the presence or absence of radiating pain. Pain intensity was assessed using a three-type NRS in which the
patients were asked to grade the current pain, the average pain
in the past 4 weeks, and the worst pain in the past 4 weeks on
an 11-point NRS. The SF-MPQ comprises two dimensions of pain
(i.e., sensory and affective). The total score of these dimensions
of the SF-MPQ is generally useful for assessing pain severity in
relation with the activities of daily living; however, the SF-MPQ
tends to be less sensitive than the simpler pain ratings (e.g., NRS). The NPSI specifically assesses NeP severity, and the psychometric
validation of the Japanese version of the NPSI has already been
performed [22]. The NPSI comprises four main components (i.e.,
spontaneous pain, paroxysmal pain, evoked pain, and sensation),
which encompass 12 questions. Introduction The International Association for the Study of Pain (IASP) defines
Neuropathic Pain (NeP) as “pain arising as a direct consequence
of a lesion or disease affecting the somatosensory system” [1]. NeP has certain characteristics that set it apart from nociceptive
pain. For example, patients with nociceptive pain caused by
cancer consistently characterize their pain as heavy, lacerating,
and suffocating, while patients with NeP tend to describe their
pain as aching, throbbing, numb, and miserable [2]. Further, such
quality of pain can indicate response to therapy. We previously
demonstrated that the two categories of NeP characteristics
(superficial-pain descriptions [burning, tingling, piercing, etc.) and
deep-somatic descriptions (squeezing, cramp-like, twisting, etc.)) are differently alleviated by mirror visual feedback treatment [3]. Thus, the quality of pain is useful for interpreting the underlying
pathophysiological mechanism(s) of pain. A variety of measurements and questionnaires are used to
evaluate pain as objectively as possible. NeP does not possess
distinct pathognomonic features, and hence, screening tools
are needed that use the most characteristic symptoms and
signs of NeP for proper identification and treatment of patients. Currently, several simple and reliable questionnaires are available 1 tive Commons Attribution 3.0 License | This article is available in: http://anaesthesia-painmedicine.imedpub.com/archive.php © Under License of Creative Commons Attribution 3.0 License | This article is available in: http://anaesthesia-painmedicine.imed 2017 2017 International Journal of Anesthesiology & Pain Medicine
ISSN 2471-982X The NPSI score is calculated
by adding the scores of these four components, with a higher
score indicating more severe NeP. The SF-36 consists of eight
subscales, namely physical functioning, role physical, bodily
pain, general health, vitality, social functioning, role emotional,
and mental health. We have previously reported that the PD-Q
and SF-36 scores demonstrate similar trends: patients with NeP
had lower physical and mental functioning than did those with
nociceptive pain [7]. For analyses, we calculated two summed
scores on the basis of these subscales: the physical component
score (PCS) and the mental component score (MCS). Lower
PCS and MCS indicate poorer health-related QOL. Each has the
same mean and standard deviation (50 and 10, respectively) in a
normal population. This article is available in: http://anaesthesia-painmedicine.imedpub.com/archive.php International Journal of Anesthesiology & Pain Medicine
ISSN 2471-982X Vol. 3 No. 1: 3 for screening NeP components [4], which support the earlier
detection and appropriate treatment of this disease. Among
these questionnaires, the PainDETECT Questionnaire (PD-Q),
developed by German pain researchers in 2004, can successfully
perform subgrouping of patients to classify the profile of
individual pain-related sensory abnormalities [5,6]. It has been
translated and validated in multiple languages, including Japanese
[7]. These translated PD-Qs generally demonstrate fair to good
criterion-related validity with an 11-point numerical rating scale
(NRS) for pain intensity, excellent internal consistency, and high
reliability with significance for patients with NeP. Many drug
studies have reported the change of the PD-Q symptom intensity
and its scores over time. Some studies have reported the PD-Q
score and used it to identify patients with NeP components at
baseline [8-11]. Other studies have used the PD-Q to assess the
response of NeP components to therapy [12,13]. These studies
showed that the PD-Q score significantly improved at the end
of the treatment period. The PD-Q seems to reliably distinguish
the severity of pain in patients with NeP [14]; however, it has
not yet been validated whether the changes of the PD-Q score
can follow changes of pain severity assessed using the NRS in
both the high and low PD-Q score subsets. In fact, one of the
largest longitudinal observational studies on a drug treatment
demonstrated significant improvement in NRS scores but not in
the PD-Q scores [15]. In the present study, we examined whether
the PD-Q score reflected pain intensity and could be used to track
the course of pain symptoms over time in both patients with NeP
components (i.e., PD-Q score ≥ 13) and without NeP components
(i.e., PD-Q score <13). for screening NeP components [4], which support the earlier
detection and appropriate treatment of this disease. Among
these questionnaires, the PainDETECT Questionnaire (PD-Q),
developed by German pain researchers in 2004, can successfully
perform subgrouping of patients to classify the profile of
individual pain-related sensory abnormalities [5,6]. It has been
translated and validated in multiple languages, including Japanese
[7]. These translated PD-Qs generally demonstrate fair to good
criterion-related validity with an 11-point numerical rating scale
(NRS) for pain intensity, excellent internal consistency, and high
reliability with significance for patients with NeP. Many drug
studies have reported the change of the PD-Q symptom intensity
and its scores over time. Experiment 1 The specific cause of NeP in the patients were brachial
plexus injury (n=12), radiculopathy (n=11); spinal cord injury
(n=10); post-herpetic neuralgia (n=11); diabetic, alcoholic, or
chemotherapy-induced polyneuropathy (n=6); post-amputation
phantom limb pain (n=5); complex regional pain syndrome (n=2);
carpal tunnel syndrome (n=1); trigeminal nerve injury pain (n=1);
and thalamic pain (n=1). Forty-three patients had a high PD-Q
score (≥ 13), and 17 had a low PD-Q score. The demographic
data of the two groups were comparable (Table 1). Despite
the difference in the PD-Q scores, the three-type NRS, SF-MPQ,
and SF-36 subscale scores were comparable between these
two patient groups. The difference in the NPSI scores reached
significance (p=0.02). Regarding criterion-related validity of the
whole dataset of patients with NeP (n=60), the total PD-Q score
exhibited statistically significant correlations with current pain
(r=0.29, p=0.03), worst pain in the past 4 weeks (r=0.36, p=0.005),
average pain in the past 4 weeks (r=0.33, p=0.009), the total NPSI
score (r=0.69, p<0.001), and the total SF-MPQ score (r=0.63, Decrease of the PD-Q score in percentage terms was significantly
associated with %decreases of current pain (r=0.31, p=0.03) and
average pain in the past 4 weeks (r=0.33, p=0.02), but showed
a tendency to be associated with a %decrease of worst pain in
the past 4 weeks (r=0.28, p=0.052) in all the patients with NeP
(n=49). These correlations were also observed in the subset of
35 patients with a high PD-Q score (≥ 13) (current pain, r=0.38,
p=0.03; average pain in the past 4 weeks, r=0.53, p=0.001;
worst pain in the past 4 weeks, r=0.28, p=0.098). However, the Table 1 Demographic data of patients with neuropathic pain who participated in experiment. Results The specific causes of NeP in the patients were brachial plexus
injury (n=15); radiculopathy (n=11); spinal cord injury (n=5); post-
herpetic neuralgia (n=8); diabetic, alcoholic, or chemotherapy-
induced polyneuropathy (n=3); post-amputation phantom limb
pain (n=3); carpal tunnel syndrome (n=2); trigeminal nerve
injury pain (n=1); and thalamic pain (n=1). Thirty-five patients
had a high PD-Q score (≥ 13), and 14 had a low PD-Q score. The
demographic data of the two groups were comparable (Table 2). Despite the significant difference in the PD-Q scores, the three-
type NRS scores were not different between these two patient
groups. International Journal of Anesthesiology & Pain Medicine
ISSN 2471-982X Vol. 3 No. 1: 3 p<0.001). The correlation between the PD-Q score and the SF-36
subscale scores (PCS and MCS) did not reach significance (p=0.55
and 0.15, respectively). In the subset of patients (n=43) with a
high PD-Q score (≥ 13) that indicates neuropathic components,
the same parameters, except for current pain (p=0.38), were
significantly correlated with the PD-Q score (i.e., worst pain
in the past 4 weeks, r=0.36, p=0.02; average pain in the past 4
weeks, r=0.32, p=0.04; NPSI, r=0.53, p=0.0003; SF-MPQ, r=0.54,
p=0.0003). In addition, correlations between the PD-Q score
and the SF-36 subscale scores (PCS and MCS) were not observed
(p=0.77 and 0.20, respectively). In the other subset of patients
(n=17) with a low PD-Q score (<13), no parameters correlated
with the PD-Q score (current pain, p=0.41; worst pain in the past
4 weeks, p=0.77; average pain in the past 4 weeks, p=0.79; NPSI,
p=0.16; SF-MPQ, p=0.89; SF-36-PCS, p=0.61; SF-36-MCS, p=0.24). and secondary endpoints [8-13,15]. The patients were asked
to complete them twice. The second survey was administered
to the patients at around 8 weeks after the first survey. The
attending physicians prescribed medicines at their discretion
for treating the patients through the period between the first
and second surveys. Decreases in the three-type NRS and PD-Q
scores from the first survey to the second survey were expressed
in percentage terms. To validate whether the %decrease of the
PD-Q score could track that of the NRS score, we calculated the
Pearson correlation coefficient between the %decreases of the
PD-Q score and three-type NRS score. Demographic data of the
two patient groups were analyzed using the Mann-Whitney test
and Chi-square test as appropriate. All statistical analyses were
performed using IBM SPSS Statistics for Windows, Version 22.0
(IBM Corp.). PD-Q = painDETECT questionnaire; NRS = numerical rating scale (an 11-point numerical rating scale on pain intensity); NPSI = Neuropathic Pain
Symptom Inventory; SF-MPQ = McGill Pain Questionnaire short-form; SF-36 = Medical Outcomes Study 36-item Short-Form Health Survey. Experiment 2 Subjects: A different patient cohort with NeP (n=49) was
eligible for participation after they provided informed consent. The inclusion and exclusion criteria were identical to those for
Experiment 1. These patients were also divided into two groups:
patients with a low PD-Q score (<13) and patients with a high PD-Q
score (≥ 13). In Experiment 2, we used a simple set of the PD-Q
and three-type NRS (i.e., current pain, average pain in the past
4 weeks, and worst pain in the past 4 weeks), because previous
studies on drug treatment used these parameters as the primary The patients were asked to complete a set of questionnaires
including the PD-Q Japanese version, a three-type NRS on pain
intensity, the Japanese version of the short-form McGill Pain
Questionnaire (SF-MPQ), the Medical Outcomes Study 36-Item 2 This article is available in: http://anaesthesia-painmedicine.imedpub.com/archive.php 2017 Data were presented as mean ± standard deviation.ii Data were presented as mean ± standard deviation.iii er License of Creative Commons Attribution 3.0 License © Under License of Creative Commons Attribution 3.0 License Data were presented as mean ± standard deviation.ii Experiment 1 3
All patients with
neuropathic pain
Patients with neuropathic
pain and PD-Q score ≥ 13
Patients with neuropathic
pain and PD-Q score <13
p-value*
Age
59.3 ± 15.2
57.3 ± 15.1
64.2 ± 14.8
0.13
Sex (male/female)
39/21
29/14
10/7
0.65
Height (cm)
163.7 ± 9.9
164.3 ± 9.4
162.0 ± 11.1
0.99
Body weight (kg)
63.8 ± 17.5
66.2 ± 17.5
57.7 ± 16.3
0.23
PD-Q
18.2 ± 6.2
21.2 ± 4.5
10.6 ± 1.7
0.001
NRS
Current pain
6.5 ± 2.3
6.9 ± 2.0
5.5 ± 2.7
0.62
Worst pain in the past 4 weeks
8.3 ± 1.7
8.5 ± 1.6
7.6 ± 1.6
0.33
Average pain in the past 4 weeks
6.7 ± 2.0
7.0 ± 1.9
6.0 ± 2.2
0.24
NPSI
4.2 ± 2.2
5.0 ± 1.9
2.4 ± 1.6
0.02
SF-MPQ
17.7 ± 8.9
20.3 ± 8.6
11.2 ± 6.1
0.23
SF-36
Physical component score
27.3 ± 16.5
26.4 ± 16.1
29.5 ± 17.7
0.95
Mental component score
41.3 ± 11.9
40.2 ± 11.2
44.1 ± 13.4
0.95
Data were presented as mean ± standard deviation. PD-Q = painDETECT questionnaire; NRS = numerical rating scale (an 11-point numerical rating scale on pain intensity); NPSI = Neuropathic Pain
Symptom Inventory; SF-MPQ = McGill Pain Questionnaire short-form; SF-36 = Medical Outcomes Study 36-item Short-Form Health Survey. Table 1 Demographic data of patients with neuropathic pain who participated in experiment. 3 Vol. 3 No. 1: 3 All patients with
neuropathic pain
Patients with neuropathic pain
and PD-Q score ≥ 13
Patients with neuropathic pain and
PD-Q score < 13
p-value*
Age
58.2 ± 15.1
56.0 ± 13.2
63.7 ± 18.4
0.09
Sex (male/female)
35/14
26/9
09/5
0.49
Height (cm)
164.5 ± 8.9
165.9 ± 8.9
160.8 ± 8.2
0.05
Body weight (kg)
62.2 ± 13.7
63.6 ± 14.8
58.8 ± 9.9
0.25
PD-Q
18.5 ± 7.4
21.8 ± 5.8
10.1 ± 2.2
<0.001
NRS
Current pain
5.7 ± 2.1
5.9 ± 1.9
5.4 ± 2.5
0.52
Worst pain in the past 4 weeks
8.2 ± 1.6
8.3 ± 1.6
8.0 ± 1.6
0.52
Average pain in the past 4 weeks
6.1 ± 1.7
6.1 ± 1.7
6.0 ± 1.8
0.7
Data were presented as mean ± standard deviation. PD-Q = painDETECT questionnaire; NRS = numerical rating scale (an 11-point numerical rating scale on pain intensity). Discussion The results of the present study revealed at least fair to moderate
validity for assessing NeP severity by using the PD-Q. Our
findings were consistent with those of a previous study in which
the patients with NeP were stratified into three pain severity
groups according to the Brief Pain Inventory Short-Form (i.e.,
mild, moderate, and severe), and the average PD-Q scores of
the three groups increased in a step-wise manner [14]. Further,
the present study revealed that changes of the PD-Q score can
follow changes of pain severity assessed using the NRS over time. Therefore, we can conclude that the PD-Q score reliably reflects
the severity of pain in patients with NeP. Analyzing subsets of patients with NeP illustrated the importance
of paying attention to the PD-Q score when evaluating NeP
severity by using the PD-Q. Patients with a high PD-Q score (≥ 13)
demonstrated the validity of severity assessment using the PD-
Q, and their PD-Q scores tracked the course of pain symptoms
over time. However, a small fraction of patients who had a low
PD-Q score (<13) did not demonstrate such validity. Previous
longitudinal studies on drug treatment used the PD-Q score as one
of the inclusion criteria (i.e., score ≥ 13) to identify patients with
NeP components [8-11], and these studies successfully revealed
that the experimental drug showed significant improvements
in the NRS as well as the PD-Q scores. Different from these
studies, a small prospective open-label study [13] used the
PD-Q in addition to the NRS to evaluate cancer pain severity. That study included 46% patients without NeP components on
the basis of their PD-Q scores (i.e., <13). The experimental drug
improved the NRS scores significantly, but the results regarding
the PD-Q scores were not reported. Another open-label study on
patients with NeP [15] used the PD-Q not only to identify NeP
components (consequently, 18.3% of the patients had low PD-Q
scores [<13]) but also to evaluate its severity. However, that
study also did not present the results of the PD-Q scores over
time, because completion of the PD-Q was optional in that study. To our best knowledge, no data are available to suggest that the The PD-Q is an easy-to-administer patient self-reported
instrument for detecting NeP components without the necessity
of limited clinical examinations. Experiment 1 other subset of 14 patients with a low PD-Q score (<13) did not
demonstrate any correlations among the PD-Q and three-type
NRS scores (current pain, p=0.14; average pain in the past 4
weeks, p=0.88; and worst pain in the past 4 weeks, p=0.21). This article is available in: http://anaesthesia-painmedicine.imedpub.com/archive.php Experiment 1 *Data of the two subsets of patients were compared using the Mann-Whitney test or Chi-square test. Table 2 Demographic data of patients with neuropathic pain who participated in experiment 2. Table 2 Demographic data of patients with neuropathic pain who participated in experiment 2. PD-Q can appropriately reflect the severity of pain in patients
with a low PD-Q score (<13). At least, our present findings
indicated that the PD-Q in patients with a low PD-Q score should
be applied cautiously when strictly assessing and tracking pain
severity in clinical trials. This might be possibly supported by a
previous finding that the PD-Q score was less sensitive to reflect
pain severity and the analgesic effect of an experimental drug
in patients with a PD-Q score between 13 and 18 than in those
with a much higher PD-Q score (≥ 19) [10]. Moreover, supporting
evidence comes from another previous finding that patients with
a much higher PD-Q score (≥ 19) showed a larger effect size of
clinically meaningful improvements in all SF-12 subscale scores
used for assessing an experimental drug, but patients with a
PD-Q score between 13 and 18 showed improvements in limited
SF-12 subscale scores [8]. We previously reported that a higher
PD-Q score reflects stronger impairments of patients’ mental and
physical states in both patients with neuropathic and nociceptive
pain [7]. However, in our present study, the PD-Q score showed
such tendency, but did not linearly correlate with the SF-
36 subscale scores not only in patients with a low PD-Q score
(<13) but also in those with a high PD-Q score (≥ 13). Previous
epidemiological studies have revealed that patients with a
higher PD-Q score demonstrated worse health-related QOL, but
these studies did not necessarily confirm the linear correlation
between the PD-Q score and impairments of the health-related
QOL [25-27]. Therefore, the PD-Q should not serve as a surrogate
for assessing the health-related QOL in some circumstances
requiring its strict assessment, such as in clinical trials. other subset of 14 patients with a low PD-Q score (<13) did not
demonstrate any correlations among the PD-Q and three-type
NRS scores (current pain, p=0.14; average pain in the past 4
weeks, p=0.88; and worst pain in the past 4 weeks, p=0.21). References 1
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Questionnaire. JJSPC 14: 9-14. 22 Matsubayashi Y, Takeshita K, Sumitani M, Oshima Y, Tonosu J, et
al. (2015) Psychometric Validation of the Japanese Version of the
Neuropathic Pain Symptom Inventory. PLoS One 10: e0143350. 9
Baron R, Kern U, Müller M, Dubois C, Falke D, et al. (2015)
Effectiveness and Tolerability of a Moderate Dose of Tapentadol
Prolonged Release for Managing Severe, Chronic Low Back Pain with
a Neuropathic Component: An Open-label Continuation Arm of a
Randomized Phase 3b Study. Pain Pract 15: 471-486. 23 Fukuhara S, Ware JE, Kosinski M, Wada S, Gandek B (1998)
Psychometric and clinical tests of validity of the Japanese SF-36
Health Survey. J Clin Epidemiol 51: 1045-1053. 24 Feise RJ, Michael Menke J (2001) Functional rating index: a new valid
and reliable instrument to measure the magnitude of clinical change
in spinal conditions. Spine (Phila Pa 1976) 26: discussion 87. 10 Baron R., Likar R., Martin-Mola E., Blanco F.J., Kennes L., et al. (2016)
Effectiveness of Tapentadol Prolonged Release (PR) Compared with
Oxycodone/Naloxone PR for the Management of Severe Chronic
Low Back Pain with a Neuropathic Component: A Randomized,
Controlled, Open-Label, Phase 3b/4 Study. Pain Pract 16: 580-599. 25 Dimopoulou C, Athanasoulia AP, Hanisch E, Held S, Sprenger T, et
al. (2014) Clinical characteristics of pain in patients with pituitary
adenomas. Eur J Endocrinol 171: 581-591 11 Baron R, Martin-Mola E, Müller M, Dubois C, Falke D, et al. (2015)
Effectiveness and Safety of Tapentadol Prolonged Release (PR) Versus
a Combination of Tapentadol PR and Pregabalin for the Management
of Severe, Chronic Low Back Pain With a Neuropathic Component: A
Randomized, Double-blind, Phase 3b Study. Pain Pract 15: 455-470. 26 Valdes AM, Suokas AK, Doherty SA, Jenkins W, Doherty M (2014)
History of knee surgery is associated with higher prevalence of
neuropathic pain-like symptoms in patients with severe osteoarthritis
of the knee. Semin Arthritis Rheum 43: 588-592. 12 Schukro RP, Oehmke MJ, Geroldinger A, Heinze G, Kress HG, et
al. International Journal of Anesthesiology & Pain Medicine
ISSN 2471-982X Vol. 3 No. 1: 3 the Ministry of Education, Science, Sports and Culture (awarded
to M. Sumitani). The authors acknowledge Drs. Tonosu J, Kato
S, Ohya J, Oichi T, and Okamoto N for their valuable assistance. the Ministry of Education, Science, Sports and Culture (awarded
to M. Sumitani). The authors acknowledge Drs. Tonosu J, Kato
S, Ohya J, Oichi T, and Okamoto N for their valuable assistance. This study was supported by a Grant for Scientific Research from 13 Mercadante S, Porzio G, Ferrera P, Aielli F, Adile C, et al. (2012)
Tapentadol in cancer pain management: a prospective open-label
study. Curr Med Res Opin 28: 1775-1779. Discussion Our findings suggest that the
PD-Q can be suitable for assessing and tracking pain severity in
patients with NeP at least in common clinical settings. However,
clinicians and clinical researchers should pay sufficient attention
when using the PD-Q as an outcome measure in clinical trials. In particular, it would not be accurate to use the PD-Q score
to assess the pain severity among patients with NeP and a low
PD-Q score (<13). This study was retrospectively analyzed for
exploratory purposes in a limited number of participants. Our
findings should be confirmed in prospective studies with a larger
number of participants. 4 This article is available in: http://anaesthesia-painmedicine.imedpub.com/archive.php 2017 Acknowledgement This study was supported by a Grant for Scientific Research from References (2016) Efficacy of Duloxetine in Chronic Low Back Pain with a
Neuropathic Component: A Randomized, Double-blind, Placebo-
controlled Crossover Trial. Anesthesiology 124: 150-158. 27 Hiyama A, Watanabe M, Katoh H, Sato M, Sakai D, et al. (2015)
Evaluation of quality of life and neuropathic pain in patients with
low back pain using the Japanese Orthopedic Association Back Pain
Evaluation Questionnaire. Eur Spine J 24: 503-512. 5 © Under License of Creative Commons Attribution 3.0 License
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Plasticity of the Systemic Inflammatory Response to Acute Infection during Critical Illness: Development of the Riboleukogram
|
PloS one
| 2,008
|
cc-by
| 11,918
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Abstract Funding: This work was supported in part by NIH GM075023 (JPC), NIH GM008795 (JEM), NSF IIS-0535257 (WZ), the American College of Surgeons Clowes Career
Development Award (JPC), a Barnes-Jewish Hospital Foundation Research Award (JPC), and an institutional grant from Washington University in St. Louis (JEM). Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. ¤a Current address: Pacific Northwest National Laboratory, Richland, Washington, United States of America
¤b Current address: Department of Mathematics and Statistics, Texas Tech University, Lubbock, Texas, United States of America .These authors contributed equally to this work. has been described as the interplay between pro- and anti-
inflammatory responses [3], although recent evidence suggests a
mixed inflammatory state is common [4,5]. While this process has
been qualitatively described, there are no quantitative diagnostic
or prognostic tools that have been validated clinically to assess
immune status in the critically ill [6]. Consequently, infectious
complications are not only common in intensive care units but also
difficult to diagnose [7]. This has contributed to inappropriate use Plasticity of the Systemic Inflammatory Response to
Acute Infection during Critical Illness: Development of
the Riboleukogram Jonathan E. McDunn1,2., Kareem D. Husain1., Ashoka D. Polpitiya1.¤a, Anton Burykin1, Jianhua Ruan3,
Qing Li4, William Schierding1, Nan Lin4, David Dixon1, Weixiong Zhang3,5, Craig M. Coopersmith1,2, W. Michael Dunne6, Marco Colonna7, Bijoy K. Ghosh8¤b, J. Perren Cobb1,5* 1 Center for Critical Illness and Health Engineering, Department of Surgery, Washington University in St. Louis, St. Louis, Missouri, United States of America, 2 Department
of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri, United States of America, 3 Department of Computer Science and Engineering, Washington
University in St. Louis, St. Louis, Missouri, United States of America, 4 Department of Mathematics, Washington University in St. Louis, St. Louis, Missouri, United States of
America, 5 Department of Genetics, Washington University in St. Louis, St. Louis, Missouri, United States of America, 6 Department of Molecular Microbiology, Washington
University in St. Louis, St. Louis, Missouri, United States of America, 7 Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, Missouri,
United States of America, 8 Department of Electrical and Systems Engineering, Washington University in St. Louis, St. Louis, Missouri, United States of America Abstract Background: Diagnosis of acute infection in the critically ill remains a challenge. We hypothesized that circulating leukocyte
transcriptional profiles can be used to monitor the host response to and recovery from infection complicating critical illness. Methodology/Principal Findings: A translational research approach was employed. Fifteen mice underwent intratracheal
injections of live P. aeruginosa, P. aeruginosa endotoxin, live S. pneumoniae, or normal saline. At 24 hours after injury,
GeneChip microarray analysis of circulating buffy coat RNA identified 219 genes that distinguished between the pulmonary
insults and differences in 7-day mortality. Similarly, buffy coat microarray expression profiles were generated from 27
mechanically ventilated patients every two days for up to three weeks. Significant heterogeneity of VAP microarray profiles
was observed secondary to patient ethnicity, age, and gender, yet 85 genes were identified with consistent changes in
abundance during the seven days bracketing the diagnosis of VAP. Principal components analysis of these 85 genes
appeared to differentiate between the responses of subjects who did versus those who did not develop VAP, as defined by a
general trajectory (riboleukogram) for the onset and resolution of VAP. As patients recovered from critical illness
complicated by acute infection, the riboleukograms converged, consistent with an immune attractor. Conclusions/Significance: Here we present the culmination of a mouse pneumonia study, demonstrating for the first time
that disease trajectories derived from microarray expression profiles can be used to quantitatively track the clinical course of
acute disease and identify a state of immune recovery. These data suggest that the onset of an infection-specific
transcriptional program may precede the clinical diagnosis of pneumonia in patients. Moreover, riboleukograms may help
explain variance in the host response due to differences in ethnic background, gender, and pathogen. Prospective clinical
trials are indicated to validate our results and test the clinical utility of riboleukograms. Citation: McDunn JE, Husain KD, Polpitiya AD, Burykin A, Ruan J, et al (2008) Plasticity of the Systemic Inflammatory Response to Acute Infection during Critical
Illness: Development of the Riboleukogram. PLoS ONE 3(2): e1564. doi:10.1371/journal.pone.0001564 Received September 24, 2007; Accepted December 12, 2007; Published February 13, 2008 Received September 24, 2007; Accepted December 12, 2007; Published February 13, 2008 Copyright: 2008 McDunn 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 | www.plosone.org Citation: McDunn JE, Husain KD, Polpitiya AD, Burykin A, Ruan J, et al (2008) Plasticity of the Systemic Inflammatory Response to Acute Infection during Critical
Illness: Development of the Riboleukogram. PLoS ONE 3(2): e1564. doi:10.1371/journal.pone.0001564 Target cRNA and gene expression signal g
g
p
g
Each RNA sample was run on one GeneChip (a total of 15
mouse blood GeneChips from 120 animals). Total RNA from
mouse blood was extracted as previously described [19]. cRNA
target for GeneChip hybridization was prepared from total RNA
(Affymetrix, Santa Clara, CA). Both total RNA and cRNA were
electrophoretically assessed for quality (Agilent Bioanalyzer). The
mouse blood cRNA samples were hybridized with the U74Av2
GeneChip (approximately 12,400 probe sets). Fluorescent hybrid-
ization signal was detected using a GeneChip Scanner 3000
(Affymetrix). These mouse microarray data (and those for patients,
see below) have been deposited in NCBI’s Gene Expression
Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are
accessible through GEO Series accession number GSE6377. Based upon our ability to diagnose abdominal sepsis in pilot
mouse studies [15], we hypothesized that the host response to
infection could not only differentiate between infected and non-
infected states, but could also be used clinically to differentiate
between the host response to infectious agents and to model the
host response to and recovery from infectious perturbations. Pneumonia was chosen as an immune system perturbation, given
its relative frequency and considerable cost in terms of patient
morbidity and health care expense [7,16]. A bench-to-bedside,
translational approach was employed to study the host response to
pneumonia in critically ill subjects, comparing the informational
content of standard clinical parameters and plasma cytokines to
changes in the RNA abundance in circulating leukocytes. Data analysis and statistical tests for differential
expression Expression values were calculated from GeneChip .cel files
using Robust Multichip Average (RMA) software [20]. Differen-
tially expressed genes were identified using a mixed-model analysis
of variance (ANOVA) and linear contrasts (PartekH InferTM
software) as previously reported [15]. Leave-one out cross-
validation (k-nearest neighbors, k = 2) was used to determine the
reproducibility within this experimental set. Principal components
analysis (PCA) was used to visually explore global effects for
genome-wide trends, unexpected effects, and outliers in the
expression data (PartekH ProTM software, www.partek.com). Expression values were calculated from GeneChip .cel files
using Robust Multichip Average (RMA) software [20]. Differen-
tially expressed genes were identified using a mixed-model analysis
of variance (ANOVA) and linear contrasts (PartekH InferTM
software) as previously reported [15]. Leave-one out cross-
validation (k-nearest neighbors, k = 2) was used to determine the
reproducibility within this experimental set. Principal components
analysis (PCA) was used to visually explore global effects for
genome-wide trends, unexpected effects, and outliers in the
expression data (PartekH ProTM software, www.partek.com). Development of Riboleukogram Development of Riboleukogram of broad-spectrum antibiotics and the emergence of multi-drug
resistant organisms [8,9]. diluted 1:1 with normal saline, pooled for the 8 animals in each
treatment group, and separated into cells and plasma. Plasma was
stored at 280uC until use. Erythrocytes were lysed hypotonically
and RNA from peripheral leukocytes was harvested using RLT
(Qiagen) and stored at 280uC until use. The 24 hour time point
after injury was chosen as a time before appreciable mortality
develops in animals with significant lung injury. A few years ago, studies employing cultured human cells
suggested that instead of a single molecule (e.g., IL-6), a
constellation
of
molecules
could
be
used
to
monitor
the
complexities of the inflammatory response, serving as markers of
infection [10,11]. Miniaturized, multiplexed assays provide a rapid
method for the unbiased screening of thousands of molecular
species in a single assay [12]. These technological advances
provided the potential for investigators to leverage high-through-
put assays to better study the host response to and recovery from
critical illness and injury [13,14]. Improved molecular diagnostics
and prognostics, a better understanding of the complexity of
systemic inflammation, and new therapeutic targets are expected
deliverables, as reviewed recently [4,12]. Introduction Critical illness is marked by organ dysfunction, the need for vital
support, and a high risk of death, occurring against a backdrop of
systemic immune activation. This immune activation may begin as
an adaptive response to the initial injury, however, as the disease
progresses, the immune response may become maladaptive or
paralyzed [1,2]. Critical illness-associated immune dysregulation February 2008 | Volume 3 | Issue 2 | e1564 1 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org Methods Mice, experimental procedures, and samples Care and use of laboratory animals were conducted in
accordance
with
a
protocol
approved
by
the
Washington
University Animal Studies Committee, in compliance with
guidelines (N01-RR-2-2135) prepared by the Committee on Care
and Use of Laboratory Animals, Division of Research Resources,
National Institutes of Health. Seven to nine week-old, male
C57BL/6 mice were purchased (Harlan, Inc. Madison WI) and
allowed to acclimatize for at least one week in a temperature- and
light-controlled, pathogen-free barrier facility. Treated animals
and concurrently studied controls were observed at 24 hour
intervals for survival over eight days. In additional cohorts, whole
blood was collected at 24 hours after injury. p
(
,
p
)
Patient studies. After obtaining informed consent, venous
blood (7 ml) was collected from mechanically ventilated, non-
septic
patients
according
to
a
protocol
approved
by
the
Washington
University Institutional
Review Board (#2004-
0294). Patients were candidates for enrollment if they were on
mechanical
ventilation
in
the
surgical
ICU
medical
ICU,
neurological ICU, or cardiothoracic ICU (CTICU) for 48 hours,
were expected by the attending ICU physician to need mechanical
ventilation for at least another 48 hours at the time of enrollment,
and could provide written informed consent (from the patient or
legal surrogate). VAP was diagnosed by the ICU attending
physician, consistent with recently reported recommendations
[16], without input from the investigators. Clinical data were
entered
into
a
VAP
database,
including
gender,
ethnic
background, age, admitting diagnosis, type of ICU, APACHE II
score, airway sampling technique and culture results, initial
antibiotic therapy, and maximal clinical pulmonary infection
score (CPIS) calculated based upon available data (several patients
lacked daily arterial blood gas measurements to calculate PaO2/
FiO2 ratios) [21]. Patient blood was processed as described
previously to minimize red blood cell RNA artifact [13]; briefly,
samples were centrifuged (4006g 109 RT) to form a buffy coat and
to separate plasma from cells. Plasma was withdrawn and stored at
280uC until use. Blood cells were diluted into EL buffer (90 ml)
(Qiagen) and incubated on ice for 159. Leukocytes were pelleted by
centrifugation (4006g 109 4uC), washed with EL buffer (30 ml)
and
lysed
into
RLT
buffer
(Qiagen)
containing
1%
b-
mercaptoethanol. Genomic DNA was sheared by repeated Patient studies. Development of Riboleukogram Online databases were used to determine
gene annotation and functional categorization (DAVID 2.0
accessed 16 November 2006) [24]. Patient
blood
leukocyte
mRNA
profiling. RNA
was
extracted, amplified and assessed for quality as described for
murine samples. cRNA was hybridized against the HG-FOCUS
array (Affymetrix, .8700 probe sets encoding ,8400 genes) and
imaged as described for murine samples. Orthologs of murine
genes were identified by comparison of the GeneChip Identifiers
using the NetAffx Toolkit (Affymetrix). Consistent with recently
published consensus statements [16,21], clinical data were judged
to determine when (if ever) each patient developed ventilator-
associated pneumonia, with each patient acting as her/his own
control. The timeline for each patient was defined such that the
day of VAP diagnosis by the ICU attending physician was defined
as day 0. A seven day time window from the gene expression time
series was chosen as days 23, 21, 0, 1, and 3, with day 0 being the
day that a patient was diagnosed as having VAP by the attending
physician. Because blood samples were collected every other day,
patients’ samples were collected either on days 22, 0, +2 or on
days 23, 21, +1, +3 relative to the VAP day of diagnosis
described as time 0. For the purpose of analyzing the data from
those patients who had samples collected on ‘‘odd’’ days, the time
0 data for these patients were interpolated. Those mRNA species
whose abundance changed concordantly among the patients
during the 7-day window surrounding the date of diagnosis were
identified
using
extraction
of
Differential
Gene
Expression
(EDGE) software [23]. Online databases were used to determine
gene annotation and functional categorization (DAVID 2.0
accessed 16 November 2006) [24]. where ai k
ð Þ~Sx k
ð Þ,yiT and Æ?,?æ stands for the standard inner
product. The orthonormal basis y1,y2,…yN can be selected as the
eigenvectors of the correlation matrix C1MRN6N, obtained as C1~ 1
NT
X
NT
k~1
x k
ð Þ
ð
Þ x k
ð Þ
ð
ÞT: The first principal mode y1 corresponds to a constant bias term. Hence the most important variation is captured by y2,y3,…yN
respectively in the decreasing order. Once the orthonormal basis is
obtained, each patient data can be projected onto this basis as
aj
i k
ð Þ~Sxj k
ð Þ,yiT for patients j = 1, 2, … . Development of Riboleukogram of the time series with respect to day 0 of VAP, we first obtained
the average expression vectors x k
ð Þ, k = 1,2,…9, by averaging the
expression values at days corresponding to 23,21,…,13 in all
patients (corresponding to the nine time points that most patients
had samples collected). The KLD method looks for a basis
y1,y2,…yN so that one can expand x k
ð Þ as passage through an 18 gauge needle and the resultant material was
stored at 280uC. Patient plasma cytokine analysis. Cytokines (GRO-a,
IFN-c; IL1-b; IL1Ra; IL1sr2; IL4; IL6; IL8; IL10; IL12; IL18;
MCP1; MIP1a; MIP1b; NGF; RANTES; TNFa; TNF-sr1; TNF-
sr2) were measured using a microarray immunoassay as previously
described [22]. Procalcitonin was measured according to the
manufacturer’s instruction (BRAHMS PCT LIA kit, Product
number 354.1). x k
ð Þ~ lim
N?? X
N
i~1
ai k
ð Þyi Patient
blood
leukocyte
mRNA
profiling. RNA
was
extracted, amplified and assessed for quality as described for
murine samples. cRNA was hybridized against the HG-FOCUS
array (Affymetrix, .8700 probe sets encoding ,8400 genes) and
imaged as described for murine samples. Orthologs of murine
genes were identified by comparison of the GeneChip Identifiers
using the NetAffx Toolkit (Affymetrix). Consistent with recently
published consensus statements [16,21], clinical data were judged
to determine when (if ever) each patient developed ventilator-
associated pneumonia, with each patient acting as her/his own
control. The timeline for each patient was defined such that the
day of VAP diagnosis by the ICU attending physician was defined
as day 0. A seven day time window from the gene expression time
series was chosen as days 23, 21, 0, 1, and 3, with day 0 being the
day that a patient was diagnosed as having VAP by the attending
physician. Because blood samples were collected every other day,
patients’ samples were collected either on days 22, 0, +2 or on
days 23, 21, +1, +3 relative to the VAP day of diagnosis
described as time 0. For the purpose of analyzing the data from
those patients who had samples collected on ‘‘odd’’ days, the time
0 data for these patients were interpolated. Those mRNA species
whose abundance changed concordantly among the patients
during the 7-day window surrounding the date of diagnosis were
identified
using
extraction
of
Differential
Gene
Expression
(EDGE) software [23]. Development of Riboleukogram The discrete derivative
of the coefficients aj
i k
ð Þ at time k is obtained as aj
i kz1
ð
Þ{aj
i k
ð Þ: Validation
of
microarray
results. Select
genes
were
subjected
to
real-time
quantitative
PCR
(RTq-PCR)
for
independent confirmation of relative expression levels. cDNA
was generated and 100 ng was subjected to routine SybrGreen
RT-PCR as per manufacturer’s instruction (Applied Biosystems,
Foster City, CA). In addition, 85 genes were selected at random
from the total number on the GeneChip. The gene expression
signal from these genes was analyzed in a manner identical to that
described above. This procedure was repeated 100 times to
estimate the informational value of randomly selected genes. Clustering algorithm. Patient genes identified by EDGE
were clustered as described previously [25]. Briefly, a gene co-
expression network was constructed by connecting every gene to
the top d genes (d = 5 in this study) to which its expression profile is
most similar. The network then was partitioned into a set of
communities, i.e., relatively densely connected sub-graphs, by a
spectral graph algorithm [26]. The genes within each community
formed a cluster. The number of clusters was determined
automatically
by
the
algorithm
in
order
to
maximize
a
modularity score [25,26]. Gene expression data were normalized
prior to clustering such that the expression levels of each gene for
each patient had a mean of zero and a standard deviation of one. Similarity between two gene expression profiles was measured by
Pearson correlation coefficient. Methods After obtaining informed consent, venous
blood (7 ml) was collected from mechanically ventilated, non-
septic
patients
according
to
a
protocol
approved
by
the
Washington
University Institutional
Review Board (#2004-
0294). Patients were candidates for enrollment if they were on
mechanical
ventilation
in
the
surgical
ICU
medical
ICU,
neurological ICU, or cardiothoracic ICU (CTICU) for 48 hours,
were expected by the attending ICU physician to need mechanical
ventilation for at least another 48 hours at the time of enrollment,
d
ld
id
i
i f
d
(f
h
i The 5 experimental groups were selected to reflect clinically
important distinctions relevant to care of ICU patients: high
mortality Gram-negative pneumonia with Pseudomonas aeruginosa
(40 ml of 0.3 McFarland culture, 90% 7-day mortality (,2–46107
organisms)),
high
mortality
Gram-positive
pneumonia
with
Streptococcus pneumoniae (60 ml of 0.5 McFarland culture, 85% 7-
day mortality (,1.8–3.66107 organisms)), and moderate mortality
Gram-negative pneumonia with Pseudomonas aeruginosa (20 ml of 0.1
McFarland culture, 50% 7-day mortality (,2–46106 organisms)). To
induce severe
systemic inflammation without infection,
intratracheal (i.t.) injection of P. aeruginosa lipopolysaccharide
(500 mg in 50 ml 0.9% normal saline; Sigma, St. Louis) was
performed (LPS group, 90% 7-day mortality). Mice injected i.t. with normal saline vehicle acted as the concurrent control group
(saline group, 0% 7-day mortality). Previously reported protocols
were used to intratracheally instill fluid into the lung [17,18]. The
census of surviving mice was recorded at 24-hour intervals for
seven days. In three additional cohorts of animals, blood was collected into
an EDTA-coated syringe from the inferior vena cava being careful
to avoid contamination of the needle with other tissues. Blood was PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 2 Validation of the riboleukogram After enrollment of the first 20 patients, a second cohort of 7
patients was analyzed to validate the informational content of the
leukocyte RNA species (genes) that changed in abundance in
response to critical illness complicated by VAP. The blood
handling, processing, and GeneChip analysis protocols were
identical to those described above. Results Karhunen-Loeve
decomposition
of
microarray
gene
expression data. To determine the dynamics of the host
response to pneumonia, we constructed first a raw gene expression
matrix corresponding to the ith pneumonia patient after RMA
normalization [20] to be Xi = [xi(1) … xi(NT)] where xi(k)MRN,
(k = 1,…,NT, i = 1,…11, and N = 8793 genes) is the gene expression
vector at k th time point. k = 1,…,NT where NT = 11 for most
patients, correspond to sample collecting days 1, 3, 5, …, 21. Note
that for few patients, only a portion of the time series (i.e., less than
11 points) was available. PLoS ONE | www.plosone.org Murine model Intratracheal (i.t.) installation of saline caused no deaths over 7-
days whereas i.t. introduction of P. aeruginosa endotoxin resulted in
death of 90% of the animals studied within 96 hours (Figure 1A). The 7-day mortality caused by live P. aeruginosa was adjusted by
varying the size of the inoculum and injuries causing 50% and
90% 7-day mortality were achieved. A dose of S. pneumoniae was
given i.t. that resulted in 85% 7-day mortality. Once these injuries
were established, three separate cohorts of mice were used for each
experimental group in the subsequent studies. For the EDGE-selected genes, the data were projected onto a
smaller dimensional space using the series expansion method
similar
to
principal
components
analysis,
Karhunen-Loeve
Decomposition (KLD) [27]. In order to preserve the alignment Peripheral blood was collected and pooled from groups of 4–7
mice 24 h after surgery, prior to appreciable mortality in any
group (Figure 1A). All murine RNA were of good quality based on February 2008 | Volume 3 | Issue 2 | e1564 February 2008 | Volume 3 | Issue 2 | e1564 3 Development of Riboleukogram Figure 1. (A) Eight-day survival curves of mice challenged with intra-tracheal injection of one of 5 solutions, each dosed to produce the observed
mortality. Significant differences (p,0.05) were observed between the 80–90% mortality (Pseudomonas bacteria, Streptococcus bacteria, and
Pseudomonas LPS) versus 50% mortality (Pseudomonas bacteria) versus 0% mortality groups. Blood samples were obtained at 24 hours (red arrow), a
time prior to appreciable mortality. (B) Clustering of the 219 probe sets that differentiated the five treatment groups separated the probe sets into six
clusters. (C) Co-expression network analysis of these six mouse gene clusters were used to explore the gene expression cartography of the leukocyte
response to pneumonia. Genes in common with the human coexpression network (Figure 3B) are circled. (D) Principal components (PC) analysis of an
algorithm-selected subset of the 219 probe sets whose microarray-measured RNA abundance in leukocytes isolated 24 h after the onset of
pneumonia. PC2 appeared to explain in part expression signal variance due to mortality rates, while PC3 explained in part the variance due to type of
insult. doi:10 1371/journal pone 0001564 g001 Figure 1. (A) Eight-day survival curves of mice challenged with intra-tracheal injection of one of 5 solutions, each dosed to produce the observed
mortality. Murine model Significant differences (p,0.05) were observed between the 80–90% mortality (Pseudomonas bacteria, Streptococcus bacteria, and
Pseudomonas LPS) versus 50% mortality (Pseudomonas bacteria) versus 0% mortality groups. Blood samples were obtained at 24 hours (red arrow), a
time prior to appreciable mortality. (B) Clustering of the 219 probe sets that differentiated the five treatment groups separated the probe sets into six
clusters. (C) Co-expression network analysis of these six mouse gene clusters were used to explore the gene expression cartography of the leukocyte
response to pneumonia. Genes in common with the human coexpression network (Figure 3B) are circled. (D) Principal components (PC) analysis of an
algorithm-selected subset of the 219 probe sets whose microarray-measured RNA abundance in leukocytes isolated 24 h after the onset of
pneumonia. PC2 appeared to explain in part expression signal variance due to mortality rates, while PC3 explained in part the variance due to type of
insult. doi 10 1371/jo rnal pone 0001564 g001 doi:10.1371/journal.pone.0001564.g001 the peak profiles of 18S and 28S ribosomal RNA. cRNA
generated from these samples had a uniform size distribution. All hybridizations were of good quality; both the number of
features present (35–40%) and the signals on each array fell within
acceptable ranges [28]. bacteria, Gram-negative bacterial toxin (LPS) and Gram-positive
bacteria. The probe sets clustered into six groups (Figure 1B) and
these groups defined the gene expression cartography of the
murine response to pneumonia (Figure 1C). Genes that fell within
clusters 2 and 3 exhibited increased RNA abundance in animals
responding to high lethality insults. Genes that fell within clusters
4–6 were transcriptionally suppressed during high lethality insults. Cluster 1 bridged the two groups. Gene ontology assignments
identified enriched molecular functions in the three distinct
groups. The bridging cluster (cluster 1) was enriched for genes
involved in the immune response (N = 10, P = 461027) and genes
with NTPase activity (N = 6, P = 561024). Clusters 2 and 3 were
enriched for genes involved in intracellular signaling pathways Analysis of normalized gene expression data identified 219
probe sets (40 unannotated ESTs, 10 redundant probe sets, 169
annotated genes (Table S1) whose expression levels differentiated
between the five groups. Leave one out cross-validation using k-
nearest neighbors (k = 2) resulted in a 93% classification accuracy. The single misclassified sample was from the low-dose P. aeruginosa
infection and was classified as ‘‘saline’’. PLoS ONE | www.plosone.org Clinical study—training cohort Based on patient demographics (Table 1 and data not shown), the
only clear difference between patients 7 and 11 and the other
patients in the study is that these two patients developed VAP later
in their ICU course (study day 18 and 14 respectively) whereas the
remaining patients developed VAP between study days 3 and 6. This was also evident in PCA analysis of the 11 individual
trajectories (data not shown). The changes in transcript abundance
of selected genes were validated by quantitative RT-PCR (Figure
S1). Finally, we tested whether host ethnicity, host gender, host
age, or the cell wall phenotype of the infectious agent had an effect
on the number of informational genes. The most informational of
these demographic variables was host ethnicity (Figure 4). g
RNA isolated from patient samples was of high quality and
hybridizations met standard performance criteria (vide supra). To
assess whether the genes identified in the murine model conveyed
information in the patient study, the microarray abundance of the
human orthologs of the 219 genes that distinguished the murine
pneumonias were numerically analyzed. Principal components
analysis of the average RMA-normalized expression levels of these
109 ortholog genes resulted in gene expression trajectories that
described the cohort of patients as they developed, were treated
for, and recovered from VAP (Figure 2A). Trajectory translocation
along the X-coordinate (principal component 2) appeared to be
informative with regard to the onset of VAP – beginning
immediately before the diagnosis and ending approximately six
days after appropriate antibiotic therapy was initiated. Principal
components analysis of plasma cytokine abundance in these
patients showed a qualitatively similar trajectory, but with large
error bars (Figure 2B). Nevertheless, translocation along the X-
coordinate (principal component 2) again appeared to coincide
with the onset of VAP. We observed in Figure 3C that the aggregate riboleukogram
variance in principal component 2 decreased as patients recovered
from acute infection. This finding suggested that principal
components analysis of microarray-measured gene expression
described an attractor, as gene expression time series can be
described in terms of dynamical system theory as trajectories in the
phase space defined by the main principal components. By
plotting the change in PC2 against PC2 over time, we found
indeed that the mapped gene expression information appeared to
converge toward a common point, suggesting that PC2 represents
the
expression
of
the
infection-inducible
genes
(Figure
5). Clinical study—training cohort y
g
After being mechanically ventilated for .48 h, 27 patients were
enrolled into the study. The first 20 patients enrolled were assigned
arbitrarily to a training cohort; the other 7 were assigned to a
validation cohort. Blood samples were taken at ,48-hour intervals
during the study period and then separated into plasma and
leukocytes (see below). Of the 20 patients in the training cohort,
eight patients either were extubated without developing VAP or
withdrew from the study. Of the 12 patients in this cohort who
developed microbiologically-confirmed VAP (Table 1), 11 met our
analysis criteria of having samples before and after the attending
physician made a diagnosis of VAP (that is, one patient, #9, was
excluded from analysis for developing VAP on the study entry
day). Clinical pulmonary infection scores (CPIS) increased in all 11
patients coincident with the diagnosis of VAP. Three of the
patients were culture positive for a Gram positive agent (S. aureus)
and the remaining eight patients were culture positive for one or
more Gram negative agents. In every case, initial intravenous
antibiotic therapy was appropriate for the cultured organism
responsible for VAP, based upon cultured organism susceptibil-
ities. Nine of the 11 patients developed VAP 3–6 days after
enrollment in the study (‘‘early VAP’’) while two patients
developed VAP after prolonged mechanical ventilation (‘‘late
VAP’’, Table 1). All of the patients survived and were discharged
from the ICU. Patient-specific timelines were aligned for analysis
by assigning ‘‘day 0’’ to the day that the attending physician
diagnosed VAP. Principal components analysis of the microarray expression
profiles of these 85 genes defined a common response to
pulmonary infection complicating critical illness (Figure 3C). Importantly, trajectory translocation in the X-coordinate (princi-
pal component 2) occurred days prior to the clinical diagnosis of
VAP. In addition, the informational content of 85 genes chosen at
random was determined iteratively 100 times for the first 11 VAP
patients. As shown in Figure 3D, only the 85 genes identified by
EDGE as significant (FDR#0.10) produced a trajectory; the other
sets of genes were scattered randomly around the origin of the
graph. Three patients exhibited contrary gene expression profiles
within two of these four clusters (Figure 3A). Patients 1, 7 and 11
showed decreased expression of genes in cluster 2 and patients 7
and 11 showed increased expression of genes in clusters 3 and 4. Development of Riboleukogram generally increased and transcript abundance in clusters 3 and 4
generally decreased around the time of VAP diagnosis. (N = 18, P = 661027). Clusters 4–6 were enriched for genes that
encode nuclear proteins (N = 24, P = 961024). Principal compo-
nents analysis of the microarray-derived transcript abundances of
the genes selected based on cross-validation clearly differentiated
the five experimental groups (Figure 1D) based on the 7-day
mortality (principal component 1) and the type of agent used
(Gram positive, sterile, Gram negative, principal component 2). Molecular cartography of the human leukocyte transcriptional
response to acute bacterial infection identified two densely
connected networks of genes, the first containing clusters 1 and
2 and the second containing clusters 3 and 4 (Figure 3B). The 26
probe sets in clusters 1 and 2 are significantly enriched with GO
biological process terms: ‘‘defense response to bacteria’’ (8 genes,
p-value = 2610211), ‘‘response to biotic stimulus’’ (12 genes,
p = 461026), and ‘‘immune response’’ (8 genes, p = 0.002) and
the cellular compartment term ‘‘extracellular region’’ (14 genes,
p = 961029). The 59 probe sets in clusters 3 and 4 are enriched
with GO molecular function terms: ‘‘ATP binding’’ (13 genes,
p = 961024), ‘‘metal ion binding’’ (22 genes, p = 0.002), and
‘‘protein binding’’ (25 genes, p = 0.002) and the cellular compart-
ment terms ‘‘cytoplasm’’ (23 genes, p = 0.007) and ‘‘plasma
membrane’’ (13 genes, p = 0.02). Clinical study—training cohort Consistent with differences in patient age, gender, ethnicity, pre-
existing co-morbidities, and nature of injury insult, each patient’s
individual trajectory started at a different point and described a
patient-specific arc (data not shown). Independent analysis of patient microarray data resulted in the
identification
of
85 probe
sets whose
abundance
changed
significantly during the course of VAP (Table 2). Of the 109
human orthologs that were used to calculate the trajectories shown
in Figure 2A, 5 probe sets (4.6%) were present in the list of human
probe sets (lactotransferrin, cathelicidin antimicrobial peptide,
phospholipid scramblase 1, inhibin beta A, and hydroxyprosta-
glandin dehydrogenase 15-(NAD)). Network analysis found that
the expression behavior of these 85 genes segregated into four
clusters (Figure 3A). Transcript abundance in clusters 1 and 2 Murine model These 219 genes
differentiated between the host responses to Gram-negative PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 4 Development of Riboleukogram PLoS ONE | www.plosone.org Validation cohort A second cohort of 7 patients was analyzed to evaluate the
informational value of the 85 genes that were identified in the first
11 patients with VAP. Two of these 7 additional patients were February 2008 | Volume 3 | Issue 2 | e1564 PLoS ONE | www.plosone.org 5 Development of Riboleukogram Table 1. Patient Characteristics. Patient
No. Gender
Ethnicity Age
Admit
Diagnosis
ICU
APACHE II
Score
VAP day
Maximum
CPIS score
Culture Results
Initial Antibiotic
Therapy
Training Set
1
M
C
65
COPD
SICU
21
6
8*
Serratia marcescens
Cefepime, Fluconazole,
Metronidazole,
Vancomycin
2
M
C
21
MVC
SICU
24
6
6*
Acinetobacter
calcoaceticus-baumanii
and Serratia marcescens
Cefazolin, Cefepime,
Clindamycin
3
M
C
69
CHI
SICU
21
4
6*
Enterobacter aerogenes
Cefepime
4
F
C
49
OD
MICU
25
3
7
Staphylococcus aureus
Cefepime, Vancomycin
5
F
AA
76
AAA
SICU
23
4
9
Stenotrophomonas
maltophilia
Trimethoprim-
sulfamethoxazole
6
F
C
20
MVC
SICU
26
6
8
Staphylococcus aureus
Cefepime, Vancomycin
7
F
C
68
CABG
CTICU
24
19
7
Stenotrophomonas
maltophilia and
Pseudomonas aeruginosa
Cefepime
8
F
AA
44
PVD
SICU
37
5
6
Acinetobacter
calcoaceticus-baumanii
Imipenem-Cilastatin
9
M
C
62
AAA
SICU
21
1
5*
Enterobacter cloacae
Cefepime, Vancomycin
10
F
C
47
MVC
SICU
22
6
8
Escerischia coli,
Haemophilus influenzae
and Streptococcus
pneumoniae
Acyclovir, Vancomycin
11
F
AA
33
GSW
SICU
17
3
7
Haemophilus influenzae
Cefazolin, Piperacillin-
Tazobactam
12
M
C
27
MVC
SICU
16
14
5*
Staphylococcus auerus
Clindamycin
Validation Set
13
M
C
29
SAH
NICU
14
n.a. 6
Haemophilus influenzae,
acinetobacter
calcoaceticus-baumanii,
and Stenotrophomonas
maltophilia
Vancomycin,
Ciprofloxacin,
Trimethoprim-
sulfamethoxazole
14
F
C
80
CABG
CTICU
21
15
6
Klebsiella pneumoniae
Vancomycin, Cefepime,
Metronidazole,
Fluconazole
15
F
C
64
stroke
NICU
21
n.a. 6
Streptococcus
pneumoniae#
Penicillin then
Vancomycin
16
F
C
30
MVC
SICU
18
16
7
Staphylococcus aureus
Vancomycin, Cefepime
17
M
C
59
SAH/SDH
NICU
29
n.a. 5
Staphylococcus aureus
n.a. PLoS ONE | www.plosone.org Validation cohort Individual cytokines do not have significant changes in abundance during the time course of disease (P . 0.05 for all individual
proteins). doi:10 1371/journal pone 0001564 g002 Figure 2. (A) Principal components analysis of the leukocyte average relative RNA abundance of the 109 human orthologs to the 219 murine genes
identified in Supplemental Table S1, plotted for all eleven patients who developed VAP. The translation along principal component (PC) 2 appears to
be associated with the development and recovery from pneumonia. The red arrow indicates the day where the attending physician diagnosed VAP. The green circle indicates the point at which the patient entered the study; the red circle is the point at which the patient exited the study. (B)
Principal components analysis of the average absolute abundance of plasma cytokines and soluble receptors during the study period across all
eleven VAP patients. Individual cytokines do not have significant changes in abundance during the time course of disease (P . 0.05 for all individual
proteins). p
doi:10.1371/journal.pone.0001564.g002 Figure 6A inset). Patient 17 grew Staphylococcus aureus from both
urine and tracheal secretions prior to withdrawal of therapy for
cure (the only death in the study). In contrast, the riboleukograms
of patients 13, 18, and 19 are atypical, in that their paths do not
start and/or do not finish with the others. Both patients 18 and 19
had pulmonary contusions secondary to polysystem trauma,
maximal CPIS scores of 7 and 9, Gram-positive cocci cultured
from airway secretions, and were treated with antibiotics, but had
a clinical course labeled by the attending physician as ‘‘possible’’
VAP. Their riboleukograms are in different portions of the graph
in Figure 6A, but have a similar shape and slope. Both patients 13
and 18 had intracranial hemorrhage. Patient 13 was not diagnosed
with VAP (no infiltrate on CXR) but was treated with antibiotics
for a fever of 39.4uC and WBC of 31,300 (day 5), tracheal
secretions that subsequently grew out Acinetobacter and Steno-
trophomonas (CPIS 6), and concern for catheter-related sepsis. and non-infectious etiologies of systemic inflammation in a de-
identified cohort [15]. Thus, the mouse circulating leukocyte
transcriptional response to infection can not only distinguish
between infectious and non-infectious inflammatory insults, but
also the type of infectious agent and its associated mortality. Validation cohort 18
F
C
50
MVC
SICU
20
"possible"
7
Staphylococcus aureus
Vancomycin,
Ciprofloxacin,
Metronidazole,
Ceftriaxone
19
F
C
25
MVC
SICU
21
"possible"
9
Streptococcus anginosus
Cefazolin
M = Male, F = Female
C = Caucasian, AA = African-American
COPD = Chronic obstructive pulmonary disease, MVC = motor vehicle crash (trauma), CHI = closed head injury, OD = acetaminophen overdose, AAA = abdominal aortic
aneurysm, CABG = coronary artery bypass grafting, PVD = peripheral vascular disease, GSW = gun shot wound, CVA = cerebrovascular accident, CABG = coronary bypass
grafting, SAH = subarachnoid hemorrhage, SDH = subdural hemorrhage
culture results are from airway specimens obtained invasively (tracheal secretions or bronchoalveolar lavage)
# indicates bacterium intermediate sensitivity or insensitive to initial antibiotic treatment
CPIS = clinical pulmonary infection scores (* indicates missing P/F ratios)
n.a. = not applicable
doi:10.1371/journal.pone.0001564.t001 left to right, that is, from critical illness to recovery (Figure 6, green
and red shaded areas, respectively). The development of an
infectious complication is typically associated with a change in
riboleukogram trajectory. For example, the paths of patients 13,
14, 15, 16, and 17 change directions abruptly coincident with
changes in clinical status and concern for VAP or sepsis (see diagnosed with ‘‘late’’ VAP by the attending physician, while
another 2 cases were described by the attending as ‘‘possible’’ VAP
(Table 1). The individual riboleukograms for these 7 patients
demonstrate
the
existence
of
immune
recovery
(basins
of
attraction) as well as the heterogeneity of the host response. In
general, the individual riboleukograms follow a path moving from PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 6 Development of Riboleukogram Figure 2. (A) Principal components analysis of the leukocyte average relative RNA abundance of the 109 human orthologs to the 219 murine genes
identified in Supplemental Table S1, plotted for all eleven patients who developed VAP. The translation along principal component (PC) 2 appears to
be associated with the development and recovery from pneumonia. The red arrow indicates the day where the attending physician diagnosed VAP. The green circle indicates the point at which the patient entered the study; the red circle is the point at which the patient exited the study. (B)
Principal components analysis of the average absolute abundance of plasma cytokines and soluble receptors during the study period across all
eleven VAP patients. Validation cohort Network analysis suggested that the pneumonia-induced tran-
scriptional
changes
reprioritized
mouse
leukocytes
for
the
initiation of an immune response, the transcriptional regulation
of intracellular signaling cascades, and the induction of numerous
transcription factors and other nuclear genes. p
g
Results from the mouse model suggested that the transcriptional
activity of buffy coat-isolated cells may be used to monitor the
onset of and recovery from acute infection. We tested this
hypothesis by calculating principal components using microarray
expression profiles of RNA isolated from mechanically ventilated
patients at risk for pneumonia. Initially, we examined the behavior
of the human orthologs to the genes identified in the mouse
pneumonia study. The onset of acute infection corresponded with
translation along PC 2 in Figure 2. Translation along PC2 ceased
5–6 days after appropriate antibiotic therapy in the patients was
started, consistent with recovery. These data show that there are
specific transcriptional programs instituted by circulating immune
cells during acute infection which have diagnostic potential in the
setting of critical illness. Principal components analysis of plasma
cytokine abundance generated a qualitatively similar trajectory;
however, in line with previous reports, plasma cytokine abundance
(including
procalcitonin)
was
insufficient
to
diagnose
acute
infection in this small cohort of critically ill patients. As with
other tissues, changes in RNA abundance observed in circulating
leukocyte do not necessarily reflect changes in protein abundance,
and vice versa. While principal components analysis of informa-
tional murine genes in authentic human disease showed there was
a conserved and informational peripheral leukocyte transcriptional
response to localized infection, the information contained in those
genes would not appear to be more useful than current clinical In Figure 6B, the aggregate riboleukogram for the training
cohort of VAP patients (aligned for day of VAP diagnosis, see also
Figure 3C) is compared to the aggregate riboleukogram for all
patients aligned for day of study entry (that is, both training and
validation cohorts, N = 11+7, irrespective of VAP day). Again
noted are the PCA domains of critical illness and recovery. The
aggregate VAP riboleukogram diverges from the aggregate critical
illness riboleukogram and rejoins the latter at the point of
recovery. PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 Discussion Using a bench-to-bedside approach, we have implemented a
mouse model of pneumonia and found that RNA abundance
profiles obtained from blood samples taken prior to appreciable
mortality were able to distinguish between the two variables tested
in the assay: lethality of the insult and type of infectious agent. These data extend our previous observations in a mouse model of
abdominal sepsis, wherein microarray-measured expression pro-
files from circulating leukocytes distinguished between infectious PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 February 2008 | Volume 3 | Issue 2 | e1564 7 Development of Riboleukogram Table 2. Leukocyte Genes Altered by Ventilator-associated Pneumonia. Probe Set ID
Gene Symbol
P-Value
Q-Value
Fold Change*
202018_s_at
LOC728320///LTF
2.27E-06
0.004432733
1.464144796
207269_at
DEFA4
2.27E-06
0.004432733
1.518640915
210244_at
CAMP
1.14E-06
0.004432733
1.409490494
212531_at
LCN2
2.27E-06
0.004432733
1.427762858
206676_at
CEACAM8
4.55E-06
0.005910311
1.504273574
212063_at
CD44
4.55E-06
0.005910311
0.936827878
218095_s_at
TMEM165
6.82E-06
0.007598972
0.929587436
205033_s_at
DEFA1///DEFA3///LOC653600///LOC728358
1.48E-05
0.012805674
1.181241234
205504_at
BTK
1.48E-05
0.012805674
0.935491658
204860_s_at
LOC728519///NAIP
2.62E-05
0.018536885
0.848248535
210254_at
MS4A3
2.62E-05
0.018536885
1.441322498
204689_at
HHEX
3.41E-05
0.022163667
0.923345636
205016_at
TGFA
3.75E-05
0.022504646
0.894038802
202076_at
BIRC2
4.44E-05
0.024696657
0.968353651
207384_at
PGLYRP1
5.00E-05
0.026005369
1.152667764
207802_at
CRISP3
5.35E-05
0.026042309
1.603981321
210452_x_at
CYP4F2
6.03E-05
0.027639396
1.099086675
200665_s_at
SPARC
8.19E-05
0.029820206
1.169183993
205557_at
BPI
8.19E-05
0.029820206
1.359468336
205896_at
SLC22A4
8.42E-05
0.029820206
0.903961604
209369_at
ANXA3
8.30E-05
0.029820206
0.945826874
214146_s_at
PPBP
7.39E-05
0.029820206
1.120758241
209205_s_at
LMO4
9.44E-05
0.031992771
0.932224575
219375_at
CEPT1
0.0001126
0.03657005
0.94093974
209193_at
PIM1
0.0001228
0.038298816
0.929182785
211200_s_at
EFCAB2
0.0001331
0.038417023
0.953743854
217788_s_at
GALNT2
0.0001331
0.038417023
0.851597039
219628_at
ZMAT3
0.0001387
0.038628105
0.934196425
202464_s_at
PFKFB3
0.0001478
0.039741748
0.90582982
204068_at
STK3
0.0001933
0.044327334
0.888056674
204554_at
PPP1R3D
0.0001786
0.044327334
0.936918267
210140_at
CST7
0.0001774
0.044327334
0.935788854
212136_at
ATP2B4
0.0001876
0.044327334
0.908700634
39402_at
IL1B
0.0001831
0.044327334
0.895031859
201798_s_at
FER1L3
0.0002138
0.046244191
0.838135068
206488_s_at
CD36
0.0002172
0.046244191
0.898756001
219607_s_at
MS4A4A
0.0002195
0.046244191
0.8064475
203757_s_at
CEACAM6
0.000232
0.047593558
1.342306036
200673_at
LAPTM4A
0.0002638
0.052738161
0.954754301
202530_at
MAPK14
0.0003548
0.059983371
0.956537364
204099_at
SMARCD3
0.0003435
0.059983371
1.108065397
204225_at
HDAC4
0.0003537
0.059983371
0.927059336
204620_s_at
VCAN
0.0003594
0.059983371
0.917399007
205074_at
SLC22A5
0.0003184
0.059983371
0.926045842
206565_x_at
SMA3
0.0003491
0.059983371
0.837007379
212592_at
IGJ
0.0003617
0.059983371
1.196011695
212737_at
GM2A
0.0003514
0.059983371
0.914644647
209288_s_at
CDC42EP3
0.0003719
0.060395992
0.96733788
205098_at
CCR1
0.0003878
0.06134903
0.925839976
205110_s_at
FGF13
0.0003935
0.06134903
0.832214064 PLoS ONE | www.plosone.org 8 Development of Riboleukogram Table 2. Cont. Discussion Probe Set ID
Gene Symbol
P-Value
Q-Value
Fold Change*
201432_at
CAT
0.0004162
0.062399247
0.96794903
205513_at
TCN1
0.0004162
0.062399247
1.301185411
202087_s_at
CTSL1
0.0004322
0.062550793
0.935466979
206838_at
TBX19
0.0004333
0.062550793
0.940074355
202437_s_at
CYP1B1
0.0004515
0.063992551
0.874011817
206493_at
ITGA2B
0.0004708
0.065541129
1.140129423
202381_at
ADAM9
0.0005061
0.066195485
0.896803767
208881_x_at
IDI1
0.0005095
0.066195485
0.919817185
214177_s_at
PBXIP1
0.0005004
0.066195485
1.061731802
222033_s_at
FLT1
0.0004947
0.066195485
1.03738168
218854_at
DSE
0.0005345
0.068307695
0.905149756
210511_s_at
INHBA
0.0005493
0.069064846
1.103622639
202187_s_at
PPP2R5A
0.0005629
0.069657239
1.041391358
201312_s_at
SH3BGRL
0.0006232
0.073610239
0.939968882
219358_s_at
CENTA2
0.0006073
0.073610239
0.911233169
220865_s_at
PDSS1
0.0006175
0.073610239
0.88394257
218699_at
RAB7L1
0.0006403
0.074496385
0.880551181
209396_s_at
CHI3L1
0.0006619
0.075877966
1.23586215
204081_at
NRGN
0.0007256
0.081973446
1.109969097
202185_at
PLOD3
0.0007642
0.085108481
0.94677975
200738_s_at
PGK1
0.0008063
0.085779381
0.977196602
202193_at
LIMK2
0.0007927
0.085779381
0.920601355
202872_at
ATP6V1C1
0.000795
0.085779381
0.900746363
202974_at
MPP1
0.0008143
0.085779381
0.963344703
202446_s_at
PLSCR1
0.0008348
0.085855047
0.953849782
204490_s_at
CD44
0.000837
0.085855047
0.88577208
203895_at
PLCB4
0.000953
0.093530674
1.031696261
206851_at
RNASE3
0.0009599
0.093530674
1.189718607
211963_s_at
ARPC5
0.0009246
0.093530674
0.980350825
215884_s_at
UBQLN2
0.0009428
0.093530674
0.906980156
201810_s_at
SH3BP5
0.0010099
0.097191784
0.921374384
211548_s_at
HPGD
0.0010224
0.097195788
0.755385963
200996_at
ACTR3
0.001077
0.099814726
0.97523089
201358_s_at
COPB1
0.0010861
0.099814726
0.96732026
203200_s_at
MTRR
0.0010884
0.099814726
0.864551148
*Change from day 3 to day 23, relative to the VAP diagnosis at day 0
doi:10.1371/journal.pone.0001564.t002 *Change from day 3 to day 23, relative to the VAP diagnosis at day 0
doi:10.1371/journal.pone.0001564.t002 *Change from day 3 to day 23, relative to the VAP diagnosis at day 0
doi:10.1371/journal.pone.0001564.t002 criteria (that is, the translation in PC2 did not begin until the day
the attending physician made the diagnosis). However, by
explicitly accounting for variance over time, a set of 85 genes
were identified subsequently in our first 11 VAP patients whose
microarray expression levels changed consistently before the
clinical diagnosis of VAP. ontology were found in cluster 2 and encode primarily granulocytic,
antimicrobial proteins, and adhesion molecules. In contrast, the
genes that decreased in apparent abundance (clusters 3–4) showed
different behavior depending on whether the patient developed
VAP early or late in the study. Although no consistent biological
theme emerged from this list of the 59 transcripts, this finding
provides some insight into the transcriptional basis of differences in
the critically ill host’s response to early- versus late-onset VAP [29]. PLoS ONE | www.plosone.org Discussion Of interest, the RNA abundance of five genes were altered by
pneumonia in both the mouse and human gene sets (Figures 1C and
3B): lactotransferrin (LTF), cathelicidin antimicrobial peptide
(CAMP), phospholipid scramblase 1 (PLSCR1), inhibin beta A
(INHBA), and hydroxyprostaglandin dehydrogenase 15-(NAD)
(HPGD). Pathway analysis indicates that these five genes connect These 85 genes clustered into four groups with their abundance
either increasing or decreasing throughout the 7-day window
bracketing the onset of infection (Figure 3A). There was a significant
association between genes known to play key roles in defense against
bacterial pathogens and those genes that increased in apparent
abundance (cluster 1 and 2 probes sets) coincident with the diagnosis
of VAP. Consistent with activation of the host response to
pneumonia, all of the genes with the ‘‘defense against bacteria’’ PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 PLoS ONE | www.plosone.org 9 Development of Riboleukogram Figure 3. Analysis of 85 genes identified as significantly changing over time during the onset of VAP. (A) The time-dependent behavior
of these genes was classified into four clusters. Shown is the normalized abundance of the average cluster member for each of the five microarrays
that bracket the attending physician’s diagnosis of VAP for each patient. (B) Co-expression network analysis of these four clusters was used to
generate the gene expression cartography of the human blood response to acute infection superimposed on critical illness. Clusters 1 and 2 are
tightly associated with one another, as are clusters 3 and 4. The red circles identify the 5 genes in common with the mouse coexpression network
(Figure 1C). (C) Principal components analysis of the abundance of these 85 genes in the training data set (11 patients with VAP). PC1 (not shown)
represents a constant bias term, PC2 and PC3 are shown. The arrow indicates where the attending physician’s diagnosed VAP. The green and red
circles indicate the points where the patients entered and exited the study, respectively. (D) Principal components analysis of microarray data
generated by 100 iterations of randomly chosen sets of 85 genes, all plotted on the same two axes. The randomly chosen sets are not informational
for healing from critical illness complicated by VAP. The only set that describes a discernable path is the list of 85 genes derived from EDGE analysis
(inset magnified view, same riboleukogram data as in panel C). Discussion doi:10.1371/journal.pone.0001564.g003
Development of Riboleukogram Figure 3. Analysis of 85 genes identified as significantly changing over time during the onset of VAP. (A) The time-dependent behavior
of these genes was classified into four clusters. Shown is the normalized abundance of the average cluster member for each of the five microarrays
that bracket the attending physician’s diagnosis of VAP for each patient. (B) Co-expression network analysis of these four clusters was used to
generate the gene expression cartography of the human blood response to acute infection superimposed on critical illness. Clusters 1 and 2 are
tightly associated with one another, as are clusters 3 and 4. The red circles identify the 5 genes in common with the mouse coexpression network
(Figure 1C). (C) Principal components analysis of the abundance of these 85 genes in the training data set (11 patients with VAP). PC1 (not shown)
represents a constant bias term, PC2 and PC3 are shown. The arrow indicates where the attending physician’s diagnosed VAP. The green and red
circles indicate the points where the patients entered and exited the study, respectively. (D) Principal components analysis of microarray data
generated by 100 iterations of randomly chosen sets of 85 genes, all plotted on the same two axes. The randomly chosen sets are not informational
for healing from critical illness complicated by VAP. The only set that describes a discernable path is the list of 85 genes derived from EDGE analysis
(inset magnified view, same riboleukogram data as in panel C). doi:10.1371/journal.pone.0001564.g003 Macroscopically, pneumonia is diagnosed when there are
symptomatic changes in clinical status, as manifested typically by
increased CPIS scores. However, microscopically pneumonia
occurs at the transition from colonization to infection concomitant
with failure of multiple host barriers protecting the bronchoalve-
olar epithelium. These events lead to tissue exposure to bacteria
and bacterial products, and in many cases to toxicosis and
bacteremia. Our data suggest that during VAP, circulating
leukocytes are exposed to bacterial products 24–72 hours prior
to diagnosis by an attending physician. Initiation of antibiotic
therapy earlier during this time window is expected to significantly
improve outcome [16]. These findings were further evaluated in a in a network rich with interactions between important mediators of
inflammation, as seen in Figure S2 and Table S2, (Ingenuity
Pathway Analysis, Ingenuity Systems, Redwood, CA). Development of Riboleukogram Figure 4. Genes informational in distinguishing clinical phe-
notypes of interest, including host gender, age, and ethnic
background, with the caveats that all African-Americans and
all middle-aged individuals were female (Table 1). Note that the
largest number of probe sets were associated with differences in ethnic
background (African-American compared to Caucasian). Of note, as
opposed to the mouse response, there we no genes that differentiated
between the human response to bacterial cell wall products (Gram
negative versus Gram-positive bacteria), suggesting that signal variance
due to ethnic background, gender, and age is greater than that due to
infecting organism. doi:10.1371/journal.pone.0001564.g004 prior to injury, nosocomial infection perturbs that system, and
after the infection cleared the system would return to it’s initial
attractor. In phase space analysis, we found that the patient-
specific trajectories appeared to converge and that the onset of
pneumonia ‘pushes’ the gene expression data away from that
basin—a perturbation that is ameliorated by appropriate antibi-
otic therapy. This result provides what we believe is the first
evidence in patients for a basin of attraction for the genetic
network associated with immunological health. Because of the heterogeneity of this patient population (for
example, age, gender, pre-existing health status, type and severity
of critical illness), it is not surprising that the error bars on the PC
analysis are initially quite large. The end-points of the trajectories,
however, appear to converge in a smaller space, suggesting that
the patients’ immune systems are returning to a health attractor
(Figures 5 and 6). As observed in the validation cohort, patient-
specific trajectories are not smooth curves, but appear to have
inflection points that may correlate with hospital intervention or
the onset of infection. Our data further suggest that differences in
host ethnic backgrounds and gender are more important than
differences in infecting organism as determinants of the host
leukocyte transcriptional response. These observations are consis-
tent with a recent study comparing GeneChip signal from cells
lines derived from Asians and Caucasians, indicating differences in
the gene expression levels of 25% of the 4000 genes studied in
these two ethnic groups [36]. Moreover, as the rate of sepsis, the
youngest age of sepsis onset, and the sepsis mortality rate are
highest for African-American males [37], our data suggest that
further study of riboleukograms is indicated to gain insight into
these health disparities. Development of Riboleukogram These profiles also suggest that there is a
transcriptional component that is conserved across that diversity of
the human population. Figure 4. Genes informational in distinguishing clinical phe-
notypes of interest, including host gender, age, and ethnic
background, with the caveats that all African-Americans and
all middle-aged individuals were female (Table 1). Note that the
largest number of probe sets were associated with differences in ethnic
background (African-American compared to Caucasian). Of note, as
opposed to the mouse response, there we no genes that differentiated
between the human response to bacterial cell wall products (Gram
negative versus Gram-positive bacteria), suggesting that signal variance
due to ethnic background, gender, and age is greater than that due to
infecting organism. d i 10 1371/j
l
0001564 004 doi:10.1371/journal.pone.0001564.g004 small validation patient cohort, confirming the value of the 85-
gene riboleukogram to quantitate the host response to and
recovery from critical illness. Individual riboleukograms changed
direction coincident with clinical findings of pneumonia or sepsis
(frequently days before the clinical diagnosis and maximal CPIS
scores). Nevertheless, marked heterogeneity was observed in some
patient responses that could not be linked to monitored variables
(e.g., patients 13 and 18, Figure 6A). In these patients, perhaps the
well-described influence of the underlying acute illnesses (pulmo-
nary contusion and intracranial hemorrhage) on host immune
responses could provide part of the explanation [30,31]. As
patients healed from critical illness, the riboleukograms converged,
a finding consistent with the existence of an immunological
‘‘attractor’’ state. Comparison of the aggregate VAP versus critical
illness riboleukograms indicated that a large portion of the signal
for the 85 genes identified is a reflection of recovery from critical
illness (PC2 in Figure 6B). Nevertheless, VAP signal became
evident among the ‘‘noise’’ of critical illness once the individual
riboleukograms were aligned for day of VAP diagnosis, evident as
a deflection of the VAP riboleukogram upward in PC3 (Figure 6B). Thus, we submit that riboleukograms are a molecular analytical
tool with substantial potential to improve diagnostics, prognostics,
and our understanding of the host response to critical illness
complicated by acute infection. In summary, our analysis demonstrates the plasticity of the blood
leukocyte response to bacteria in vivo, extending previous in vitro
studies [10,11] into the clinical realm. Development of Riboleukogram For the first time, we provide
evidence that riboleukogram gene expression analysis can be applied
to a heterogeneous clinical population to monitor the host response
to and recovery from critical illness complicated by acute infection. Moreover, we identify new, conserved gene targets that appear to be
informational for recovery at the transcriptional level, many of
which are involved with granulocyte maturation and chemokine
(not cytokine) responsiveness. This conclusion is supported by the
relative weak diagnostic information provided by the plasma
cytokine data, consistent with conclusions reached at recent a
consensus conference [16]. In particular, plasma procalcitonin levels
were not informational. There are, however, important limitations
to our study, most notably, the preliminary nature of the work, the
small number of patients, and the well-known difficulty of ruling out
false-positive and false-negative diagnoses of VAP based on clinical
parameters. This uncertainty is both the motivation for (and
challenge of) developing novel molecular diagnostics for VAP. In
addition, there is no consensus on how to leverage the dynamic
nature of the clinical, RNA, and protein data collected to build
hybrid models that improve diagnostics and prognostics. Thus, we
conclude that our data demonstrate the technical feasibility and
clinical potential of the riboleukogram approach, but proof of
clinical utility will require further study. An important consequence of our observation that patient-
specific riboleukograms converged is that the variance in leukocyte
gene expression for these 85 genes decreased significantly over the
time course in patients with VAP. Studies of physical systems
suggest that the stable states of networks can be represented as
attractors, a set of points in the phase space to which the genetic
network evolves over time [32,33]. In particular, every trajectory
initiated within the bounds of an attractor terminates inside the
attractor. Recently this hypothesis has been confirmed experi-
mentally in vitro [11,34]. Our findings suggest that the immune
system of a critically ill patient who recovers from disease returns
back to a stable state and that the immune response trajectory can
be a considered formally as a dynamic system. Discussion Principal
components analysis of the microarray expression data for the 85
transcripts generated a curve with qualitative similarities to curves
derived from either cytokines or the 109 human orthologs; however,
the patient-to-patient variance in the measured abundance of these
genes was significantly smaller and translation along the second
principal component preceded the diagnosis of VAP by 24 to
72 hours (Figure 3C). In contrast, equivalent analysis of randomly
selected sets of genes provided no information about the host
response to critical illness complicated by pneumonia. in a network rich with interactions between important mediators of
inflammation, as seen in Figure S2 and Table S2, (Ingenuity
Pathway Analysis, Ingenuity Systems, Redwood, CA). Principal
components analysis of the microarray expression data for the 85
transcripts generated a curve with qualitative similarities to curves
derived from either cytokines or the 109 human orthologs; however,
the patient-to-patient variance in the measured abundance of these
genes was significantly smaller and translation along the second
principal component preceded the diagnosis of VAP by 24 to
72 hours (Figure 3C). In contrast, equivalent analysis of randomly
selected sets of genes provided no information about the host
response to critical illness complicated by pneumonia. February 2008 | Volume 3 | Issue 2 | e1564 February 2008 | Volume 3 | Issue 2 | e1564 PLoS ONE | www.plosone.org 10 PLoS ONE | www.plosone.org Development of Riboleukogram Borrowing from
concepts in the physical sciences [35], we hypothesized that an
individual’s baseline immune system is within a basin of attraction Thus, as graphs of myocardial electrical information (electro-
cardiograms) were tapped over a century ago to provide an
objective means to aid heart diagnostics, we submit that
riboleukograms will aid in the diagnosis and prognosis of acute
infectious and inflammatory disease. The diagnostic potential of
riboleukograms is supported by two very recent independent
reports that corroborate our mouse data (Figure 1D), indicating February 2008 | Volume 3 | Issue 2 | e1564 PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 11 Development of Riboleukogram Figure 5. Phase space analysis of the average ICU patient riboleukogram trajectories as they develop VAP, respond to antibiotics,
and recover. (A) Decrease of variance and the convergence of individual trajectories to a common small region in the phase space (‘‘immunological
attractor’’) associated with health. The green and red circles indicate where the patients entered and exited the study, respectively. (B) Decreases in
variance (standard deviation, STD) over time for the phase space trajectory in panel A, consistent with the existence of an attractor. The diagnosis of
VAP was made by the attending physician on Day 0. doi:10.1371/journal.pone.0001564.g005 Figure 5. Phase space analysis of the average ICU patient riboleukogram trajectories as they develop VAP, respond to antibiotics,
and recover. (A) Decrease of variance and the convergence of individual trajectories to a common small region in the phase space (‘‘immunological
attractor’’) associated with health. The green and red circles indicate where the patients entered and exited the study, respectively. (B) Decreases in
variance (standard deviation, STD) over time for the phase space trajectory in panel A, consistent with the existence of an attractor. The diagnosis of
VAP was made by the attending physician on Day 0. doi:10.1371/journal.pone.0001564.g005 the six genes depicted above, the RT-PCR correlation coefficients
(R2 values) for the microarray expression values are 0.60, 0.67,
0.66, 0.86, 0.85, and 0.43, respectively. Found at: doi:10.1371/journal.pone.0001564.s001 (0.32 MB TIF) the six genes depicted above, the RT-PCR correlation coefficients
(R2 values) for the microarray expression values are 0.60, 0.67,
0.66, 0.86, 0.85, and 0.43, respectively. Development of Riboleukogram Found at: doi:10.1371/journal.pone.0001564.s001 (0.32 MB TIF) that circulating leukocyte RNA signatures in patients differentiate
between the host responses to sterile versus infectious causes of
systemic inflammation and between the host response to Gram-
negative versus Gram-positive pathogens [38,39]. Prospective
clinical trials are indicated to validate our results and determine
the value of this new technology; to optimize gene selection
methods that account for differences in patient ethnicity, gender,
and age; and to develop computational approaches that integrate
clinical and molecular data to improve diagnostics [40]. Figure S2
Hypothetical leukocyte network based on reported
gene-gene interactions. Starting with the 5 genes altered by
pneumonia in both mice and patients (grey shapes), the
Ingenuity Pathway Analysis tool was used to build an interaction
network based upon gene-gene interactions reported in the
literature. Note the rich network of interactions and the
consistent inflammation theme of the genes added by the
software. Table S2 lists the individual genes and their reported
connections. Figure S2
Hypothetical leukocyte network based on reported
gene-gene interactions. Starting with the 5 genes altered by
pneumonia in both mice and patients (grey shapes), the
Ingenuity Pathway Analysis tool was used to build an interaction
network based upon gene-gene interactions reported in the
literature. Note the rich network of interactions and the
consistent inflammation theme of the genes added by the
software. Table S2 lists the individual genes and their reported
connections. Supporting Information Figure S1
The changes in transcript abundance of selected
genes from Table 2 were validated by quantitative RT-PCR. For PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 12 Development of Riboleukogram Figure 6. Principal components analysis of 85 leukocyte genes in the training and validation patient cohorts. (A) The solid black curve
depicts the aggregate riboleukogram of the first 11 VAP patients (training cohort, same data as in Figure 3C). The other 7 curves are the individual
riboleukograms of the patients in the validation cohort. The inset magnifies the trajectories of patients 13–17 (see Table 1) and demonstrates abrupt
changes in riboleukogram course typically coincident with an increase in CPIS score (first occurrence of maximal CPIS value is indicated by the
arrows). The paths of patients 13, 18 and 19, are atypical (see text for additional details). (B) The aggregate 11 patient VAP riboleukogram (black curve,
same as panel A) is compared to the aggregate riboleukogram of all patients aligned by study day (that is, training and validation cohorts,
irrespective of VAP day of diagnosis, dotted blue curve). Note that the VAP riboleukogram deviates from the ‘‘critical illness’’ riboleukogram (black
arrows) prior to VAP diagnosis (lighting bolt) but after treatment the VAP riboleukogram converges with the critical illness riboleukogram at the Figure 6. Principal components analysis of 85 leukocyte genes in the training and validation patient cohorts. (A) The solid black curve
depicts the aggregate riboleukogram of the first 11 VAP patients (training cohort, same data as in Figure 3C). The other 7 curves are the individual
riboleukograms of the patients in the validation cohort. The inset magnifies the trajectories of patients 13–17 (see Table 1) and demonstrates abrupt
changes in riboleukogram course typically coincident with an increase in CPIS score (first occurrence of maximal CPIS value is indicated by the
arrows). The paths of patients 13, 18 and 19, are atypical (see text for additional details). (B) The aggregate 11 patient VAP riboleukogram (black curve,
same as panel A) is compared to the aggregate riboleukogram of all patients aligned by study day (that is, training and validation cohorts,
irrespective of VAP day of diagnosis, dotted blue curve). Note that the VAP riboleukogram deviates from the ‘‘critical illness’’ riboleukogram (black
arrows) prior to VAP diagnosis (lighting bolt), but after treatment, the VAP riboleukogram converges with the critical illness riboleukogram at the
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219 mouse genes: probe/gene mean fold change
Found
at:
doi:10.1371/journal.pone.0001564.s003
(0.08
MB
XLS) The authors wish to thank Drs. Daniel G. Remick and Derek C. Angus for
providing cytokine and procalcitonin measurements, respectively. We also
acknowledge the technical and clinical expertise of Sandra MacMillan,
Douglas Oppedal, Diane Salamon, Barbara Anderson, and Sharon Daube. Table S2
Rich network connections for 5 common mouse/
human genes (Ingenuity Pathway Analysis)
Found
at:
doi:10.1371/journal.pone.0001564.s004
(0.02
MB
XLS) Supporting Information The green and red circles indicate where the patients entered and exited the study, respectively. doi:10.1371/journal.pone.0001564.g006 February 2008 | Volume 3 | Issue 2 | e1564 PLoS ONE | www.plosone.org 13 Development of Riboleukogram Found at: doi:10.1371/journal.pone.0001564.s002 (3.24 MB TIF)
Table S1
219 mouse genes: probe/gene mean fold change
Found
at:
doi:10.1371/journal.pone.0001564.s003
(0.08
MB
XLS)
Table S2
Rich network connections for 5 common mouse/
human genes (Ingenuity Pathway Analysis)
Found
at:
doi:10.1371/journal.pone.0001564.s004
(0.02
MB
XLS) Found at: doi:10.1371/journal.pone.0001564.s002 (3.24 MB TIF) Author Contributions Conceived and designed the experiments: JC MC JM CC WD BG. Performed the experiments: JM KH. Analyzed the data: JC JM KH AP
NL QL AB JR WZ BG DD WS. Contributed reagents/materials/analysis
tools: JM AP NL QL AB JR WZ BG WS. Wrote the paper: JC JM AP AB
WZ CC WS. Other: Designed the study: JC. References Nat Genet 39: 226–231. 17. Coopersmith CM, Stromberg PE, Dunne WM, Davis CG, Amiot DM, et al. (2002) Inhibition of intestinal epithelial apoptosis and survival in a murine model
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Illness and Injury: Fourth NIH Symposium. submitted. PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org February 2008 | Volume 3 | Issue 2 | e1564 14
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Impact of COVID-19 on Fintech with Reference to Youngster
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Journal of multidisciplinary cases
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Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
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DOI: https://doi.org/10.55529/jmc.32.26.38 Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
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DOI: https://doi.org/10.55529/jmc.32.26.38 Journal of Multidisciplinary Cases
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http://journal.hmjournals.com/index.php/JMC
DOI: https://doi.org/10.55529/jmc.32.26.38 Keywords: COVID-19, Youngsters, Fintech, Review Study. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 26 Email: 1rj88chauhan@gmail.com, 2rajanish285@gmail.com, 4nabilakv@gmail.com
Corresponding Email: 3*maselenoandino@gmail.com Received: 24 October 2022 Accepted: 09 January 2023 Published: 11 February 2023 Rahul Chauhan1, Rajpurohit Anish Kumar2, Andino Maseleno3*, Nabila Kharimah
Vedy4 1Assistant Professor, Parul Institute of Business Administration, Parul University, India. 2Student, Parul Institute of Business Administration, Parul University, India. 3*,4Institut Bakti Nusantara, Lampung, Indonesia. Received: 24 October 2022 Accepted: 09 January 2023 Published: 11 February 2023 Abstract: “In Wuhan, China, in December 2019, the Novel Coronavirus (2019-nCoV) was
discovered for the first time. Covid-19, the virus-caused disease, had been reported in 205
countries by the end of March 2020. There had been about 700,000 cases of infection and
33,000 fatalities worldwide.” Kerala, India, reported the first case of 2019-nCoV in the
final week of January 2020. Since that time, 27 States and Union Territories have reported
about 1353 cases. More than 300 cases have been reported by the State of Tamil Nadu, and
at the time of writing, 110 samples were being investigated. Around 290 people in Kerala
are infected, and 120,000 more are being monitored. Children, families, and entire
communities could be at risk from the 2019 n-CoV. In addition to the risk of secondary
morbidity and mortality, the virus infection itself has direct health effects. It also has a
negative impact on the economy and way of life of marginalised people. Basic services like
health, education, and social protection programmes will inevitably be disrupted. Children
and parents experienced fear, panic, anxiety, and stress as a result of the 21-day total
lockdown that the Indian government had announced. Correct information must be given,
myths, misconceptions, and false information must be dispelled, social isolation should be
encouraged, hand and personal hygiene should be promoted, and flu-like symptoms should
be treated as soon as possible. Keywords: COVID-19, Youngsters, Fintech, Review Study. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 26 26 26 Journal of Multidisciplinary Cases
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DOI: https://doi.org/10.55529/jmc.32.26.38 COVID-19 The crucial duty of effective risk communication falls on the media and other concerned
organisations. By disseminating fair and accurate reports, the media can significantly
contribute to increasing awareness, busting myths, exposing false information, and
encouraging kindness, preventing stigmatisation, and boosting public confidence. It can also
help to foster a positive environment by publishing tales of successful coping and recovery. S. No
Author Name
Topic Name
Results
1. (Dipinder S
Randhawa, 2018)
“India Singapore
FinTech Cooperation:
Opportunities and
Challenges”
Through the use of a single account,
interoperability enables seamless fund
transfers between geographic regions. India and Singapore are at the
forefront of FinTech development. 2. (Gurung, Siddhanth,
2018)
“FinTech: A Messiah
for the ailing Banking
Industry in India”
A partnership with the failing
traditional financial institutions would
aid in giving India's financial sector a
new direction. 3. (Dubey, Vivek, 2019)
“FinTech Innovations
in Digital Banking”
Currently, AR technologies are used
to streamline processes, cut costs, and
generate a wide range of profitable
outcomes. 4. (Mittal, Varun, 2019)
“India FinTech
Landscape”
Due in large part to government
initiatives, rising mobile and internet
penetration, and other factors, India
has seen a significant shift away from
cash and toward digitization. Results Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 27 27 Journal of Multidisciplinary Cases
ISSN: 2799-0990
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DOI: https://doi.org/10.55529/jmc.32.26.38 5. (Kapadia, Sunil, 2020)
“How Digitization Is
Impacting Banking
Transactions and
Financial Markets in
India?”
Digitization automates both the
product and the process, raising both
the standard and productivity. Digitalization has facilitated
innovations like credit cards, ATMs,
and online banking. 6. (Buchak, Matvos,
Piskorski, & Seru,
2018)
Fintech makes the job
becomes more
effective and efficient
According to customer reviews,
fintech companies can reduce labour
costs and office space costs while
obtaining significantly more
comfortable workspace. 7. (Kudinska, Romānova
&, 2016)
Fintech is easy to
adapt
There is no need for geographic
concentration in the fintech industry
because it is simple to adapt to
changing consumer demands and the
availability of affordable financial
services. 8. (Shin, Lee and, 2018)
“The internet and the
world wide web had
changed the finance
industry's face”
The financial sector has undergone a
transformation thanks to the internet
and the world wide web; transactions
for banking services (savings,
transfers), insurance, and stock
trading can now be made online. 9. COVID-19 (al, Arner et, 2015)
“Three stages of
fintech development.”
1866–1987 saw the earliest phase (or
fintech 1.0), between 1987 and 2008,
the second stage (or fintech 2.0), The
third stage, from 2008 to the present
(also known as fintech 3.0). 10. (Kuzmina-Merlino,
Saksonova and, 2017)
“The global financial
crisis 2008-2009 is
the milestone for
developing the fintech
sector.”
The financial sector had been severely
impacted by the crisis. For the bank to
maximise performance, new
technologies must be used. 11. (Vives, 2019)
The disruptive
technologies
The most recent technologies used for
the fintech platform are distinct from
the fintech business models or
segments. The fintech company's
success demonstrates the significance
of disruptive technologies in the
fintech-outside. 12. Atri D, 2020
“The coronavirus
disease-
2019(COVID-19)”
“A global pandemic that started in the
Chinese province of Wuhan was
brought on by coronavirus-2 causing
severe acute respiratory syndrome Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 28 28 Journal of Multidisciplinary Cases
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DOI: https://doi.org/10.55529/jmc.32.26.38 (SARS-CoV2)”
13. Atri D, 2020
The number of
confirmed cases
worldwide. According to the WHO COVID -19
Dashboard, there were 66,63,204
confirmed cases worldwide as of 6
June 2020, and there were more than
392802 fatalities. 14. Huang C, 2020
The origin of the
virus. The SARS-CoV2 epidemic in China
was largely attributed to early cases'
frequent exposure to the Huanan
seafood market. 15. Lu R, 2020
Phylogenetic and
virus genome
analysis. Chinese horseshoe bats in the
Zhejiang province in China. 16. Paraskevis D, 2020
Genome evolutionary
analysis. With a 96.3% sequence similarity to
the Bat CoVRaTG13 sequence, the
SARS-CoV-2 and similarly
discovered findings were confirmed. 17. Sahu KK, 2020
“Disease Control and
Prevention.”
“Incubation period of COVID-2019 is
approximately 5.1 days (range 2-14
days).”
18. Yang HY, 2020
Mode of transmission. “Close or direct contact with infected
secretions or large aerosol droplets is
the primary method of transmission.”
19. Zhang W, 2020
Spread of Virus. Corona virus spreads via the faecal-
oral route because the epithelium of
the intestinal lumen contains the ACE
2 receptor, which the corona virus
binds to. 20. Van Doremalen N,
2020
Detection of SARS-
CoV-2 viral nucleic
acids. The faecal samples and anal swabs of
COVID19 patients contained SARS-
CoV-2 viral nucleic acids, according
to Zhang et al. from Wuhan
University. 21. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 30 COVID-19 Liang H, 2020
Vertical
Transmission. There hasn't been any evidence of
vertical transmission from mother to
child in breast milk, vaginal
secretions, or pregnant women. 22. (al., Pohan et al. Sembiring et al. Yanti
et, 2020)
Effects of Covid -19. The Covid 19 pandemic period has an
impact on both the level of economic
activity and human health. 23. (LS, Wang, 2020)
COVID-
19 characteristic. It is characterised by a high fever, a
cough, exhaustion, breathlessness,
pneumonia, and other symptoms of
the respiratory tract, and it frequently Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 29 29 Journal of Multidisciplinary Cases
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DOI: https://doi.org/10.55529/jmc.32.26.38
progresses to death. 24. (Q, Bi, 2020)
“Child and
adolescents.”
“SARS-COV-2 have mostly caused
mild respiratory symptoms rather than
severe forms like in adults and old age
people.”
25. (Archna,
2020)Shubhi&Archna,
2020
COVID -19. COVID-19 is a large group of viruses
that creates illness. 26. (al., Nagaoka et,
2015)
“Many youths in need
of care and protection
rely on institutional
and alternative care to
meet their
developmental needs. “Such facilities must now provide
comprehensive services in order to
promote healthy development. Western youth developmental models
emphasise this all-encompassing
perspective of youth and highlight the
assets, connections, and institutional
supports required for a smooth
transition to adulthood.”
27. (al., Albuquerque et,
2015)
Individual strengths
Provide safeguards and aid in the
healthy development of
institutionalised youth, contributing to
attitudes like self-worth and self-
concept. 28. (Matos, Mota and,
2015)
Resilience or the
development of
competencies
a sense of positive self-worth and
being appreciated by others in the
face of adversity
29. (al., Gearing et, 2015)
Individual Factors. promoted by positive interactions
with peers and other individuals
within institutional settings
30. (James, 2011)
Environment
“Resilience among children in foster
care can be improved by the
institutional environment. Reviewing
American institutional group care
models brought out the importance of
environments in fostering healthy
relationships between children and
adults.”
31. (Saraswathi, Parikh, &
Prakash, 1999)
Peer Groups
Though they frequently share the
same gender and now exert more
power over higher social classes,
Indian families are frequently seen as
having the final say in matters of
importance like career choice and
mate choice. 32. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 31 COVID-19 (Charnes, 1972)
Financial technology
Explains what a fintech company is Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 30 30 Journal of Multidisciplinary Cases
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Vol : 03, No. 02 , Feb-Mar 2023
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DOI: https://doi.org/10.55529/jmc.32.26.38 (FinTech) has
attracted global
attention. and categorises it into different
fintech businesses, like fintech
lending and fintech payment
businesses. 33. (Nasution, 2020)
impact of COVID-19
on the development of
FinTech
Due to the shift in people's lifestyles
to digitalization, COVID-19 has
created opportunities for Islamic
FinTech. 34. (Thakor, 2019)Anjan
VThakor 2019
Effective and
Efficient
“Fintech is the practise of using
technology to carry out financial
activities more quickly and
efficiently.”
35. (Sharma, Vinod,
2016)
tool which supports
all financial services
Finance technology will manage
money automatically for societal
improvement. Business opportunities
will grow as a result of these
innovations. 36. (Gilligan, 2020)
Technological
development
Technology advancement is made not
only to cut costs but also to complete
tasks. The majority of tasks still
require manual labour. Therefore,
they must create technology that
complies with regulations and
performs work quickly while also
reducing costs. 37. (Agarwal, 2020 )
increase in the usage
of smartphones
One of the biggest opportunities came
about as a result of people using
smartphones more frequently, starting
to work from home, and the
government discouraging the use of
cash. 38. (Belgavi, 2020 )
Web aggregators
Start-ups are using technology to offer
a wide range of best-suited customers’
options through marketplaces and
digitally streamlined purchasing. 39. (Demirguc-Kunt et al.,
2018)
Global Findex
That while getting easier, finding
comparable data on fintech activity
across countries is still difficult. 40. (Dhawan, 2020)
COVID-19 Pandemic. The pandemic has given us the chance
to prepare the ground for the
introduction of digital learning. 41. Murgatrotd, 2020
Challenges of E-
Learning. “Accessibility, affordability,
flexibility, learning pedagogy, life-
long learning, and educational policy Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 31 Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 31 31 31 Journal of Multidisciplinary Cases
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DOI: https://doi.org/10.55529/jmc.32.26.38 DOI: https://doi.org/10.55529/jmc.32.26.38
are the main issues with e-learning.”
42. Aafreen et al. (2018)
investigated one
effect of stress. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 32 COVID-19 According to research on the
academic performance of students in
various streams, those majoring in
science experienced higher levels of
stress than those majoring in other
subjects. They experience mental,
physical, and emotional effects from
it. 43. Laxmi and Kaur
(2017)
Study Habits and
Attitudes. The study's findings showed a
significant difference between
P.S.E.B. and C.B.S.E. secondary
school students in terms of gender
among secondary school students. 44. Sarwer (2019)
Study on Secondary
School Students. Academic anxiety and English
proficiency achievement, with an
indication that the study's goal is to
determine how these two factors
relate to various demographic factors. The effect of academic anxiety on
secondary school students' English
achievement is also estimated. 45. Bhatt et al., 2016
Family or Individual
Classifications. When classifying a family or an
individual into one of these groups,
any or all of the three factors—
income, education, and occupation—
can be looked into and assessed. 46. Islam and Khan, 2017
Socio-economic
Status. a person's or family's combined social
and economic status as determined by
their income, level of education,
occupation, possessions, and other
factors relative to others in the
community. Socio-cultural factors,
economic factors, educational factors,
and possession of goods and services
that are available in a family all fall
under the general heading of socio-
economic status. 47. Suleman et al. (2012),
Saifi (2011)
Rich source of
literature and Socio-
Economic status. According to research on the impact
of socioeconomic status on academic
achievement, kids with a good
socioeconomic status perform
academically better than kids with a
bad socioeconomic status, who Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 32 32 32 Journal of Multidisciplinary Cases
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perform academically poorly and
below expectations. Examined how
socioeconomic status affected
students' performance. 48. Clemente-González
(2016)
Relevance of
grandparent–
grandchild
relationships
The former's importance in the social
and emotional growth of the child
gives their grandparents great
significance because of the admiration
they have been shown and because of
how important they are to the family
unit. 49. Founaud and
González-Audicana,
2020
Subject based on
skills. “This subject is not just based on
motor skills; it also has an impact on
many aspects of students' daily lives
and helps teachers better understand
students in all of their varied
dimensions.”
50. Petrie, 2020
Online Platforms. COVID-19 The unified communication and
collaboration platforms that have been
used up to this point include
Microsoft Teams, Google Classroom,
Canvas, and Blackboard, which give
teachers the ability to design training
and skill-development programmes as
well as educational courses. 51. Doucet et al., 2020
Strategy for Learning
Resources. “The flipped classroom is a
straightforward method for
distributing learning materials before
a class, such as articles, pre-recorded
videos, and YouTube links. The
following step in the online classroom
process is to use discussion with peers
and faculty to further understanding.”
52. Murgatrotd, 2020
Challenges Identified. “Accessibility, affordability,
flexibility, learning pedagogy,
lifelong learning, and educational
policy are all generally acknowledged
as challenges with e-learning.”
2. CONCLUSION Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 33 2. CONCLUSION Although the fintech sector is still in the early stages of adoption, we think it is well-
positioned to experience long-term growth in the years to come. The changes will place a
greater emphasis on open banking and digital lending (alternative finance). Growth in the Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 33 Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 33 33 33 Journal of Multidisciplinary Cases
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DOI: https://doi.org/10.55529/jmc.32.26.38
fintech industry will ultimately give businesses enormous opportunities and give them power
in the digital era. Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
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DOI: https://doi.org/10.55529/jmc.32.26.38 fintech industry will ultimately give businesses enormous opportunities and give them power
in the digital era. fintech industry will ultimately give businesses enormous opportunities and give them power
in the digital era. “Fundamentally, fintech is used to assist organisations, business owners, and consumers in
better managing their financial operations, processes, and lives through the use of specialised
software and algorithms that are used on computers and, increasingly, smartphones. This
technology is independent of the type of product being provided. The survey reveals
significant differences between men and women who are digitally active: women report
worrying more about their privacy when interacting with businesses online, while men report
worrying less. The financial technology sector will create original and cutting-edge methods
for evaluating risks. The penetration of financial services in India will increase by utilising
big data, machine learning, and alternative data to underwrite credit and develop credit scores
for customers with little credit history.” In the reported use of fintech services, a gender gap has been found by this study. In almost
every nation, there is a gap. Although the gap can be partially closed when using country and
individual characteristics, these controls do not fully explain it. It exists regardless of whether
newcomers or established businesses are willing to share their data with fintech companies in
exchange for better offers and less willing to use fintech companies to produce better or more
inventive products. The gender gap in fintech is significantly reduced when attitudes toward
new financial technology and willingness to use fintech competitors if they provide less
expensive services are taken into account. 2. CONCLUSION The causes of the gender gap in fintech will need
to be addressed by policies that seek to increase financial inclusion. There may not be much
room for policy if the gender gap is accounted for by disparities in preferences, such as risk
aversion. However, policy interventions may be required to improve the inclusiveness of
fintech services if the gap is caused by gender-based discrimination or by social norms and
circumstances that disadvantage women. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 34 3. REFERENCES Western
youth developmental models emphasise this holistic view of youth and identify
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DOI: https://doi.org/10.55529/jmc.32.26.38 Journal of Multidisciplinary Cases
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commentators suggest that the decision of individuals and firms to adopt new products
and services from a given financial institution. 23. Guiso et al. Gennaioli et al.A. V. Thakor . ((2004); (2015); (2020)). Adoption of new
products and services from a given financial institution. 3. REFERENCES a number of studies and
commentators suggest that the decision of individuals and firms to adopt new products
and services from a given financial institution. 24. Gurung, Siddhanth. (2018). FinTech: A Messiah for the ailing Banking Industry in
India. a collaboration with the ailing traditional financial institutions would help
provide a new direction to India’s financial sector. 24. Gurung, Siddhanth. (2018). FinTech: A Messiah for the ailing Banking Industry in
India. a collaboration with the ailing traditional financial institutions would help
provide a new direction to India’s financial sector. p
25. Hale et al. (2020). Government Response Tracker (OxCGRT). OxCGRT provides daily
updates on the number of confirmed cases and confirmed deaths, as well as various
policies being implemented at the country−daily level. 26. Higgins; al., Crouzet et. ((2019)). fintech adoption. Fintech adoption studies thus far can
be broadly grouped into those focusing on i) network effects, ii) individual−level
determinants, and iii) country−level predictors. y
p
27. Hornuf, Haddad and. (2019). component and venture capital in the fintech company's
success. the macroeconomic condition, internet service infrastructure, smartphone, and
venture capital witness the fintech start-up formulation. 28. James. (2011). Environment. the environment within institutions can enhance resilience
among youth in care. A review of American institutional models of group care
underscored how settings can encourage positive relationships amongst peers and
adults. 29. Jayakar, Sangwan et 401 al Miao &. (2016). regulatory sandbox. Hong Kong, the UK,
and Singapore are pioneering regulations to facilitate fintech companies' development
called the “regulatory sandbox”. 30. Kapadia, Sunil. (2020). How Digitization Is Impacting Banking Transactions and
Financial Markets in India? Digitisation mechanizes both product and process through
which standard and productivity increases. This digitization has contributed to advances
like online banking, ATMs, and credit cards. 30. Kapadia, Sunil. (2020). How Digitization Is Impacting Banking Transactions and
Financial Markets in India? Digitisation mechanizes both product and process through
which standard and productivity increases. This digitization has contributed to advances
like online banking, ATMs, and credit cards. 31. Kudinska, Romānova &. (2016). Fintech is easy to adapt. Fintech is easy to adapt with
the needs of consumers who are fast changing and the existence of low-cost financial
services that make Fintech more attractive to the public, so there is no need for
geographical concentration. 31. Kudinska, Romānova &. (2016). Fintech is easy to adapt. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 36 3. REFERENCES Defined FinTech companies and classifies it according to FinTech business types, such
as FinTech payment businesses and FinTech lending businesses. 15. Claessens et al. Frost et al. Frost. ((2018); (2019); (2020)). analyze predictors of fintech
adoption. the third group includes a number of academic and policy studies using
country−level data (typically cross−sectional) to analyze predictors of fintech adoption. 15. Claessens et al. Frost et al. Frost. ((2018); (2019); (2020)). analyze predictors of fintech
adoption. the third group includes a number of academic and policy studies using
country−level data (typically cross−sectional) to analyze predictors of fintech adoption. y
( yp
y
)
y
p
p
16. Dapp, Slomka, & Hoffmann. (2014). Digitalization of the financial sector. Fintech is a
tool used for advanced technology, mostly internet-based in the financial sector. y
( yp
y
)
y
p
p
16. Dapp, Slomka, & Hoffmann. (2014). Digitalization of the financial sector. Fintech is a
tool used for advanced technology, mostly internet-based in the financial sector. 16. Dapp, Slomka, & Hoffmann. (2014). Digitalization of the financial sector. Fintech is a
tool used for advanced technology, mostly internet-based in the financial sector. 17. Demirguc-Kunt et al. (2018). Global Findex. that finding comparable data on fintech
activity across countries is still a challenge, although improving. 17. Demirguc-Kunt et al. (2018). Global Findex. that finding comparable data on fintech
activity across countries is still a challenge, although improving. 17. Demirguc-Kunt et al. (2018). Global Findex. that finding comparable data on fintech
activity across countries is still a challenge, although improving. y
g
g
p
g
18. Dhawan. (2020). COVID-19 Pandemic. The pandemic has provided us with an
opportunity to pave the way for introducing digital learning. 18. Dhawan. (2020). COVID-19 Pandemic. The pandemic has provided us with an
opportunity to pave the way for introducing digital learning. 19. Dipinder S Randhawa, C. J. (2018). India Singapore FinTech Cooperation:
Opportunities and Challenges. Interoperability allows for seamless fund transfers across
geographical zones via a single account. Singapore and India lead developments in the
use of FinTech. 35 35 Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
http://journal.hmjournals.com/index.php/JMC
DOI: https://doi.org/10.55529/jmc.32.26.38 Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
http://journal.hmjournals.com/index.php/JMC
DOI: https://doi.org/10.55529/jmc.32.26.38 20. Doucet et al. (2020). Variety of Subject and Needs. There are a variety of subjects with
varying needs. 3. REFERENCES It characterized by fever, cough, fatigue,
shortness of breath, pneumonia, and other respiratory tract symptoms and, in many
cases, progress to death. 36. M. Guruprasad. (2020). financial sector growth, privatization, and liberalization in
India. Several private and foreign players entered the Indian market with superior
technologies that allowed them to effectively service their customers across multiple
channels such as ATMs and online banking. 36. M. Guruprasad. (2020). financial sector growth, privatization, and liberalization in
India. Several private and foreign players entered the Indian market with superior
technologies that allowed them to effectively service their customers across multiple
channels such as ATMs and online banking. g
37. Matos, Mota and. (2015). Resilience or the development of competencies. adverse
circumstances is associated with positive self-esteem and a sense of being
acknowledged by others. 37. Matos, Mota and. (2015). Resilience or the development of competencies. adverse
circumstances is associated with positive self-esteem and a sense of being
acknowledged by others. 38. Matthew Blake, P. V. customers choice and inconvinience. Fintech is a wider concept
which use technology to plan and delivery the financial products to customers as per
their choice and convenience. 38. Matthew Blake, P. V. customers choice and inconvinience. Fintech is a wider concept
which use technology to plan and delivery the financial products to customers as per
their choice and convenience. 39. McCall. (2013). Caregivers trained. to engage with children in a family-like
environment by adopting parental roles, along with bringing in structural changes such
as reducing group size, reducing the ratio of caregivers per group of children and
providing more consistent in-house monitorin. 39. McCall. (2013). Caregivers trained. to engage with children in a family-like
environment by adopting parental roles, along with bringing in structural changes such
as reducing group size, reducing the ratio of caregivers per group of children and
providing more consistent in-house monitorin. 40. Mello. (2018). Fintech has many meanings according to experts. Fintech includes all of
financial products and services that were traditionally supplied by the financial industry. 40. Mello. (2018). Fintech has many meanings according to experts. Fintech includes all of
financial products and services that were traditionally supplied by the financial industry. 41. Mittal, Varun. (2019). India FinTech Landscape. India has experienced a huge shift
from cash towards digitization, primarily due to Government initiatives and increasing
mobile and internet penetration. 41. Mittal, Varun. (2019). India FinTech Landscape. 3. REFERENCES Fintech is easy to adapt with
the needs of consumers who are fast changing and the existence of low-cost financial
services that make Fintech more attractive to the public, so there is no need for
geographical concentration. 32. 32. Kundariya. (2019). India stands in 2nd place to adapt the Fintech. Fintech start-up
funding was $3.6 billion whereas in 2018 it stood up at $39.57 billion which means it
increased at 1000% within a short span. 32. Kundariya. (2019). India stands in 2nd place to adapt the Fintech. Fintech start-up
funding was $3.6 billion whereas in 2018 it stood up at $39.57 billion which means it
increased at 1000% within a short span. 32. Kundariya. (2019). India stands in 2nd place to adapt the Fintech. Fintech start-up
funding was $3.6 billion whereas in 2018 it stood up at $39.57 billion which means it
increased at 1000% within a short span. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 36 36 36 Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
http://journal.hmjournals.com/index.php/JMC
DOI: https://doi.org/10.55529/jmc.32.26.38 Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
http://journal.hmjournals.com/index.php/JMC
DOI: https://doi.org/10.55529/jmc.32.26.38 33. Kuzmina-Merlino, Saksonova and. (2017). the global financial crisis 2008-2009 is the
milestone for developing the fintech sector. The crisis had significantly affected the
finance industry. The bank must apply new technologies for leveraging performance. 33. Kuzmina-Merlino, Saksonova and. (2017). the global financial crisis 2008-2009 is the
milestone for developing the fintech sector. The crisis had significantly affected the
finance industry. The bank must apply new technologies for leveraging performance. 34. Loo, Van. (2018). financial solutions for connecting peer-to-peer customers. The
peerto-peer transaction and the decentralized exchange market without authorities'
monitoring create new risks and threaten the finance industry's sustainability. 34. Loo, Van. (2018). financial solutions for connecting peer-to-peer customers. The
peerto-peer transaction and the decentralized exchange market without authorities'
monitoring create new risks and threaten the finance industry's sustainability. 35. LS, Wang. (2020). COVID-19 characteristic. It characterized by fever, cough, fatigue,
shortness of breath, pneumonia, and other respiratory tract symptoms and, in many
cases, progress to death. 35. LS, Wang. (2020). COVID-19 characteristic. It characterized by fever, cough, fatigue,
shortness of breath, pneumonia, and other respiratory tract symptoms and, in many
cases, progress to death. 35. LS, Wang. (2020). COVID-19 characteristic. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 37 3. REFERENCES India has experienced a huge shift
from cash towards digitization, primarily due to Government initiatives and increasing
mobile and internet penetration. p
42. Nasution, R. &. (2020). impact of COVID-19 on the development of FinTech. COVID-
19 has opened up opportunities for Islamic FinTech due to the change in people's
lifestyle to digitalization. p
42. Nasution, R. &. (2020). impact of COVID-19 on the development of FinTech. COVID-
19 has opened up opportunities for Islamic FinTech due to the change in people's
lifestyle to digitalization. p
42. Nasution, R. &. (2020). impact of COVID-19 on the development of FinTech. COVID-
19 has opened up opportunities for Islamic FinTech due to the change in people's
lifestyle to digitalization. y
g
43. Ozili. (2018). positive effects toward financial inclusion. Digital finance through
Fintech providers has positive effects toward financial inclusion in emerging and
advanced economies. 43. Ozili. (2018). positive effects toward financial inclusion. Digital finance through
Fintech providers has positive effects toward financial inclusion in emerging and
advanced economies. 43. Ozili. (2018). positive effects toward financial inclusion. Digital finance through
Fintech providers has positive effects toward financial inclusion in emerging and
advanced economies. 44. Philippon Das Frost et al. ((2016); (2019); (2019)). competitive advantages. many
scholars in this area emphasize that providers leveraging more innovative and novel
financial technologies should have competitive advantages in terms of lower costs,
creating more targeted products and services, and reducing time to develop and scal. 44. Philippon Das Frost et al. ((2016); (2019); (2019)). competitive advantages. many
scholars in this area emphasize that providers leveraging more innovative and novel
financial technologies should have competitive advantages in terms of lower costs,
creating more targeted products and services, and reducing time to develop and scal. 44. Philippon Das Frost et al. ((2016); (2019); (2019)). competitive advantages. many
scholars in this area emphasize that providers leveraging more innovative and novel
financial technologies should have competitive advantages in terms of lower costs,
creating more targeted products and services, and reducing time to develop and scal. 45. 45. Priya & Anusha. (2019). New emerging technology all over the world. Financial
technology widely known as Fintech; this brings all the financial companies to use
technology to betterment of services provided by them. this is new emerging technology
all over the world. 45. Priya & Anusha. (2019). New emerging technology all over the world. 3. REFERENCES Financial
technology widely known as Fintech; this brings all the financial companies to use
technology to betterment of services provided by them. this is new emerging technology
all over the world. 46. 46. Q, Bi. (2020). Child and adolescents. SARS-COV-2 have mostly caused mild
respiratory symptoms rather than severe forms like in adults and old age people. 46. Q, Bi. (2020). Child and adolescents. SARS-COV-2 have mostly caused mild
respiratory symptoms rather than severe forms like in adults and old age people. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 37 37 Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
http://journal.hmjournals.com/index.php/JMC
DOI: https://doi.org/10.55529/jmc.32.26.38 Journal of Multidisciplinary Cases
ISSN: 2799-0990
Vol : 03, No. 02 , Feb-Mar 2023
http://journal.hmjournals.com/index.php/JMC
DOI: https://doi.org/10.55529/jmc.32.26.38 47. Qu, C. (2020). high security. fintech is a kind of software which enables modern and
automated financial services with high security, fintech majorly started in 1990 when
importance for internet and e-commerce raised. 47. Qu, C. (2020). high security. fintech is a kind of software which enables modern and
automated financial services with high security, fintech majorly started in 1990 when
importance for internet and e-commerce raised. 48. Saraswathi and Oke. (2013). Identity in India. is evolving and multi-faceted, India has
traditionally been an interdependent society based on collectivist values, close family
relationships and highly prescribed roles determined by gender, caste and position in the
family. 48. Saraswathi and Oke. (2013). Identity in India. is evolving and multi-faceted, India has
traditionally been an interdependent society based on collectivist values, close family
relationships and highly prescribed roles determined by gender, caste and position in the
family. 49. Saraswathi, Parikh, G. a., & Prakash. ( 1999). Peer Groups. Tend to be same-gendered
and have become a stronger influence for higher social classes, but important decisions
such as career choice and mate selection are often seen as the prerogative of Indian
families. 49. Saraswathi, Parikh, G. a., & Prakash. ( 1999). Peer Groups. Tend to be same-gendered
and have become a stronger influence for higher social classes, but important decisions
such as career choice and mate selection are often seen as the prerogative of Indian
families. 50. Saraswathi, V. a. (2002). Adolescence in India. situated within the socio-cultural
context of Indian youth. 3. REFERENCES Adolescence in India is a gendered and class-based
phenomenon that is marked by a discontinuity between childhood and adulthood. 50. Saraswathi, V. a. (2002). Adolescence in India. situated within the socio-cultural
context of Indian youth. Adolescence in India is a gendered and class-based
phenomenon that is marked by a discontinuity between childhood and adulthood. 51. Scott, Van Reenen, and Zachariadis. (2017). Innovation in digital finance can have
long-term positive effects for banking performance. the adoption of SWIFT has large
effects on profitability in the long-term and these profitability effects are greater for
small banks than for large banks and exhibits significant network effects on
performance. p
52. Sharma, Vinod. (2016). tool which supports all financial services. Fintech will
automatically manage money for betterment of society. These innovations will increase
business opportunities more and more. 52. Sharma, Vinod. (2016). tool which supports all financial services. Fintech will
automatically manage money for betterment of society. These innovations will increase
business opportunities more and more. 53. Shin, Lee and. (2018). the internet and the world wide web had changed the finance
industry's face. the internet and the world wide web had changed the finance industry's
face; banking services (savings, transfers), insurance, stock trading was transacted
without the physical location. 53. Shin, Lee and. (2018). the internet and the world wide web had changed the finance
industry's face. the internet and the world wide web had changed the finance industry's
face; banking services (savings, transfers), insurance, stock trading was transacted
without the physical location. 53. Shin, Lee and. (2018). the internet and the world wide web had changed the finance
industry's face. the internet and the world wide web had changed the finance industry's
face; banking services (savings, transfers), insurance, stock trading was transacted
without the physical location. 54. Subedi et al. (2020). E-learning tools. E-learning tools have played a crucial role during
this pandemic, helping schools and universities facilitate student learning during the
closure of universities and schools. 54. Subedi et al. (2020). E-learning tools. E-learning tools have played a crucial role during
this pandemic, helping schools and universities facilitate student learning during the
closure of universities and schools. 54. Subedi et al. (2020). E-learning tools. E-learning tools have played a crucial role during
this pandemic, helping schools and universities facilitate student learning during the
closure of universities and schools. 55. Thakor, A. V. (2019). Effecctive and Efficient. 3. REFERENCES fintech is the use of technology to carry
out financial activities more effectively within a short span of time. 55. Thakor, A. V. (2019). Effecctive and Efficient. fintech is the use of technology to carry
out financial activities more effectively within a short span of time. 55. Thakor, A. V. (2019). Effecctive and Efficient. fintech is the use of technology to carry
out financial activities more effectively within a short span of time. 56. Thompson. (2017). Fintech could facilitate higher payment frequencies. It became
threat for traditional banking service because the easier people to lend money from
Fintech, the more often they will do it. Therefore, it led to reduce in bank deposits. 56. Thompson. (2017). Fintech could facilitate higher payment frequencies. It became
threat for traditional banking service because the easier people to lend money from
Fintech, the more often they will do it. Therefore, it led to reduce in bank deposits. 56. Thompson. (2017). Fintech could facilitate higher payment frequencies. It became
threat for traditional banking service because the easier people to lend money from
Fintech, the more often they will do it. Therefore, it led to reduce in bank deposits. 57. Vijai. (2019). Increase in digital money value like crypto currency, bitcoins and etc. Many start-ups are being benefited by the government policies and the regulations
framed or giving by the RBI. Modernisation will lead to increase in customers, provide
better services in less time. 57. Vijai. (2019). Increase in digital money value like crypto currency, bitcoins and etc. Many start-ups are being benefited by the government policies and the regulations
framed or giving by the RBI. Modernisation will lead to increase in customers, provide
better services in less time. 57. Vijai. (2019). Increase in digital money value like crypto currency, bitcoins and etc. Many start-ups are being benefited by the government policies and the regulations
framed or giving by the RBI. Modernisation will lead to increase in customers, provide
better services in less time. 58. Vives. (2019). The disruptive technologies. The latest technologies that have been
applied for the fintech platform are different from the fintech segments or fintech
business models. The success of the fintech company shows the role of disruptive
technologies in the fintech-outside. 58. Vives. (2019). The disruptive technologies. The latest technologies that have been
applied for the fintech platform are different from the fintech segments or fintech
business models. Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 38 3. REFERENCES The success of the fintech company shows the role of disruptive
technologies in the fintech-outside. 58. Vives. (2019). The disruptive technologies. The latest technologies that have been
applied for the fintech platform are different from the fintech segments or fintech
business models. The success of the fintech company shows the role of disruptive
technologies in the fintech-outside. 59. Website Link https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9759262/ 59. Website Link https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9759262/ 59. Website Link https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9759262/
60. https://journals.sagepub.com/doi/10.1177/2349301120160208 60. https://journals.sagepub.com/doi/10.1177/2349301120160208 Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 38 Copyright The Author(s) 2023.This is an Open Access Article distributed under the CC BY
license. (http://creativecommons.org/licenses/by/4.0/) 38 38 38
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Türkisch-Deutsche Studien
Jahrbuch 2016 Türkisch-Deutsche Studien
Jahrbuch 2016 The Transcultural Critic:
Sabahattin Ali and Beyond herausgegeben von
Şeyda Ozil, Michael Hofmann,
Jens-Peter Laut, Yasemin Dayıoğlu-Yücel,
Cornelia Zierau und Kristin Dickinson Universitätsverlag Göttingen Şeyda Ozil, Michael Hofmann, Jens-Peter Laut,
Yasemin Dayıoğlu-Yücel, Cornelia Zierau, Kristin Dickinson (Hg.)
The Transcultural Critic: Sabahattin Ali and Beyond
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0
International License. This work is licensed under a Creative Commons Attribution-ShareAlike 4.0
International License. This work is licensed under a Creative Commons Attribution-ShareAlike 4.0
International License. Türkisch-deutsche Studien. Jahrbuch 2016
erschienen im Universitätsverlag Göttingen 2017 Türkisch-deutsche Studien. Jahrbuch 2016
erschienen im Universitätsverlag Göttingen 2017 Herausgegeben von
Şeyda Ozil, Michael Hofmann,
Jens-Peter Laut,
Yasemin Dayıoğlu-Yücel,
Cornelia Zierau
und Kristin Dickinson
in Zusammenarbeit mit Didem Uca
Türkisch-deutsche Studien.
Jahrbuch 2016 Universitätsverlag Göttingen
2017 Universitätsverlag Göttingen
2017 Bibliographische Information der Deutschen Nationalbibliothek Bibliographische Information der Deutschen Nationalbibliothek
Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen
Nationalbibliographie; detaillierte bibliographische Daten sind im Internet über
<http://dnb.dnb.de> abrufbar. Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen
Nationalbibliographie; detaillierte bibliographische Daten sind im Internet über
<http://dnb.dnb.de> abrufbar. Türkisch-deutsche Studien. Jahrbuch
herausgegeben von
Prof. Dr. Şeyda Ozil (Istanbul Universität)
Prof. Dr. Michael Hofmann (Universität Paderborn)
Prof. Dr. Jens-Peter Laut (Universität Göttingen)
Dr. Yasemin Dayıoğlu-Yücel (Istanbul Universität)
Dr. Cornelia Zierau (Universität Paderborn) Türkisch-deutsche Studien. Jahrbuch
herausgegeben von
Prof. Dr. Şeyda Ozil (Istanbul Universität)
Prof. Dr. Michael Hofmann (Universität Paderborn)
Prof. Dr. Jens-Peter Laut (Universität Göttingen)
Dr. Yasemin Dayıoğlu-Yücel (Istanbul Universität)
Dr. Cornelia Zierau (Universität Paderborn) Dieses Buch ist auch als freie Onlineversion über die Homepage des Verlags sowie über
den Göttinger Universitätskatalog (GUK) bei der Niedersächsischen Staats- und
Universitätsbibliothek Göttingen (http://www.sub.uni-goettingen.de) erreichbar. Es gelten die Lizenzbestimmungen der Onlineversion. © 2017 Universitätsverlag Göttingen
https://univerlag.uni-goettingen.de
ISBN: 978-3-86395-297-6
ISSN: 2198-5286 Vorwort ....................................................................................................................... 3
Sabahattin Ali’s Translingual Transnationalism
Kristin Dickinson ................................................................................................... 5
Sabahattin Ali’s Views on the Arts and Literature
Sevengül Sönmez ................................................................................................. 13
World Literature (Already) Wrote Back: Sabahattin Ali after Germany
David Gramling/Martina Schwalm ..................................................................... 25
Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations
Kristin Dickinson ................................................................................................. 45
The Political behind the Fur Coat: Sabahattin Ali’s The Madonna in the Fur Coat
and Leopold Sacher-Masoch’s Venus in Furs in an Intertextual Context
İlker Hepkaner ..................................................................................................... 63
Between Languages: Translative Acts in Sabahattin Ali’s Comprehensive
Germanistan Travelogue
Zeynep Seviner ..................................................................................................... 81
Translators’ Introduction to The Comprehensive Germanistan Travelogue
Kristin Dickinson/Zeynep Seviner ...................................................................... 97
The Comprehensive Germanistan Travelogue
Sabahattin Ali ..................................................................................................... 103
Mufassal Cermenistân Seyâhatnâmesi
Sabahattin Ali ...................................................................................................... 111
Deutschsein: Zafer Şenocak’s Poetic and Enlightened Vision of a Cosmopolitan
German Identity
Vera Stegmann .................................................................................................... 119
Transkulturelles Lernen mit mehrsprachigen Bilderbüchern
Serap Atagül/Christian Müller .......................................................................... 139
Interview mit Selim Özdoğan. Erfüllen und Verweigern von Erwartungshaltungen ... Daniel Schreiner.................................................................................................. 161 Inhaltsverzeichnis 2 Almut Küppers/Barbara Pusch/Pınar Uyan Semerci (Hg.) (2016): Bildung in
transnationalen Räumen. Education in Transnational Spaces,
Wiesbaden (297 S.)
Emre Arslan........................................................................................................ 173
Beiträgerinnen und Beiträger ................................................................................... 177
Wissenschaftlicher Beirat ......................................................................................... 179 Vorwort Seit der ersten Ausgabe des Jahrbuches Türkisch-deutsche Studien ist es unser Anliegen,
zu türkisch-deutschen Themen arbeitende Wissenschaftlerinnen und Wissen-
schaftler sowie Nachwuchswissenschaftlerinnen und Nachwuchswissenschaftler
international und interdisziplinär zu vernetzen. An den zurückliegenden sechs
Ausgaben lässt sich erkennen, dass dieses Anliegen erfolgreich umgesetzt werden
konnte und das Jahrbuch auch außerhalb des deutschen Wissenschaftsraums Re-
sonanz fand. Um noch mehr Wissenschaftlerinnen und Wissenschaftlern erleich-
terten Zugang zum Jahrbuch Türkisch-deutsche Studien zu gewähren und damit dem in
den unterschiedlichen Voraussetzungen in Bildungssystemen begründeten Un-
gleichgewicht zu begegnen, haben wir uns mit dieser Ausgabe dazu entschieden,
das Jahrbuch sowohl online frei zugänglich (open access) als auch weiterhin in
Buchform zu publizieren. Als Partner hierfür konnten wir Jens Peter Laut und mit
ihm den Universitätsverlag Göttingen gewinnen. Mit Jens Peter Laut und Cornelia Zierau stoßen mit dieser Ausgabe ein weite-
rer Herausgeber und eine weitere Herausgeberin zum Team, die durch ihre Exper-
tise in ihrem jeweiligen Fachgebiet – der Turkologie und der interkulturellen Lite-
ratur und Didaktik – das Profil des Jahrbuches stärken. Wir sind uns sicher, dass
wir mit diesen Neuerungen ein international noch größeres Publikum erreichen
und dem Anspruch des Jahrbuches noch besser gerecht werden können. Wenn die
jüngsten weltpolitischen Ereignisse eines zeigen, dann ist es, dass der transkulturel-
le Dialog nicht abbrechen darf, auch nicht auf wissenschaftlicher Ebene. Diese Ausgabe hat einen Schwerpunkt zum türkischen Autor Sabahattin Ali,
dessen posthumer Erfolgsroman Kürk Mantolu Madonna (Die Madonna im Pelz-
mantel) zum Großteil in Deutschland spielt. Was sich vordergründig als türkisch-
deutsche Liebesgeschichte liest, hat – wie die Beiträge eindringlich zeigen – viel
weitreichendere Implikationen. Sabahattin Alis Werk, das in den letzten Jahren
immer breiter international rezipiert wird, hat auch über diesen Roman hinaus Vorwort 4 einen Deutschlandbezug, was sich unter anderem in dem hier sowohl im türki-
schen Original als auch erstmals in englischsprachiger Übersetzung abgedruckten
humoristischen Reisebericht aus „Cermenistan“ zeigt. Ali war aber auch als literari-
scher Übersetzer aus dem Deutschen tätig. Kristin Dickinson, die Gastherausgebe-
rin des Sabahattin Ali-Teils, geht in ihrer Einleitung zum Thementeil ausführlicher
auf diese Zusammenhänge ein. Abgerundet wird die diesjährige Ausgabe mit einem Beitrag zu Zafer Şenocaks
Essaysammlung Deutschsein von Vera Stegmann, einem Review Essay zum transkul-
turellen Lernen mit Bilderbüchern von Serap Atagül und Christian Müller, einem
Interview mit dem Autor Selim Özdoğan und einer Rezension zum Band Bildung in
transnationalen Räumen von Emre Aslan. Şeyda Ozil, Michael Hofmann und Yasemin Dayıoğlu-Yücel Vorwort Wir danken sowohl v & r unipress, wo wir bislang kompetent betreut wurden,
als auch unserem neuen Verlag, dem Universitätsverlag Göttingen, für den unkompli-
zierten, fließenden Übergang. Weiterhin gilt unser Dank Katja Korfmann und
Didem Uca für ihren anhaltenden Einsatz bei der redaktionellen Mitarbeit. Şeyda Ozil, Michael Hofmann und Yasemin Dayıoğlu-Yücel Istanbul, Paderborn
Oktober 2016 Istanbul, Paderborn
Oktober 2016 Sabahattin Ali’s Translingual Transnationalism Kristin Dickinson A seminal author of early 20th-century Turkish literary modernism, Sabahattin
Ali’s (1907-1948) life and work attest to his multifaceted interests and talents; in
addition to poetry, short stories, and novels, Ali published satirical journalistic
prose, worked as a literary translator and simultaneous interpreter, and produced a
significant portfolio as an amateur photographer. While newly edited volumes of
Ali’s articles (2009), court documents and prison notes (2004), and letters (2008),
together with an exhibition of his photographs (2012) have begun to shed light on
the complexity of his career, secondary scholarship on his literary output remains
limited. Following literary critic Berna Moran’s lead, scholarship has tended to empha-
size Ali’s centrality to the development of social realist literature in Turkey (Moran
1990). The 2014 edited volume Sabahattin Ali: Anılar, İncelemeler, Eleştiriler offers a
welcome array of new approaches to this significant but largely under-researched
figure, with contributions on the progressive nature of his female characters, fa-
mous settings of his poetry to music, and his satirical contributions to the journal
Markopaşa. This special issue of the Jahrbuch Türkisch-deutsche Studien aims to open
new avenues of interpretation by situating Ali’s life and work in a transnational
context, with particular foci on his relationship to German literature and culture,
and the centrality of translation and intertextuality to his literary legacy. In explor-
ing precisely these curiously understudied aspects of Ali’s oeuvre, this issue also
aims to create a new body of criticism around this seminal figure for readerships
that are are unable to access his texts in Turkish. Kristin Dickinson 6 In her introductory article for this special issue, Sevengül Sönmez examines
Ali’s understanding of literature and the arts as agents of social change. Drawing
on Ali’s interviews and personal correspondence, Sönmez demonstrates his refusal
to cater to the public and highlights a crucial shift across the arc of his career from
social realism to critical realism. As such, Sönmez provides critical background
material for the articles that follow. The remaining articles in this special issue fo-
cus on the wider implications of Ali’s work for Turkish and German Studies, world
literature, and translation theory; in doing so, each article recognizes Ali as both an
author marked by the turbulent political context in which he was born, as well as
an intellectual who was well ahead of his time. 1 Reforms included but were not limited to the adoption of the European 24-hour day, a new system
of secular primary and secondary schools, creation of a family law, increased women’s rights, abol-
ishment of the Şeriat courts, and adaptation of the Swiss Civil Code. 2 For an insightful discussion of this term in relation to Ali’s work, see Erika Glassen’s afterword to
the German translation of Içimizdeki Şeytan (2007) (translated as Der Dämon in uns). Sabahattin Ali’s Translingual Transnationalism Recent translations into French (2007), German (2008), Russian (2010),
Albanian (2010), Croatian (2012), Arabic (2012), and English (2016) further attest
to a noticeable turn in this reception history and a burgeoning international interest
in Ali’s work. All contributions to this issue recognize that, as a semi-autobiographical ac-
count of Ali’s experiences in Weimar Berlin, Madonna is significant for understand-
ing Ali’s relationship to Germany. With their diversity of interpretations, articles
reveal the wide-ranging implications of Madonna’s cross-cultural love story; due to
the subtleties of its form and storyline, which actively reflect on processes of mod-
ernization in the Republic of Turkey (Hepkaner) and the ethics of translation in an
era of nationalized monolingualism (Gramling / Schwalm), this novel fell through
the cracks of state-censorship. As such, Madonna provides a refreshing intervention
into ongoing debates about the status of ‘non-Western’ translated texts in a canon
of World Literature with a historically Eurocentric basis. At the same time, contributors prod beyond Madonna to explore how Ali’s en-
gagement with German language and culture allowed him to critically reflect on the
fast-paced restructuring of Turkish society vis-à-vis the model of a monolingual,
Western European nation-state. Zeynep Seviner examines the little-known text
Mufassal Cermenistan Seyahatnamesi (1929) (The Comprehensive Germanistan Travelogue),3 a
private letter detailing Ali’s first impressions of Germany. Modeled on 17th-century
Ottoman travelogues and composed in the Ottoman riqa style, the Seyahatname
describes an alcohol-ridden New Year’s Eve party in Potsdam; Seviner reads this
mismatch of form and content as a method of making visible the act of translation,
understood not simply as an act of linguistic transfer, but rather as a process of
estrangement. Written on the heels of Turkey’s alphabet reform—which replaced
the Perso-Arabic script of Ottoman Turkish with Latin letters—Ali’s letter calls
attention to language as an arena of power contest in early Republican Turkey,
within which translators functioned as crucial agents of change. Kristin Dickinson and Zeynep Seviner’s English translation of the Seyahatname
appears alongside Seviner’s article in this special issue. With its ironic adaptation of
genre conventions and its skillful implementation of word play, Ali’s Seyahatname is
a challenging text to translate. While the English translation cannot always do jus-
tice to the linguistic and historical specificities of the original, it does provide a
strong metaphorical extension of the interlingual and intercultural elements of the
Seyahatname. Sabahattin Ali’s Translingual Transnationalism In his youth and adolescence, Ali
experienced World War I (1914-1918), the Turkish War of Independence (1919-
1923), and the transformation of the Ottoman Empire into the modern Republic
of Turkey (1923); in his late teens and early 20s, the newly founded Republic un-
dertook significant legal, political, cultural, and linguistic reforms aimed at moder-
nizing and westernizing Turkish society.1 Ali was thus of a generation trained in
the Ottoman language and its literary traditions, which also actively partook in
modernization processes that sought to overwrite them. While Ali was a seminal author of the early Republican period, his diverse oeu-
vre attests to his ambivalent stance toward the large-scale cultural reforms at hand. As a committed socialist well read in Marxist literature, Ali’s short stories and first
novel, Kuyucaklı Yusuf (1937), were indeed central to the establishment of social
realism (toplumsal gerçekcilik) in Turkey. Yet his literature covers a diverse range of
subject matters, from the social fabric of rural Anatolian life to the intellectual and
bohemian circles of pre-World War II Istanbul. Drawn to social outsiders and
lonesome figures on the margins of society, Ali weaves socially critical information
into his characters’ inner monologues, identity crises and ill-fated love stories, cre-
ating a form of social commentary his good friend and fellow author Pertev Naili
Boratav described as psychological realism.2 In this regard, his novels İçimizdeki
Şeytan (1940) (The Devil Within Us) and Kürk Mantolu Madonna (1943) (Madonna in the
Fur Coat), were both central to the development of literary modernism in Turkey. At the same time, his work critically engages with issues that transcend the
boundaries of a national literary canon. Just as Ali utilized his knowledge of Ger-
man to explore the classics of Russian literature and Greek antiquity, his own liter-
ary output attests to the mutual mediation of literatures and cultures through their
international circulation and translation. Madonna is a case in point. At the time of
its serialized publication in Hakikat newspaper (1940-41), this novel received little
to no critical attention. The novel’s initial non-reception was compounded by Ali’s
mysterious murder on the Bulgarian border in 1948, which led to a ban on the sale 7 Sabahattin Ali’s Translingual Transnationalism and further publication of his literature until 1965. This novel has made a recent
comeback and has been among the top ten bestselling books in Turkey for the past
five years. 3 This letter was first published in transliteration in the edited volume Sabahattin Ali (1979). 4 Ali was one of 15 intellectuals sent abroad; five scholarships were granted for Germany, France, and
England, respectively. Sabahattin Ali’s Translingual Transnationalism By making this text available to an English-speaking audience, this
issue further aims to highlight Ali’s witty experimentation with Ottoman literary
forms, an aspect of his diverse oeuvre that has received little critical attention to
date. Kristin Dickinson 8 In contrast to the Seyahatname—which was never intended for publication—Ali
maintained a highly visible role as literary translator for the state-run translation
bureau from 1940-1943. Through a case study of Ali’s engagement with Kleist,
Dickinson argues that his actual translation practice undermined the dominant
paradigm of civilizational transfer that undergirded larger humanist reforms at the
time; Ali’s legacy thus provides fertile ground for sketching an alternative history
of translation in the Republic of Turkey that worked against the official form of
culture planning endorsed by the state. In her analysis, Dickinson shows how Ali’s
translation practice informed and overlapped with his literary production, both of
which participated in the humanist reform process, while also critiquing them from
within. Situating Ali in a Transnational Context As part of a larger initiative aimed at creating a new Turkish intellectual youth edu-
cated in Western European languages, Ali received a four-year government grant
to study language, literature, and philosophy in Berlin and Potsdam in 1928 at the
age of 21.4 While Ali broke off his studies in Germany after only one and a half
years, his experiences abroad made an indelible impact on his life and work. The
German language served not only as a point of departure into German literature
and culture; Ali also read the great works of Russian literature—such as those by
Gogol, Tolstoy, Turgenev, Chekhov and Gorky—in German translation, and
translated works of antiquity—such as Sophocles’ Antigone—into Turkish from
the German. It was precisely Ali’s unbridled passion for literature and language learning that
gained him a position as a government employee for the fledging Republic of Tur-
key. Even as he was later imprisoned for his politically critical writings, his German
skills did not go unnoticed by the state: he worked as a German teacher in Aydın
(1930), Konya (1931), and Ankara (1935), and was often called upon as an expert
of German literature for government sponsored projects, such as the İnönü Ansi-
klopedisi (Inönü Encyclopedia); he was further employed as a simultaneous interpreter
for the German-Jewish exile and dramaturg Carl Ebert at the Ankara State Con-
servatory, and he was a founding member of the state-funded translation bureau in
1940. The diverse positions Ali held as a civil servant were enabled by wide-reaching
humanist cultural reforms that targeted the publishing and education sectors. Initi-
ated by Minister of Education (Maarif Vekili) Hasan Ali Yücel in 1939, reforms
included the establishment of village institutes that trained and enabled teachers to
establish local schools (1940), a translation bureau (1940), a state conservatory
(1941), and a national library (1946). Reforms enacted in the 1940s were preceded 9 Sabahattin Ali’s Translingual Transnationalism 9 by a crucial overhaul of the Darülfünun (House of Knowledge, established in 1863),
the first institution of higher education in the Ottoman Empire modeled on the
European university system. Refounded as İstanbul Üniversitesi (Istanbul Univer-
sity) in 1933, the restructuring of this university to promote the Europeanization of
scholarship and disciplinary practices was greatly aided by prominent German-
Jewish academic émigrés escaping National Socialism. Situating Ali in a Transnational Context Amid rampant state censorship, Ali’s presumed love-story thus Kristin Dickinson 10 subtly protests the use of art as a yardstick for measuring one’s loyalty as a citizen. In conclusion, Hepkaner considers the stakes of Ali’s engagement with German
and Austrian literary culture at a time when the Turkish government was openly
sympathetic to Nazi Germany. subtly protests the use of art as a yardstick for measuring one’s loyalty as a citizen. In conclusion, Hepkaner considers the stakes of Ali’s engagement with German
and Austrian literary culture at a time when the Turkish government was openly
sympathetic to Nazi Germany. On the flipside of this discourse, Gramling and Schwalm consider the stakes of
reading Madonna from a contemporary standpoint, at the interstices of compara-
tive, translation, postcolonial, and multilingualism studies. In their analysis of Ma-
donna’s oft-ignored frame narrative, they show how Ali’s final novel actively theo-
rizes the concept of world literature from its pre-1945 standpoint. Rather than
reading Madonna retroactively through the lens of postcolonial and globalization
studies, they ask how the novel itself theorizes debates regarding world literature
and translation as they were still in the process of being institutionalized. This
question is implicit in all articles presented here; by reading Ali’s translation prac-
tice against the grain of the very nation-building process in which it participated
(Dickinson), or highlighting the processes of translation and intertextuality that
mark Ali’s literary legacy (Seviner and Hepkaner), the articles in this special issue
press us to critically rethink the categories of Turkish, German, and world litera-
ture that Ali worked both within and against. Situating Ali in a Transnational Context Significant research has
been devoted to this aspect of the reform process; the kind of comparative philo-
logical scholarship generated by émigrés and their Turkish colleagues in this time
period has been heralded by scholars such as Emily Apter as representative of
“transnational humanism or global translatio” (2006: 46) and a foundational mo-
ment for the contemporary field of Comparative Literature. In her detailed analysis of the time period, Kader Konuk nevertheless reveals
how the kind of humanism that emerged in Turkey during the 1930s and 40s
served primarily national, rather than transnational interests. Transnationalism, she
writes, implies “the outcome of an exchange between individuals and communities,
independent of the interests of nation-states” (2010: 75), through which individual
actors exercise their agency to transgress national borders. On the contrary, the
Notgemeinschaft der Deutschen Wissenschaft (Emergency Association of German Sci-
ence), which facilitated the hiring of German-Jewish émigrés at both Istanbul Uni-
versity and later diverse institutions throughout Turkey, negotiated directly with
representatives of the Turkish nation-state. Throughout this process, German
academics were not “rescued” on humanitarian grounds, but rather were often
instrumentalized for larger political processes, as they were carefully selected for
their academic qualifications and potential to modernize and Europeanize the sec-
ondary education system in Turkey (Konuk 2010: 75). Overall, Konuk argues, the
humanist reforms were part of a national agenda that linked its success to its capacity for
overcoming cultural differences between East and West. The moderniza-
tion reforms promoted sameness with Western Europe but simultaneously
maintained a notion of national particularity (Konuk 2010: 74). As the articles in this issue show, Ali’s engagement with German literature and
culture goes beyond the kind of Europhilia officially endorsed during the time
period to offer a critical view of both Germany and Turkey’s relationship to it. İlker Hepkaner’s article in particular considers the political implications of the
complex formal and thematic connections between Madonna and Leopold von
Sacher-Masoch’s Venus im Pelz (1870). Writing in an ultranationalist context, Ali
was frequently imprisoned for his artistic representations of political dissidents or
social outcasts deemed to cast the state in a negative light; it is precisely by engag-
ing with the ekphrastic and intertextual elements of Venus in relation to Madonna,
Hepkaner argues, that Ali is able to critique the Turkish government’s conflation
of art with reality. Moran, Berna (1990): “Soylu Vahşi Olarak Kuyucaklı Yusuf,” in: Türk Edebiyatına
Eleştirel Bir Bakış 2: Sabahattin Ali’den Yusuf Atılgan’a, İstanbul, 24-30. Works Cited Apter, Emily (2006): The Translation Zone: A New Comparative Literature, Princeton Apter, Emily (2006): The Translation Zone: A New Comparative Literature, Princeton. Ali, Filiz / Özkırımlı, Atilla / Sönmez, Sevengül (eds.) (2014): Sabahattin Ali:
Anılar, İncelemeler, İstanbul. Ali, Filiz / Özkırımlı, Atilla / Sönmez, Sevengül (eds.) (2014): Sabahattin Ali:
Anılar, İncelemeler, İstanbul. Ali, Sabahattin (2008): Kürk Mantolu Madonna, İstanbul. Ali, Sabahattin (2008): Hep Genç Kalacağım: Bütün Yapıtları Mektup, edited by
Sevengül Sönmez, İstanbul. Ali, Sabahattin (2004): Mahkemelerde: Belgeler, edited by Nüket Esen and Nezihe
Seyhan, İstanbul. Ali, Sabahattin (2009): Markopaşa Yazıları ve Ötekiler: Bütün Yapıtları Yazılar, edited
by Sevengül Sönmez and Hikmet Altınkaynak, İstanbul. Ali, Sabahattin (1979): “Mufassal Cermenistan Seyahatnamesi,” in: Ali, Filiz Laslo /
Özkırımlı, Atilla (eds.): Sabahattin Ali: Anılar, İncelemeler, Eleştiriler, İstanbul, 386-
396. Glassen, Erika (2007): “Nachwort,” in: Ali, Sabahattin: Der Dämon in Uns,
translated by Ute Birgi-Knellessen, Zürich, 331-434. Konuk, Kader (2010): East West Mimesis: Auerbach in Istanbul, Stanford. Sabahattin Ali’s Translingual Transnationalism Sabahattin Ali’s Translingual Transnationalism 11 1 This article stems from a keynote given by Sevengül Sönmez at the conference Transnational Perspe-
citives on the Life and Work of Sabahattin Ali at New York University in 2015. The keynote was originally
given in Turkish; many thanks to Ali Bolcakan and İlker Hepkaner for their translation of this speech
into English. 2 While Sabahattin Ali took the surname Ali, he chose to not use it as such. For this reason, I refer to
him with his full name Sabahattin Ali throughout this article. 1 This article stems from a keynote given by Sevengül Sönmez at the conference Transnational Perspe-
citives on the Life and Work of Sabahattin Ali at New York University in 2015. The keynote was originally
given in Turkish; many thanks to Ali Bolcakan and İlker Hepkaner for their translation of this speech
into English.
2 While Sabahattin Ali took the surname Ali, he chose to not use it as such. For this reason, I refer to
him with his full name Sabahattin Ali throughout this article Sabahattin Ali’s Views on the Arts and Literature1 Sevengül Sönmez A significant amount of scholarship on Sabahattin Ali2 revolves around his life
story, or rather, the sorrowful story of his death. As a result, we still lack a com-
prehensive study on Sabahattin Ali’s corpus as a whole. Additional research on
Sabahattin Ali’s diverse body of work is thus imperative for our understanding of
the contributions he made to, and the changes he affected within, modern Turkish
literature. While recent critical editions such as Sabahattin Ali: Anılar, İncelemeler,
Eleştiriler (2014) have begun to address this lacuna, there is much work left to be
done. Within a critical body of scholarship still in the making, special issues such as
“Sabahattin Ali’s Translingual Transnationalism” play a crucial role in introducing
Sabahattin Ali to an English speaking readership and helping us to understand the
unique impact Sabahattin Ali had on the field of modern Turkish literature via an
emphasis on the transnational aspects of his work. This article offers a broad and
comprehensive introduction to Sabahattin Ali’s views on literature and the arts in
order to provide key background information for the articles that follow. Sabahattin Ali’s close friends tell us that he worked on his novels diligently and
that he desired to write a number of additional novels (Ergün 2000). These sugges-
tions are corroborated by the fact that a list of planned novels replete with titles
and brief notes was among Sabahattin Ali’s possessions when he was murdered. 14 Sevengül Sönmez Even though Sabahattin Ali was never able to bring these novels to fruition, the
diverse body of work he did leave behind made a profound impact on the field of
modern Turkish literature. Indeed, Sabahattin Ali wrote two novels that changed
the course of Turkish literature: Both Kuyucaklı Yusuf (Yusuf from Kuyucak) and Kürk
Mantolu Madonna (The Madonna in the Fur Coat) take a different approach than previ-
ous Turkish-language novels, as their realist narrative and sincere style succeed in
capturing their audiences and differentiating the novel from the romance genre. Despite their resemblance to romance due to formal and affective patterns, these
two novels can be distinguished from the genre’s conventions thanks to the in-
depth formation of their characters. In a limited number of studies, scholars have observed how Sabahattin Ali’s
perception of literature evolved from social realism to critical realism over the
course of his career (Bezirci 1987: 168-171). 4 “Sanatın gayesi de her içtimai fiil gibi, cemiyet olduğuna göre, gelecek büyük sanatkârların içtimai
taraflarının çok kuvvetli olacağı ve mazinin ve halin zengin hazinelerinden toplanarak kendi
kafalarının genişliği, dehalarının derinliği içinde yoğuracakları büyük meseleleri insanlığa ileri ve yukarı
doğru birer adım daha attıracak eserler halinde ortaya koyacakları şüphesizdir. Benim kanaatimce
sanat, insana insanı ve hayatı ve bunların manasını öğretmekle muvazzaftır.” (Yazar 1938: 372-373) 3 “Ben hiçbir zaman sanatın maksatsız olduğuna kani olmadım. Sanatın bir tek ve sarih bir maksadı
vardır: İnsanları daha iyiye, daha doğruya, daha güzele yükseltmek; insanlarda bu yükselme arzusunu
uyandırmak.” (Reşit 1936) 3 “Ben hiçbir zaman sanatın maksatsız olduğuna kani olmadım. Sanatın bir tek ve sarih bir maksadı
vardır: İnsanları daha iyiye, daha doğruya, daha güzele yükseltmek; insanlarda bu yükselme arzusunu
uyandırmak.” (Reşit 1936)
4 “Sanatın gayesi de her içtimai fiil gibi, cemiyet olduğuna göre, gelecek büyük sanatkârların içtimai
taraflarının çok kuvvetli olacağı ve mazinin ve halin zengin hazinelerinden toplanarak kendi
kafalarının genişliği, dehalarının derinliği içinde yoğuracakları büyük meseleleri insanlığa ileri ve yukarı
doğru birer adım daha attıracak eserler halinde ortaya koyacakları şüphesizdir. Benim kanaatimce
sanat, insana insanı ve hayatı ve bunların manasını öğretmekle muvazzaftır.” (Yazar 1938: 372-373) Sabahattin Ali’s Views on the Arts and Literature1 While this analysis is well-founded for
his short stories, we still need new interpretations of his novels. In the following, I
draw on Sabahattin Ali’s personal correspondences to provide a starting point for
this much-needed scholarly work. Throughout his life, Sabahattin Ali read con-
stantly and vocally shared his ideas with others; he openly expressed his ideas on
art and literature and answered questions addressed to him regarding this subject
with great sincerity. In an interview published in Varlık magazine in 1936, for ex-
ample, Sabahattin Ali clearly explains his approach to the arts: “I have never been a
partisan of purposeless arts. The arts have a single and clear reason: elevating peo-
ple towards the better, more accurate and more aesthetic; awakening this desire of
elevation.”3 Two years after this interview, he expressed the purpose of the arts
more clearly in a letter he penned for a book that Mehmet Behçet Yazar was pre-
paring: Since art is aimed, like all other social activities, at society, all future major
artists will undoubtedly have a very strong social quality. They will pro-
duce artworks expressing important issues, woven with the rich treasures
of the past and the present within the depth of their geniuses and within
the breadth of their mind. These artworks will strive to give humanity a
leap upward and forward. In my opinion, the arts have the duty of teach-
ing people about humanity, life, and their meaning.4 Sabahattin Ali’s Views on the Arts & Literature 15 Sabahattin Ali viewed both the arts and literature as socially purposeful and politi-
cal activities: “Literature,” he stated, “is also a service and a struggle. And it is a
struggle that brings us to the aforementioned goal, to the better and more aesthet-
ic; and it is never for the entertainment of the bourgeoisie.”5 In this same interview
Sabahattin Ali also ridicules self-proclaimed authors of the people and the idea of
catering to the public. Indeed, the inauthenticity of literati was a major issue for
him and he advocated strongly for the production of artworks that take issue with
society and create change: Our most ridiculous authors are the ones who think they are writing for
the people. 5 “Edebiyat da bir hizmet ve bir mücadeledir. Ve yukarıda söylediğim şeye, daha iyiye, daha güzele
götüren bir mücadele ve hiçbir zaman yüksek ruhlu bay üdebanın gönül eğlencesi değil.” (Reşit 1936)
6 “Kitle için yazdıklarını zanneden muharrirlerimiz ise en gülünç olanlarıdır. Kitle ile beraber ıstırap
çekmeyen, halkın sevinci ile yüzü gülüp onun isyanı ile şaha kalkmayan, nabzı kitlenin nabzıyla aynı
tempoda atmayan adamın kitleye ‘sen’ diye hitap etmesi hatta gülünçten de ileri bir şeydir. Hâlâ
köylüyü Amerikalı bir seyyah gözüyle seyredip onda ya mistik karanlık bir ruh veya iptidai bir hayvan
gören büyük romancılarımız var. Halktan bahsediyorum diyen yabancı ve ucuz esprili hikâyelerle halı
maskaraya çeviren meşhur muharrirlerimiz var. Cinsi ihtiraslardan histeriye uğramış yarım tahsilli genç
kızlar için yazdığı sulu romanının ciltlerine dayanarak kendisine ‘en çok okunan halk muharriri’ sıfatını
takan şımarık şarlatanlar var. Edebiyatımızla okurlar kitlesi arasındaki boşluğu bunlar mı dolduracak.”
(Reşit 1936) (
)
7 “Sanatta bulunmamasını lüzumlu gördüğüm bu hayır, muayyen bir manada belki de ‘tez’dir.” (Nazif
1938) Sabahattin Ali’s Views on the Arts and Literature1 When someone who doesn’t suffer with the masses, who nei-
ther celebrates nor rebels with the people, who has a totally different
heartbeat than theirs, addresses the society with the singular you, it is
something beyond ridiculous. We still have novelists who look at the vil-
lager from an American tourist’s point of view, and see a dark and myste-
rious soul or a primitive animal in them. We have famous authors who
claim to be narrating society while turning them into laughingstocks with
stories of cheap and strange humor. There are spoiled charlatans who pro-
claim themselves “the most read author by the public” based on volumes
of wishy-washy novels they wrote for half-educated young girls going
through hysteria due to their sexual desires. Are these novelists the ones
who will fill the gap between our literature and the masses?6 Over the course of Sabahattin Ali’s career, he shifted from advocating for social
realism to advocating for critical realism, and held a distinct position among au-
thors who thought similarly and wrote for society. Sabahattin Ali describes the
difference between social and critical realism as the following: “This prosperity that
I see as unnecessary in the arts is perhaps a ‘thesis’ in a determinate sense.”7 This
statement embodies the quality that distinguishes him from his contemporary and
successor social realist literati and gives Sabahattin Ali the unique place he holds in
Turkish literature. 16 Sevengül Sönmez For Sabahattin Ali, the arts must be objective, accessible, sincere, and compre-
hensible. This understanding of the arts put him in a privileged position in another
field in which he became successfully involved: photography. In his evaluation of
Sabahattin Ali’s photography, İsa Çelik explains the similarity between his literary
works and his photographs as such: In his photographs, just as is widely observed in his literary works, we
can see that instead of relying on literary artistry, he prioritized the ‘mar-
ginal benefit’ that the reader -the society- will acquire from the social and
political analysis of a condition or occasion recounted in simple lan-
guage.8 Taking a clear stance toward the definition and function of literature, Sabahattin
Ali made the following statement in an interview he gave in 1935: Today, we don’t have a corpus we can call ‘literature.’ There are a couple
of individuals who write well or poorly, but they are not even affiliated
with each other. 8 “Yazılarında belirgin biçimde görülen, edebi sanatlara yaslanmak yerine yalın, duru bir dille olayın ya
da durumun sosyal ve politik çözümlemelerinden okuyucunun—toplumun—alacağı ‘marjinal
fayda’nın önde tutulması fotoğraflarında da görülüyor.” (Çelik 1984) 9 “Bugün edebiyat denecek toplu bir şeyimiz yoktur. İyi veya fena yazan birkaç şahıs var ki birbiriyle
münasebettar bile değiller. Şiir olsun, nesir olsun, yazanın kafasının dar ve ukala hududunu aşabilip
halka yükselen ve şekil, ruh, fikir itibarıyla bir kuvvet ve başarma gösteren ve etrafında bir fikir grubu
toplayabilecek olan Türkçe bir forma bile okumadım.” (Aygün 1935)
10 “Bugünkü roman ve şiirimizi ele alınca şiirimizde tek tük ileri hamleler görüyoruz. Fakat romanda
daha doğrusu alelumum epik sahada, üstüne düşen vazifeleri kısmen dolsun yapmış bir eser ortada
yoktur. Biraz acı ama bu böyle.” (Reşit 1936) p y
ç
( yg
)
10 “Bugünkü roman ve şiirimizi ele alınca şiirimizde tek tük ileri hamleler görüyoruz. Fakat romanda
daha doğrusu alelumum epik sahada, üstüne düşen vazifeleri kısmen dolsun yapmış bir eser ortada
yoktur. Biraz acı ama bu böyle.” (Reşit 1936) “Bugün edebiyat denecek toplu bir şeyimiz yoktur. İyi veya fena yazan birkaç şahıs var ki birbiriyl
ünasebettar bile değiller. Şiir olsun, nesir olsun, yazanın kafasının dar ve ukala hududunu aşabili
lka yükselen ve şekil, ruh, fikir itibarıyla bir kuvvet ve başarma gösteren ve etrafında bir fikir grub
playabilecek olan Türkçe bir forma bile okumadım.” (Aygün 1935) 8 “Yazılarında belirgin biçimde görülen, edebi sanatlara yaslanmak yerine yalın, duru bir dille olayın ya
da durumun sosyal ve politik çözümlemelerinden okuyucunun—toplumun—alacağı ‘marjinal
fayda’nın önde tutulması fotoğraflarında da görülüyor.” (Çelik 1984)
9 “Bugün edebiyat denecek toplu bir şeyimiz yoktur. İyi veya fena yazan birkaç şahıs var ki birbiriyle
münasebettar bile değiller. Şiir olsun, nesir olsun, yazanın kafasının dar ve ukala hududunu aşabilip
halka yükselen ve şekil, ruh, fikir itibarıyla bir kuvvet ve başarma gösteren ve etrafında bir fikir grubu
toplayabilecek olan Türkçe bir forma bile okumadım.” (Aygün 1935)
10 “Bugünkü roman ve şiirimizi ele alınca şiirimizde tek tük ileri hamleler görüyoruz. Fakat romanda
daha doğrusu alelumum epik sahada, üstüne düşen vazifeleri kısmen dolsun yapmış bir eser ortada
yoktur. Biraz acı ama bu böyle.” (Reşit 1936) Sabahattin Ali’s Views on the Arts and Literature1 Whether poetry or prose, I haven’t read even a pam-
phlet in Turkish that can reach out to the public over its author’s limited
mind and smart-alecky measures, constitute a strength and success re-
garding form, essence, and thought, and is capable of having an opinion-
ated group rally around it.9 In the interview cited above with Varlık magazine in 1936, Sabahattin Ali offers a
milder view on the contemporary novel and poetry: “We see a few progressive
moves in our contemporary novel and poetry. But in the novel or specifically in
the epic, there’s currently no work that fulfills its responsibilities even partially. It’s
disheartening, but that’s the way it is.”10 In the interview cited above with Varlık magazine in 1936, Sabahattin Ali offers a
milder view on the contemporary novel and poetry: “We see a few progressive
moves in our contemporary novel and poetry. But in the novel or specifically in
the epic, there’s currently no work that fulfills its responsibilities even partially. It’s
disheartening, but that’s the way it is.”10 In his letter to the owner of the newspaper Hakikat (Truth), Cemal Hakkı Se-
lek, Sabahattin Ali claims that the serialized publication of his novels in newspapers
did not diminish the value of his writings, and that he would never let his ideas be
compromised; just because his writings appeared in the newspaper, this did not
mean he diverged from his sense of art, and unlike some other novelists, he wasn’t
writing popular novels to appease the public. Sabahattin Ali composed this letter Sabahattin Ali’s Views on the Arts & Literature 17 on February 10, 1941, after he did not receive payment for his novel Kürk Mantolu
Madonna, which was serialized in Hakikat: on February 10, 1941, after he did not receive payment for his novel Kürk Mantolu
Madonna, which was serialized in Hakikat: For the first time in my life as an author, I was told that my writings didn’t
generate interest. Why? Nobody felt the need to investigate this. Was it re-
ally just that the novel didn’t stick? If it indeed did not, does the fault lie
with the novel or with the quality of the readers of Hakikat newspaper? 11 “Yazı hayatımda ilk defa olarak yazımın tutmadığı suratıma çarpıldı. Neden? Bunu araştırmaya
lüzum bile hissedilmedi. Acaba roman hakikaten tutmadı mı? Tutmadı ise kabahat romanda mı
Hakikat gazetesi karilerinin seviyesinde mi? Benim şimdiye kadar intişar etmiş bulunan eserlerim
meydanda olduğuna göre, benden gazeteniz için yazı isterken İskender Fahrettin, Esat Mahmut
beylerden veya Peride Celal, Kerime Nadir, Mükerrem Kamil hanımlardan bekleyeceğiniz neviden bir
roman istemiş olamayacağınız aşikardır. Akşam gazeteleri karileri ancak bu nevi yazıları tutuyorlarsa
kabahat bende mi? Sanatı üzerine benim kadar titreyen ve bunu “talebe muvafık emtia” haline
getirmekten benim kadar kaçan bir insana, eliniz titremeden “roman maalesef tutmamıştır” diye
yazarken ne yaptığınızın farkında mı idiniz?” (Sabahattin Ali 2008: 366) 12 “Romanın ne demek olduğunu anlatmaya çalışacak değilim. Yalnız şunu söyleyeyim ki; roman
Türkiye’de kari bulamamış değildir. İyi roman daima hitap edecek bir kitleye malik olagelmiştir. Türki-
ye’de, kötü muharrirlerin karilerin zevkine her gün yaptıkları suikastlara rağmen bir kari buhranı
yoktur. Eser buhranı vardır. İyi eseri kari tutmuyor demek için evvela iyi eseri ortaya atmak lazımdır.
Hani? Bunu yapmadıkça karii kabahatli bulmak, aczimize pek acemice bir bahane bulmaktan ileri
geçmez.” (Nazif 1938) Sabahattin Ali’s Views on the Arts and Literature1 Since my work has been publicly available for some time, when you asked
me to write for your newspaper, it’s perfectly obvious that you wouldn’t
ask for a novel in the vein of Iskender Fahrettin, Esat Mahmut, Peride
Celal, Kerima Nadir and Mükerrem Kamil. If the readers of the evening
newspapers only like this kind of writing, does the fault lie with me? Did
you know what you were doing to someone like me who cares a great deal
about his art and takes pains to not turn it into a “commodity that meets
demand” when you unabashedly wrote “sadly people didn't care for it.”11 Not believing that Kürk Mantolu Madonna did not attract attention, Sabahattin Ali
described readership and the state of the Turkish novel in the following way in an
interview from 1938: I’m not going to try to explain what the novel means. But I will say this;
it’s not that the novel didn’t find a readership in Turkey. A good novel was
always able to reach the masses. In Turkey, despite constant attacks on
readers by bad writers, there’s not a crisis of readership. There’s a crisis of
work. To be able to say that a good work can’t find a readership, it is nec-
essary to produce a good work to begin with. But where is it? Without
producing such a work, placing blame on the readership is nothing more
than coming up with an inept excuse for our own incompetence.”12 18 Sevengül Sönmez Despite this dismal portrayal of the Turkish literary scene, it is notable that Sa-
bahattin Ali included Nâzım Hikmet in the list of five works that he loved the
most and read repeatedly. These included: “Gorky, Life of Klim Samgin; Mikhail
Sholokhov, The Quiet Don; Nâzım Hikmet, Letters to Taranta-Babu; Andre Malraux,
Man’s Fate; Dostoyevsky, The Idiot” (Aygün 1935). Nâzım Hikmet himself remarked
on Sabahattin Ali's favorite foreign authors as such: He was in awe of the German Romantics until the end of his life. He liked
French literature, especially the French Realists. But we can’t say that
French literature influenced him deeply. His encounter with classical Rus-
sian literature, especially with Gogol, Tolstoy, Turgenev, Chekhov and
Gorky had impacts on not only his literary but also his social activities as
well. 13 “Ömrünün sonuna kadar Alman romantiklerinin hayranı kaldı. Fransızları hele Fransız realistlerini
çok severdi. Ama üzerinde Fransız edebiyatının büyük bir etkisi olmuştur denemez. Klasik Rus
edebiyatıyla hele Gogol, Tolstoy, Turgenyev, Çehov ve Gorki ile tanışması yalnız edebiyat değil,
sosyal çalışmaları üstünde de etkili olmuştur. Sovyet yazarlarından Şolohov’u çok sever, onu büyük
Rus klasikleri değerinde sayardı.” (Ali-Laslo / Özkırımlı 1979: 13) 14 “Koltuğunun altında her zaman kitaplar vardı ve çoğu defa kalın bir sözlük… Durmadan okuyor-
du. Koltuğunun altındaki kitaplar ile kurstaki arkadaşların alay konusu olmuştu. Ama bunlara hiç
aldırmaz, alayların üzerinde bile durmazdı.” (Ali-Laslo / Özkırımlı 1979: 63) Sabahattin Ali’s Views on the Arts and Literature1 From the Soviet authors, he loved Sholokhov and considered his
works a part of the great Russian classics.13 The diverse range of authors with whom Sabahattin Ali engaged point to his deep
curiosity for world literature and his passion for reading. Indeed, one of Sabahattin
Ali’s most significant characteristics was his fondness of books. One of the most
basic realities everyone agreed upon in his life was that he read constantly, and
often carried a book in his pocket. Melahat Tolgar, who studied in Germany with
Sabahattin Ali, mentions this in her memoirs: “There were always books under his
arm, or a thick dictionary… He read ceaselessly. The books under his arms became
a running joke among classmates. But this never bothered him; he didn’t even
mind the jokes.”14 Indeed, this joke came to define his life. Years later, in a letter
written from Üsküdar Paşakapı Prison in 1947, he asks—in addition to pajamas,
old yellow shoes, a bathing suit and some underwear—for his spouse Aliye to
bring him the following books: Bros, Der Pharao; Ehrenburg, Der Fall von Paris;
Steinbeck, The Grapes of Wrath; Lofts, White Hell of Pity (Sabahattin Ali 2008: 510). Sabahattin Ali was also murdered while reading a book; among his possessions at
the time were a Balzac novel and Pushkin’s Eugene Onegin. g
g
In her memoirs, Filiz Ali recounts visits to bookstores with her father: As soon as he got his paycheck, he would go to Akba Bookstore in Ulus [An-
kara.] I, too, joined him in this festivity of browsing and purchasing books. He
would let me go to the children’s section and leave me by myself saying “you
pick whatever you want.” Then he would review the books I chose, pick the Sabahattin Ali’s Views on the Arts & Literature 19 useless ones out, but still would say “alright” and buy most of them. By that
time my father would have looked at every new book and magazine in Ger-
man, and couldn’t stop himself from buying most of them. Our bookstore ad-
ventures would last at least two hours.15 useless ones out, but still would say “alright” and buy most of them. By that
time my father would have looked at every new book and magazine in Ger-
man, and couldn’t stop himself from buying most of them. 15 “Aybaşında maaşını alır almaz ilk iş Ulus’taki Akba Kitabevi’ne giderdi. Bu kitap bakma ve satın
alma şenliğine arada sırada ben de katılırdım. Beni çocuk kitapları bölümüne salar, ‘sen istediklerini
seçedur’ diye kendi halime bırakırdı. Sonra da seçtiğim kitapları gözden geçirir, işe yarayan ve
yaramayanları ayırır, yine de pek çoğuna peki deyip alırdı. Babam o sırada yeni gelen Almanca kitap ve
dergilerin hemen hepsini gözden geçirmiş olur, pek çoğunu da dayanamayıp alırdı. Bizim bu kitapçı
serüvenimiz en azından iki saat sürerdi.” (Ali-Laslo / Özkırımlı 1979: 52) 18 “Düşündürücü bir gülümsemeyi boş kahkahalardan üstün tutanlar ve bir tiyatro eserinde kıymet ve
meziyet sahibi gerçek insanlar bulmak isteyenler Lessing’in dehasına ve sanatına hayranlık duymaktan
kendilerini alamazlar” demektedir.” (Lessing 1942) 17 “Odamda Turgenyev’in veya Theodor Storm’un hikâyelerine kapanacağımı düşündükçe”
(Sabahattin Ali 2005: 63). 16 “Üzerimde en çok tesir yapanlar Rus muharrirleriydi. Turgenyev’in koskocaman hikâyeleri
defada sonun kadar okuduğum oluyordu.” (Sabahattin Ali 2005: 55) 15 “Aybaşında maaşını alır almaz ilk iş Ulus’taki Akba Kitabevi’ne giderdi. Bu kitap bakma ve satın
alma şenliğine arada sırada ben de katılırdım. Beni çocuk kitapları bölümüne salar, ‘sen istediklerini
seçedur’ diye kendi halime bırakırdı. Sonra da seçtiğim kitapları gözden geçirir, işe yarayan ve
yaramayanları ayırır, yine de pek çoğuna peki deyip alırdı. Babam o sırada yeni gelen Almanca kitap ve
dergilerin hemen hepsini gözden geçirmiş olur, pek çoğunu da dayanamayıp alırdı. Bizim bu kitapçı
serüvenimiz en azından iki saat sürerdi.” (Ali-Laslo / Özkırımlı 1979: 52)
16 “Üzerimde en çok tesir yapanlar Rus muharrirleriydi. Turgenyev’in koskocaman hikâyelerini bir
defada sonun kadar okuduğum oluyordu.” (Sabahattin Ali 2005: 55)
17 “Odamda Turgenyev’in veya Theodor Storm’un hikâyelerine kapanacağımı düşündükçe”
(Sabahattin Ali 2005: 63).
18 “Düşündürücü bir gülümsemeyi boş kahkahalardan üstün tutanlar ve bir tiyatro eserinde kıymet ve
meziyet sahibi gerçek insanlar bulmak isteyenler Lessing’in dehasına ve sanatına hayranlık duymaktan
kendilerini alamazlar” demektedir.” (Lessing 1942) Sabahattin Ali’s Views on the Arts and Literature1 Our bookstore ad-
ventures would last at least two hours.15 When Sabahattin Ali went to Germany in late 1927, one of the most exciting as-
pects of his trip was learning German and reading books in this language. He be-
came proficient in German within a very short period of time. Reading in German
was like a new world for Sabahattin Ali, and once he had cracked the door open,
he never closed it. Kürk Mantolu Madonna carries traces of his years in Germany and
gives us clues about Sabahattin Ali’s reading habits. The books Sabahattin Ali read
in his boarding house room are the books that Raif Efendi reads in his own board-
ing house room: “The Russian authors were the ones that influenced me the
most;”16 “I could read Turgenev’s lengthy stories to the end in one sitting.”17 g
g
y
g
As Sabahattin Ali’s German improved, his interest in German authors in-
creased. Heinrich von Kleist, whose life and death are narrated by Maria Puder in
Kürk Mantolu Madonna, deeply affected Sabahattin Ali. Upon returning to Turkey,
Sabahattin Ali translated Kleist’s “The Betrothal in St. Domingo” (Sabahattin Ali
1943). As Bulgarian Turcologist Ibrahim Tatarlı mentions, Theodor Storm (1817-
1888) and Jakob Wassermann (1873-1934) also had a tremendous effect on Sa-
bahattin Ali’s literature (Tatarlı 2014). Jakob Wassermann’s “Der niegeküßte
Mund” (A Mouth that Had Never Been Kissed) is among the stories that Raif
Efendi reads in Kürk Mantolu Madonna. Gotthold Ephraim Lessing (1829-1881), a name forgotten in Turkish intellec-
tual circles in discussions of German literature, is an author Sabahattin Ali de-
scribed as a “genius.” In the foreword to his translation of Lessing’s play, Minna
von Barnhelm, Sabahattin Ali says: Those who value a thought-provoking smile above empty laughter and strive
to find real humans with dignity and virtue in a theater piece will not be able to
keep themselves from admiring the genius and artistry of Lessing.18 20 Sevengül Sönmez Thomas Mann, a prominent figure in German literature, is also among the authors
Sabahattin Ali greatly appreciated and who influenced his work. German Tur-
cologist Otto Spies identified this influence and mentioned it while translating
Sabahattin Ali’s short stories into German (Spies 1943). Indeed, Sabahattin Ali’s
interest in German literature never lessened. Throughout his life he translated
works by Rainer Maria Rilke, Heinrich Heine, and Gottfried Keller into Turkish. 21 “Güzel Sanatlar Umum Müdürlüğü tarafından gönderilen okulumuz repertuarına alınmaya değeri
olup olmadığı hakkında mütalaa istenilen (G.A. de Gaillavet ve Robert de Flers’in) dilimize çevrilen
Kalp Mantığı adlı eseri ilişiktir. İstenilen mütalaanın tez elden gönderilmesini saygılarımla rica ederim.”
(Sabahattin Ali 2008: 373) b g s yd
(
s
/
9 9
)
20 “Çalışmaları ilerleyen İnönü Ansiklopedisi için mühim Garp eserlerinin birer madde halinde
hazırlanmaları sırasında Alman edebiyatındaki vukufunuzdan istifade edilmek arzusunda bulunulduğu
cihetle bu hususta görüşülmesi ve bir çalışma programı hazırlanılması için büroya teşrifinizi
saygılarımla dilerim.” (Sabahattin Ali 2008: 391) 19 “Esas dili Almanca idi. Alman edebiyatını derinlemesine bilirdi. Beni en çok hayrete düşüren yönü
yalnız uzmanlarca yakından tanınabilecek eserlerin ve diğer dünya edebiyatlarının üzerindeki yoğun
bilgisiydi.” (Ali-Laslo / Özkırımlı 1979: 13) 19 “Esas dili Almanca idi. Alman edebiyatını derinlemesine bilirdi. Beni en çok hayrete düşüren yönü
yalnız uzmanlarca yakından tanınabilecek eserlerin ve diğer dünya edebiyatlarının üzerindeki yoğun
bilgisiydi.” (Ali-Laslo / Özkırımlı 1979: 13)
20 “Çalışmaları ilerleyen İnönü Ansiklopedisi için mühim Garp eserlerinin birer madde halinde
hazırlanmaları sırasında Alman edebiyatındaki vukufunuzdan istifade edilmek arzusunda bulunulduğu
cihetle bu hususta görüşülmesi ve bir çalışma programı hazırlanılması için büroya teşrifinizi
saygılarımla dilerim.” (Sabahattin Ali 2008: 391)
21 “Güzel Sanatlar Umum Müdürlüğü tarafından gönderilen okulumuz repertuarına alınmaya değeri Sabahattin Ali’s Views on the Arts and Literature1 His series of writings titled “Shakespeare Meselesi” (The Question of Shake-
speare), for example, exhibit Sabahattin Ali’s deep knowledge of Western literature,
to the extent that he was able to follow and comment on complex discussions
surrounding it. After discovering Shakespeare through Lessing, Sabahattin Ali
remained in awe of Shakespeare’s psychological analyses and artful mastery, and
often expressed a desire to write literature in the vein of his works (Sabahattin Ali:
1934b / 1934c / 1934d). )
Sabahattin Ali’s stories were also influenced by Gorky, to the extent that he
was called the “Turkish Gorky.” Nurullah Ataç further likens Sabahattin Ali’s
“Birdenbire Sönen Kandilin Hikâyesi” (The Story of a Candle that Suddenly Burnt
Out) in the short story collection Değirmen to Edgar Allen Poe’s frightful stories
such as “The Fall of the House of Usher,” “Black Cat,” and “M. Waldemar.”22
When “Birdenbire Sönen bir Kandilin Hikâyesi” came out in 1931, Sabahattin Ali
was very familiar with Edgar Allen Poe’s works. In fact, in his critique of Ahmet
Kutsi Tecer’s poem “Nerdesin’’ (Where Are You) he writes, “The desperate cry of
‘oh where are you’ comes through sleep plagued with nightmares, as is the case for
the stories of Edgar Poe.”23 In addition to these diverse literary influences, I personally believe the author
who affected Sabahattin Ali the most was Knut Hamsun. In an essay published by
Varlık in 1934, he writes about Knut Hamsun at length: “His writings are free of
any noise and excess and as expansive as nature. One need not look for anything
other than the wide, borderless, and deep human soul when reading his books.”24 p
g
Sabahattin Ali shared his interest in world literature with Turkish readers by
way of translation. In addition to the translations of short stories, poems, and es-
says that were published in magazines, he also translated many books into Turkish. His translation of Max Kemmerich’s Aus der Geschichte der menschlichen Dummheit
(History of Human Stupidity) was published in 1936 and Gotthold Ephraim Lessing’s
Minna von Barnhelm in 1942. Sabahattin Ali’s translations of Heinrich von Kleist’s
Die Verlobung in St. Domingo (The Betrothal in St. Domingo), Albert von Cham-
isso’s Peter Schlemihls wundersame Geschichte (Peter Schlemihl's Miraculous Story), and
E.T.A. Hoffmann’s “Doge und Dogaresse” (The Duke and the Duchess), ap-
peared together under the title Üç Romantik Hikâye (Three Romantic Stories) in 1943. Sabahattin Ali’s Views on the Arts and Literature1 When discussing their cooperative work in the Translation Bureau, Erol
Güney stated: His real language was German. He had a deep knowledge of German literature. What amazed me most was his profound knowledge of works that would only
be familiar to specialists and his knowledge of other world literatures.19 This deep knowledge did not go unnoticed by the state. By the 1940s, Sabahattin
Ali had become an important figure whom other people consulted in the field of
German Studies. In a letter from 1943, Nahit Sırrı Örik says: I kindly ask you to come the bureau to discuss the issue of benefiting from
your expertise regarding the entries for German literature in the ongoing pro-
ject of the İnönü Encyclopedia and its focus on important works of Western
literature, and preparing a work program for this purpose.20 As is evident in the Director of the State Conservatory Orhan Şaik Gökyay’s letter
from June 19, 1941, Sabahattin Ali’s views and evaluations were also often consult-
ed for the repertoire efforts of the State Conservatory: Attached is the Turkish translation of G.-A. de Caillavet and Robert de Flers’
Primerose: Comédie en Trois Actes, which has been translated into Turkish as The
Heart has its Reasons, sent by the General Directorate of Fine Arts. It needs to
be determined whether it should be included in our repertoire or not. I re-
spectfully ask the requested opinion to be sent in a swift manner.21 Sabahattin Ali’s Views on the Arts & Literature 21 In addition to his engagement with German literature, Sabahattin Ali’s stories ex-
hibit numerous literary influences that reach beyond the German cultural realm. His series of writings titled “Shakespeare Meselesi” (The Question of Shake-
speare), for example, exhibit Sabahattin Ali’s deep knowledge of Western literature,
to the extent that he was able to follow and comment on complex discussions
surrounding it. After discovering Shakespeare through Lessing, Sabahattin Ali
remained in awe of Shakespeare’s psychological analyses and artful mastery, and
often expressed a desire to write literature in the vein of his works (Sabahattin Ali:
1934b / 1934c / 1934d). In addition to his engagement with German literature, Sabahattin Ali’s stories ex-
hibit numerous literary influences that reach beyond the German cultural realm. geliyor. (Sabahattin Ali 1930)
24 “Gürültüsüz, patırtısız ve tabiat kadar büyüktür. Kitaplarını okurken orada geniş, hudutsuz ve derin
bir insan ruhundan başka bir şey aranmamalıdır.” (Sabahattin Ali 1934a) 22 “Uslier Konağının Yıkılışı, Kara Kedi, M. Waldemar gibi iç ürperten hikâyelerine benzetmektedir.”
(Ataç 1935)
23 “‘Ah nerdesin’ sayhası burada Edgar Poe’nun hikâyelerinde olduğu gibi kâbuslu uykular arasından 22 “Uslier Konağının Yıkılışı, Kara Kedi, M. Waldemar gibi iç ürperten hikâyelerine benzetmektedir.”
(Ataç 1935)
23 “‘Ah nerdesin’ sayhası burada Edgar Poe’nun hikâyelerinde olduğu gibi kâbuslu uykular arasından
geliyor.” (Sabahattin Ali 1930)
24 “Gürültüsüz patırtısız ve tabiat kadar büyüktür Kitaplarını okurken orada geniş hudutsuz ve derin (
)
23 “‘Ah nerdesin’ sayhası burada Edgar Poe’nun hikâyelerinde olduğu gibi kâbuslu uykular arasından
geliyor.” (Sabahattin Ali 1930)
24 “Gürültüsüz, patırtısız ve tabiat kadar büyüktür. Kitaplarını okurken orada geniş, hudutsuz ve derin
bir insan ruhundan başka bir şey aranmamalıdır.” (Sabahattin Ali 1934a) Sabahattin Ali’s Views on the Arts and Literature1 Sabahattin Ali furthermore co-translated Friedrich Hebbel’s Gyges and his Ring and
Pushkin’s The Captain’s Daughter with Erol Güney; both were published in 1943. 22 Sevengül Sönmez Sabahattin Ali’s translations also introduced a previously unknown author to
Turkish readers: in 1943, he translated Ignazio Silone’s Fontamara.25 This text, in
which Silone criticizes racism with irony, received accolades via Sabahattin Ali’s
translation. Nâzım Hikmet thanks Sabahattin Ali for his translation in an undated
letter: The language of the translation was magnificent. The language in the ro-
mantic stories was also very successful in terms of stylization. I now know
how difficult and demanding translation is, so I understand very well the
great difficulty you surmounted with great success.26 In addition to his knowledge of diverse European literatures, Sabahattin Ali was
also familiar with the rules of Ottoman divan poetry. Many people who knew Sa-
bahattin Ali well remark that his knowledge of Aruz prosody was so thorough that
he noticed a flaw in the meter immediately. He was furthermore able to compose
in the divan style himself. He began the long and witty poem titled “Terkib-i Bend”
in 1928, and later mailed it to Pertev Naili Boratav after finishing it in Germany. Sabahattin Ali’s knowledge of Ottoman literary forms also included the seyahatname. He penned “Seyahatname-i Sudlice,” which is based on a tour of the Bosphorus
with friends, as a divan in the style of Evliya Çelebi. And, finally, he wrote about his
travels to Berlin in the same witty manner and Çelebi-style in a piece called
“Mufassal Cermenistan Seyahatnamesi,” which Kristin Dickinson and Zeynep
Seviner have translated into English for this special issue as “The Comprehensive
Germanistan Travelogue.” Finally, it is worth noting the influence of folk poetry on some of the poems in
Sabahattin Ali’s collection Dağlar ve Rüzgâr (Mountains and Wind). Moreover, the fact
that songs like “Leylim Ley” and “Aldırma Gönül” (Don’t Worry My Heart),
which have been set to music, have come to be seen as anonymous and an integral
part of folk culture attests to this influence. The fable form in “Sırça Köşk” (Glass
Pavillion) also demonstrates Sabahattin Ali’s proximity to folk literature, a genre
which Sabahattin Ali describes as follows: Folk literature is more beneficial as it indicates its ways of reaching the
people. But it’s also wrong to take this at face value. There is a strong con-
servative aspect of folk literature. 25 Ali translated this work from German. It is notable that Ali was strongly opposed to translating
from a secondary language; when introducing Fontamara's author he notes that Silone was a powerful
voice of Italian literature and that he wrote and published this work in Switzerland in German (Sa-
bahattin Ali 1941).
26 “Tercüme dili fevkaladeydi. Romantik hikâyelerdeki dil de istilizasyonu bakımından çok muvaffak.
Tercümenin ne zor, ne kadar mesuliyetli bir iş olduğunu tecrübeyle artık bildiğim için ne büyük bir
zorluğu nasıl başarıyla yendiğini gayet iyi anlıyorum.” (Sabahattin Ali 2008: 398). 27 “Halk edebiyatı ise, halka varabilmek yollarını işaret edeceği için daha istifadelidir. Fakat bunu da
olduğu gibi almak yanlıştır. Halk edebiyatının geri tarafları çoktur. Mahsullerinin ekserisi din ve tasav-
vuf karanlığının, derebeylik zihniyetinin tersleri ile doludur. Bu materyali kullanacak olanlar, ayıkla-
masını bilen insanlar olmalıdır.” (Reşit 1936) Sabahattin Ali’s Views on the Arts and Literature1 Most of these works are filled with feu- Sabahattin Ali’s Views on the Arts & Literature 23 dal mentality and the darkness of religion and mysticism. The people who
use this material should be those with the ability to discern.27 dal mentality and the darkness of religion and mysticism. The people who
use this material should be those with the ability to discern.27 As this citation shows, Sabahattin Ali drew upon folk literature very carefully and
consciously, and made an indelible impression on public discourse with his unfor-
gettable verses and heroes. As this citation shows, Sabahattin Ali drew upon folk literature very carefully and
consciously, and made an indelible impression on public discourse with his unfor-
gettable verses and heroes. Throughout this article, I have tried to convey Sabahattin Ali’s outlook on art
and literature by making use of his own words; through recourse to Sabahattin
Ali’s interviews and his personal correspondences, I have indicated that Sabahattin
Ali was a dedicated follower of world literature, a meticulous translator, and a per-
severant author; the works he left us—and, in particular, his translations—should
be examined carefully once more and brought to the attention of a contemporary
readership. To this end, detailed studies of Sabahattin Ali’s works are crucial for
showing the reflection of his views on art within his works, and of utmost im-
portance for determining the progress of his artistic development and his place
within Turkish literature. Works Cited Ali-Laslo, Filiz / Özkırımlı, Atilla (eds.) (1979): Sabahattin Ali, İstanbul. Ataç, Nurullah (1935): “Değirmen,” in: Akşam, 21.09.1935, 8. Aygün, İhsan (1935): “Gençler Diyor ki: Sabahattin Ali,” in: Yücel 8, 57. Bezirci, Asım (1987): Sabahattin Ali, İstanbul. Çelik, İsa (1984): “Fotoğraf Sanatçısı Sabahattin Ali,” in: Hürriyet Gösteri 41,
04.1984, 6-8. Ergün, Mehmet (2000): “Sabahattin Ali’nin Yazamadıkları,” in: E Dergisi 13,
04.2000, 54-58. Lessing, Gotthold E. (1942): Minna von Barnhelm, translated by Sabahattin Ali,
Istanbul. Nazif, Umran (1938): “Sabahattin Ali İle Bir Konuşma,” in: Varlık 108,
01.01.1938, 573. Reşit, Muzaffer (1936): “Sabahattin Ali İle Bir Konuşma,” in: Varlık 65, 15.03. 1936, 264-265. Sevengül Sönmez 24 Sabahattin Ali (1930): “Görüş Mecmuası Hakkında Şahsi Bazı Mütalaalar,” in:
Resimli Ay 6, 08.1930, 25, 40. Sabahattin Ali (1934a): “Knut Hamsun,” in: Varlık 21, 15.05.1934, 329-330. Sabahattin Ali (1934b): “Shakespeare Meselesi –Hakiki Shakespeare-,” in: Varlık
32, II. Teşrin, 138-139. Sabahattin Ali (1934c): “Shakespeare Meselesi –Sahte Shakespeare’ler: Bacon-,” in:
Varlık 34, I. Kânun, 151-152. Sabahattin Ali (1934d): “Shakespeare Meselesi – Sahte Shakespeare’ler: Eduard de
Vere,” in: Varlık 37, Kânun 1935, 138-139. Sabahattin Ali (1941): “İkinci Dilden Tercüme Meselesi ve Bir Misâl,” in: Tercüme. 6, 581-585. Sabahattin Ali (1943): Üç Romantik Hikâye, Ankara. Sabahattin Ali (1943): Üç Romantik Hikâye, Ankara. Sabahattin Ali (2005): Kürk Mantolu Madonna, İstanbul. Sabahattin Ali (2005): Kürk Mantolu Madonna, İstanbul. Sabahattin Ali (2008): Hep Genç Kalacağım: Bütün Yapıtları Mektup, edited by
Sevengül Sönmez, İstanbul. Spies, Otto (1943): Die Türkische Prosaliteratur der Gegenwart, Leipzig. Tatarlı, Ibrahim (2014): “Sabahattin Ali, Hayatı, Kişiliği ve Yaratıcılığına Genel Bir
Bakış,” in: Ali, Filiz / Özkırımlı, Atilla / Sönmez, Sevengül (eds.): Sabahattin
Ali, 151-183. Yazar, Mehmet Behçet (1938): “Bir Mektubundan,” in: Edebiyatımız ve Türk Ede-
biyatı, İstanbul, 372-373. World Literature (Already) Wrote Back:
Sabahattin Ali after Germany David Gramling/Martina Schwalm The age of late neoliberal modernity has prompted new discussions on World Literature and
translation, which—but for a handful of classic exceptions—inadvertently tend to exclude literary
texts written before 1945 in the Global South, colonial settings, and settings beyond Western /
Central Europe and North America. In his 1943 novel, The Madonna in the Fur Coat, the
Turkish-speaking author Sabahattin Ali critically problematizes the theorization of world-
literary translation, as well as such latter-day conceptions as transnational aesthetics. This article
sets itself to reveal how Ali, already in 1943, espouses a theory of world literature-in-translation,
responds to ambient questions about world literature and the invisibilization of translating, long
prior to the political debates on these themes in the 1980s and 1990s. As a novel written in
Turkish, and therefore long relegated to the sidelines of anglophone and francophone scholarship
on World Literature and translation, the Madonna nonetheless anticipates the discourse about
World Literature long before the so-called age of globalization, and offers critical understanding
about literary translation and monolingualism in the process of nation building. David Gramling/Martina Schwalm 26 The study of world literature can very readily
become culturally deracinated, philologically
bankrupt, and ideologically complicit with the
worst tendencies of global capitalism. Other
than that, we’re in good shape. —David Damrosch, in conversation with
Gayatri Chakravorty Spivak (2011) Nothing on earth was more appalling than be-
ing obliged to correct someone else’s mis-
judgment about oneself.1
—The Madonna in the Fur Coat,
Sabahattin Ali (2014 [1943]) Sabahattin Ali’s novel The Madonna in the Fur Coat (1943) is not post-colonial litera-
ture, nor is it in any way properly categorizable under the aegis of “the empire
writ[ing] back” (Ashcroft et al. 1989). Composed twenty years after the dissolution
of the Ottoman Empire and the founding of the Republic of Turkey, it is a text
whose author was nonetheless raised in the twilight of empire, in territories beyond
the national holdings of post-1923 Republican Turkey (present-day Bulgaria and
northern Greece). When a fellowship from the young republic’s Ministry of Edu-
cation sent him to the (also young) republic of Germany from 1928-1930, Ali ar-
rived there as a citizen of a political entity that differed sharply from the country
and territory of his own youth. 1 “Hiç bir şey beni, hakkımdaki bir kanaati düzeltmek mecburiyeti kadar korkutmazdı.” (Ali 1998: 49) Theoretical Context We begin by noting the still habitual temptation to overlook extra-European, early
20th-century novels’ critical interventions into geopolitical questions, and to view
these narratives reductively as representative national portraiture or allegory. As
Alexander Beecroft has noted in his essay “World Literature without a Hyphen,”
prevailing grand models for the study of World Literature have had little to say
about non-European literatures prior to decolonization and 1945 (Beecroft 2008:
88). If primarily out of a need to stop somewhere, several leading voices in the
reinvigorated 21st-century project of theorizing World Literature have even made
the era of decolonization an explicit historical limit criterion (Casanova 1999: 62-
63). One of the results of this spirit of pragmatism seems to be that most pre-1945
literary texts emerging from Global South contexts, colonial holdings, and Eu-
rope’s West Asian peripheries continue to remain in the implicit scholarly remand
of religious studies, historical anthropology, social history, folklore, and anthropo-
logical philology (Hymes 1965)—but do not yet accrue their own load-bearing
function in current World Literature models which, as Pheng Cheah has also
claimed, now tend to hew to a presentist spatial picture: [T]he defining characteristic of the world in recent accounts of world liter-
ature is spatial extension. It refers to the extensive scope and scale of the
production, circulation, consumption, and evaluation of literature. Simply
put, “world” is extension on a global scale, where world literature is con-
ceived through an analogy with a world market’s global reach. What is
worldly about literature is its locomotion or movement in Mercatorian
space according to the mathematical coordinates of Euclidean geometry. Where literary history is broached, time is viewed in similarly spatial terms. (Cheah 2014: 306-307) What Cheah sees as the brisk and vigorous “locomotion” of post-colonial literary
texts trafficking across multilingual, transnational frontiers is the result of a regular-
ized translative free market in what Ashcroft et al. described in 1989 as “the em-
pire writ(ing) back.” What role literary translators might be seen as playing in this
spatialist conception of world-literary markets remains something of an undertheo-
rized question, as the practice and products of literary translating still run counter
to some of comparative literature’s most deeply held convictions about its own
mandate. World Literature (Already) Wrote Back:
Sabahattin Ali after Germany Madonna is thus a novel that confounds not only
today’s disciplinary boundaries and philological conceptions of national persis-
tence, but one that itself exudes a particular mood of political, linguistic, and narra-
tive chronodiversity (Gramling / Hepkaner 2016). Against the backdrop of this complex historical setting and production con-
text, the current article sets for itself three related tasks: 1) to understand how the
“world literature in translation / Dünya Edebiyatından Tercümeler” initiatives contem-
porary to the publication of The Madonna in the Fur Coat in 1943 both presage and
relativize today’s models for world-literary study in comparative literature; 2) to
understand how Ali’s novel critically intervened, avant la lettre, in scholarly discours-
es about transnationalism, multilingualism, and the (invisibility of the) translator;
and 3) to think more broadly about the critical role that novels such as Ali’s Ma-
donna might play in adumbrating a more historically capacious theory of World
Literature, beyond Europe and prior to 1945. How did texts such as Madonna offer
their own theoretical accounts of concepts, experiences, and predicaments that
would be described, a half-century later, under the auspices of hybridity and trans- World Literature (Already) Wrote Back: Sabahattin Ali after Germany 27 nationalism? What do the specific historical answers to these questions offer to-
ward a rethinking of world-literary presuppositions and models in 2017? Theoretical Context Prominent voices in Translation Studies have recently been suggesting,
for instance, that the contemporary endeavor of comparative literary study “con-
tinues to suppress translated texts” (Venuti 2016) by promoting new versions of
old prejudices against translated literature in the humanistic disciplines. The stigma David Gramling/Martina Schwalm 28 associated with translating in the United States was so strong over the 1980s and
1990s, according to Lawrence Venuti, that: “With rare exceptions, a scholar’s deci-
sion to translate or to study translations was likely to jeopardize an academic ca-
reer.” (Venuti 2016: 181) associated with translating in the United States was so strong over the 1980s and
1990s, according to Lawrence Venuti, that: “With rare exceptions, a scholar’s deci-
sion to translate or to study translations was likely to jeopardize an academic ca-
reer.” (Venuti 2016: 181) When it comes to the structural and epistemological role of translating and
translators generally, world literature debates and comparative literature discourses
(at least on the Western shores of the North Atlantic) appear to have some ex-
plaining left to do. Particularly in relation to non-European literature prior to 1945,
comparative literature scholars have only recently begun to set forth theoretical
questions about the differential practices that constituted literary translation across
the “hard multilingual” borders of—say—Arabic, Persian, and Ottoman courtly
poetry prior to the 20th century, and what these translingual procedures might re-
veal about literary study under latter-day conditions of globalization (Noorani
2013). A recent collection edited by Michelle Woods under the title Authorizing
Translation (2016) offers further indications that translation and translators may
indeed be slowly moving out from their back-of-the-house positionality in the
comparative literature world—by way not only of massive collaborative projects
like Cassin’s Vocabulaire européen des philosophies (2004), but also through a critical
reengagement with (once or currently) living, working literary translators. Rendering the Multilingual Translator Visible As we noted above, The Madonna in the Fur Coat was composed by an Ottoman-
educated author, amid but somewhat ill at ease with the nation-building efforts
into which state actors conscripted his learned and well-travelled imagination. It is
an example of a novel that, long ago, before the canon debates of the 1980s,
spurned any symbolic duty to “be” national literature on a global market of trans-
lated texts, and turned its figural ambitions rather toward a keen-eyed theorization
of translation, multilingualism, and hybridity. More than just a tale of border-
crossing between Germany and Turkey, Ali’s novel is just as eager to document
how translators intervene in the high modern order of parallel nationalisms and
nation-building projects as it is in conveying one source culture to an awaiting
world-literary public. This meta-national critique would bring great hardship and
precarity to Ali over the course of the acutely nationalist 1940s in Turkey, a fact
that is foreshadowed in the novel itself. Serialized in 1940–1941 and published in
book form in 1943, The Madonna in the Fur Coat would indeed be Sabahattin Ali’s
last novel of three. His previous, domestic-realist novel The Devil Within Us had
been ill received among political elites, and he found himself increasingly in the
political forcefield of the emerging world-literary translation industry in Ankara,
which sought to systematically deliver world literature (in Turkish) to an upcoming
generation of republican citizens, the first generation born under the official West-
ernization programs of post-Ottoman Turkey. Though Ali’s ‘Madonna novel’ has
long been a mainstay of literary bibliophilia in Turkey, it bears the uneasy imprint
of a fraught relationship with the early world-literary translation projects of Turk-
ish republican cultural programming elites, like Hasan Ali Yücel’s Dünya Edebiyatın-
dan Tercümeler (Albachtan 2013, Aksoy 2010, Yücel 1939, 125, Szurek 2015). (
y
)
The goal of the Dünya Edebiyatından Tercümeler (hereafter “DET”) series was to
bring as many Western classics to Turkish readers as possible and to Turkify their
literary features for domestic accessibility. Ali’s German-Turkish novel irritated
that model and its presumed vectors of domestication and representational tute-
lage. That is, in composing a transnational story with volatile and ambivalent alle-
giances to any national community, Madonna competed with the international spirit
of the DET program, which was nationalist in its incorporative, educational strate-
gies (Aytürk 2004). Theoretical Context David Damrosch traces these lingering forms of disciplinary méconnaissance
(Bourdieu / Wacquant 1992: 171-172) back to the bitter objections in 1960 of the
Chapel Hill-based, Swiss-educated comparatist Werner Friedrich—once referred to
as the “Christoph Colomb du comparatisme américain” (Leonard 2010: 191)—
namely, that world literature curricula tend to attract: bricks [...] flying from the left and from the right, from the solid language
departments snorting that this is the flimsiest kind of sheer amateurism,
and from the solid comparatists complaining that it is because of such
[sweeping survey courses on “The Novel in World Literature”] that Com-
parative Literature, ever since the 1920s, has gotten a black eye and an ill
repute from which it has yet not completely recovered.” (Friedrich 1960:
15) Such a mood of ill repute, suppression, and multidirectional flying bricks casts a
long shadow on today’s debates about the ideal division of labor between world
literature, comparative literature, translation studies, literary translation, postcolo-
nial studies, and multilingualism studies (see, for instance, Schwalm 2015, Gram-
ling / Hepkaner 2016, Noorani 2013, Cheah 2014). These questions appear at first
glance to be primarily academic dilemmas, of interest to those in the professoriate
who tend to participate in discussions ‘about’ world literature and ‘about’ literary
translation. What remains to be fully investigated, however, is the extent to which
literary texts themselves—and, indeed, those composed prior to 1945—have par-
ticipated knowingly and presciently in the creative theorization of “world litera- World Literature (Already) Wrote Back: Sabahattin Ali after Germany 29 ture,” as well as of the often rancorously politicized and mercilessly suppressed
“task of the translator,” and of the complicated, precarious relationship between
world literature, literary translation, and “the worst tendencies of global capital-
ism.” (Damrosch 2011: 456, see also Damrosch 2005) The Precarity of the Translator under Conditions of Monolingualism It is easy to overlook these historical contingencies and contexts for Madonna and
other pre-1945 novels, because a tendency still prevails to characterize world litera-
ture-in-translation as something that comes ‘from’ places like Turkey, rather than
as something that a place like Turkey has gone to great lengths to produce, theo-
rize, and criticize for itself in its own (translated) language—pursuant to a worlded,
Westernizing, and yet Turkocentric civic sensibility and a domestically consistent
cultural politics (Aytürk 2004: 15). The Madonna in the Fur Coat, as we shall see in
the analysis that follows, ran quite stridently afoul of this kind of instrumental con-
sistency expected of authors writing in Turkish in the third decade of the Republic. Previous work has sought, in greater detail, to account for the ways in which
Sabahattin Ali dealt with his own positionality as a literary translator and author
among the quickly shifting sands of Turkey’s early language and alphabet reform
endeavors in the 1920s through 1940s (Dickinson 2013, Gramling / Hepkaner
2016). An active member of the state’s Translation Bureau, Ali was intensively
involved in the practice, planning, and debate around the endeavor to deliver to
Turkish readers, in their own language, the classics of Western belles-lettres. The
Minister of Education heading up the initiative, Hasan Ali Yücel, described the
mission of the Translation Bureau and Yücel’s DET series three years prior to the
publication of Madonna in these terms: Republican Turkey, which aspires to and is determined to become a dis-
tinguished member of Western culture and thinking, is obliged to translate
into its own language the works of the old and new thinking of the mod-
ern world and thus to strengthen its own existence with their perception
and thought. This obligation invites us to start a full-scale translation pro-
ject. Rendering the Multilingual Translator Visible Ali was thus living and working amid the instrumentalist dy-
namics of his own era’s world-literature-in-translation debate, one in which he
vigorously participated, and one that was itself in the implicit service of domestic
nationalization and monolingualization endeavors in Turkey. Madonna’s critical
attention to the ethical allegiances of the translator-as-citizen and the translator’s David Gramling/Martina Schwalm 30 complex stance toward nation-building efforts meant that the novel was unlikely to
find eager reception among mid-century political elites, when the ‘Anatolian’ heart-
land novel genre was gaining in cultural capital. The Precarity of the Translator under Conditions of Monolingualism (Yücel 1939: 125, see also Aksoy 2010) Ali—a political dissident, an on-again, off-again state functionary, a multilingual
translator from the German, and a legacy of Ottoman humanistic educational tra-
ditions—remains throughout his life an ‘unredeemed captive’ of his own
translingual, transnational experiences, and appears poised in 1941 to take the oc-
casion of his third novel to intervene critically in the politics of “world literature in
translation.” The remainder of the article will canvas these domains by counterpos-
ing Madonna’s interventions alongside critical questions that have been animating
very recent discussions about world literature and translation, amid what are often
presumed to be unprecedented conditions of late neoliberal globalized modernity. Ali’s novel will thus not only help us to imagine the subjectivity of translated world World Literature (Already) Wrote Back: Sabahattin Ali after Germany 31 literary texts as they actively renegotiate the theoretical terms of the debates that
interpellate them in complicated ways, but will also illustrate how texts such as The
Madonna in the Fur Coat, speaking to us as they do from the 1940s and prior, can
expand our historical awareness about the critical animus at work in texts of earlier
periods. How can we better entertain the possibility that so-called world-literary
texts from 1945 and prior have their very own ideas about world literature, trans-
latedness, and the institutionalization of both, rather than serving primarily as the
retrospective proving ground for others’ ideas about the same? Ali’s late novel The Madonna in the Fur Coat—‘late’ because political suppression
and violence ended his career before his fiftieth birthday—pursues precisely such a
phenomenological account of translated world literature, doing so beyond the
cartographic parameters Cheah describes as characteristic of the world literature
debate (Cheah 2014). Though the novel appears at first to thematize primarily an
axis that contemporary Germanists understand as Turkish-German transnational-
ism, the narrative is in many ways utterly unconcerned with nationally understood
histories or ethnically understood cultures. Its engagement with the historical spec-
tacle of nation-building and its political assemblages is decidedly formal and dram-
aturgical, lacking any propositional investment in one endeavor of cultural nation-
alism over another, whether that of (Republican) Turkey, (Weimar-Republican)
Germany, or any other. 2 There are several options for how to translate the main character’s nickname “Raif Efendi.” “Sir
Raif,” as Hepkaner and Gramling (Ali 2014) have chosen, has the advantage of connoting not only
the old aristocratic order with which Raif is metonymically associated by his age, employment history,
and his overall uncontemporary habitus, but also the scornful mockery with which the other employ-
ees of Raif’s firm treat his special “dominated dominant” status as the firm’s translator.
3 “Para düşkünlüğü yüzünden servetini kaybeden ihtiyar bir kadın, zabitlere kızıyor, zabitler grev
yapan ameleyi ve harbe devam etmek istemeyen askerleri kabahatlı buluyor, müstemleke tüccarı durup
dururken, harp açan imparatora küfür ediyordu. Sabahları odamı düzelten hizmetçi kız bile benimle
siyasetten konuşmaya kalkar, boş zamanlarında derhal gazetesini okumaya koyulurdu. Onun da
kendine göre ateşli kanaatleri vardı ve bunlardan bahsederken yüzü büsbütün kızarır, yumruğunu
sıkarak havada sallardı.” (Ali 1998: 53) The Precarity of the Translator under Conditions of Monolingualism A typical descriptive passage in this regard arises as Sir
Raif2, the main subject of the embedded narrative that constitutes the last 80% of
the novel, describes the histrionic partisan excesses of the cast of characters at his
boarding house in mid-1920s Berlin: An old, once-wealthy lady was bitter at the officer corps, though she’d lost
her fortune due to her own sheer avarice. The officers found fault with the
striking workers and with the soldiers who’d been no longer willing to
keep up the fight. And totally out of the blue, the colonial tradesman curs-
ed the emperor for starting a war in the first place. Even the maid who
cleaned my room in the mornings tried to drum up political conversations
with me, and she would read her newspaper whenever she had a spare
moment. She had her own ardent opinions, and while talking about them,
her face would flush red and she’d swing her fist in the air.3 (Ali 2014: 25) David Gramling/Martina Schwalm 32 Throughout the novel, the frame narrator and Sir Raif’s embedded storyline treat
nation-building as a comical, spastic sideshow, grasping for attention in settings
where deeper aesthetic and epistemological questions are being foregrounded. The
narrator’s apparent anationalism, or at least ironic agnosticism vis-à-vis national
projects, is a remarkable feature for a text composed so acutely within a nation-
building ethos and moment, such as that in which Sabahattin Ali found himself
circa 1940. Eliding the imperative to do representational work on behalf of the
nation in compliance with the controlled forms of heteroglossic diversity that ani-
mate imagined communities was itself a strategic political move that incurred for
the author and his text a more than disobliging response from his powerful peers
in government. g
More than mere scene-setting or pathos-building, we contend that these carica-
tures of contemporary civic posturing (whether in Raif’s pension in Berlin or at the
frame narrator’s firm in Ankara) serve a broader purpose: to reflect on the formal
and structural predicaments of multilingual border-crossers and translators amid
aggressive mobilizations of national spirit. Ali could have chosen to write a realist
novel about either one of these highly dramatic, crisis-fueled national moments, or
he could have written a political novel that interwove the two Republican projects
into one another. But he did not. The Precarity of the Translator under Conditions of Monolingualism To do so may have guaranteed Ali’s novel the
kind of political legibility and contemporary relevance that would have ushered it
into the canon of translated world literature much earlier than the 2000s. Madonna,
like its main character, is oddly and inexplicably ill at ease with contemporary polit-
ical commentary and the soaring urgencies of national becoming. Images of Ger-
manness or Turkishness play a muted, even derided role in this novel, which turns
its sights to a different sphere of modernity altogether. Rather than the ‘subject-
becoming-citizen,’ Madonna is at pains to understand the ‘translator-becoming-
subject’ amid accelerated conditions of modernist nationalism. The Purpose of the Frame Narrative It should be noted that Sabahattin Ali could have entirely foregone the device of a
frame narrative, delivering instead what has become the official focus of attention
for most literary reception of the novel: the love story between Sir Raif and the
Czech-German-Jewish painter Maria Puder, which indeed does well enough as a
stand-alone narrative. It is important therefore to ask: What compelled Ali to for-
mulate the first fifth of the novel as a frame narrative, and—more importantly—
why did he infuse the frame narrative with such philosophical complexity and so-
cial critique of 1930s Ankara, all of which then appears at first tangential to the
ensuing embedded narrative? Once the novel cedes its narrative voice to Sir Raif
and his notebook manuscript at the threshold between frame and embedded narra-
tive, it does not return thematically to the bureaucratic absurdities that character-
ized the social ethos of the frame. We readers are left to presumptively grasp the
relationship between the two stories as one of vague mutual affinity, banal World Literature (Already) Wrote Back: Sabahattin Ali after Germany 33 happenstance, or pragmatic verisimilitude. Why, after a scathing sketch of Ankara’s
upwardly mobile clerk classes in the frame narrative, are we—or more importantly,
are early 1940s citizens of the young Republic of Turkey—prompted to then read a
tale about an old-fashioned, self-effacing translator and his star-crossed, mostly
platonic love affair with a German Jewish painter and her mythically aestheticized
self-portrait? The key lies, perhaps, in the habitus of the narrator as he undergoes a trans-
formation from normative citizen to aspiring critic, through a translational mode
of cultural defamiliarization. When readers first come in contact with him, the
frame narrator is a creative writer down on his luck in an Ankara characterized by
commercial ambition, bureaucratic jockeying, and—so the narrator complains—
utter social banality. Here is how the frame narrator appears to see nearly all of his
contemporaries, with the exception of Sir Raif the translator: They were not bad people at all. They were just empty, dead-empty crea-
tures. All their inappropriate behavior resulted from that fact. 4 “Hiç de fena insan değillerdi. Yanlış boş, bomboş mahluklardı. Yaptıkları münasebetsizlikler hep
buradan geliyordu. İçlerinin esneyen boşluğu karşısında ancak başka başka insanları istihfaf ve tahkir
etmek, onlara gülmek suretiyle kendilerini tatmin edebiliyorlar[dı].” (Ali 1998: 28-29)
5 “Makine vesaire komisyonculuğu yapan, aynı zamanda orman ve kereste işleriyle uğraşan bir şirkette
müdür muavini olduğunu ve oldukça iyi bir para aldığı[nı biliyordum].” (Ali 1998: 13) “Makine vesaire komisyonculuğu yapan, aynı zamanda orman ve kereste işleriyle uğraşan bir şirket
müdür muavini olduğunu ve oldukça iyi bir para aldığı[nı biliyordum].” (Ali 1998: 13) buradan geliyordu. İçlerinin esneyen boşluğu karşısında ancak başka başka insanları istihfaf ve tahkir
etmek, onlara gülmek suretiyle kendilerini tatmin edebiliyorlar[dı].” (Ali 1998: 28-29)
5 “Makine vesaire komisyonculuğu yapan, aynı zamanda orman ve kereste işleriyle uğraşan bir şirkette
müdür muavini olduğunu ve oldukça iyi bir para aldığı[nı biliyordum].” (Ali 1998: 13) The Purpose of the Frame Narrative Faced with
the ever-growing emptiness of their character, they could only ever find
out who they were when they disdained others and took satisfaction from
laughing at them.4 (Ali 2014: 11) It is among this cast of contemporary republican citizens that the frame narrator
happens upon his first interlocutor and benefactor in the novel, the “vice president
of a forestry and lumber business that also procured mechanical building materials,
and for this he was getting paid quite well indeed.”5 (Ali 2014: 2) This old ac-
quaintance, Hamdi, is important to the overall theoretical arc of the novel, not
merely a device propelling readers into a plausible narrative course to the frame
story. Hamdi is, to the frame narrator, and most probably for Sabahattin Ali, too,
the ultimate poster-child for republican citizenship—successful in his international
commercial pursuits, compliant with the one-party social doctrines of his time, and
both dependent on and disdainful of translation, which reminds him of the worldly
multilingualism that neither he nor the state can control. One of Hamdi’s exempla-
ry triumphs early on in the novel comes to pass in the following, very humorous
scene, which is tone-setting for the bureaucratic satires to come: “I’ve found a job for you!” Then, he stared at my face with his dauntless
and important eyes, adding: “I mean, I invented one. It isn’t too taxing. You’ll keep track of our business at different banks, particularly at our
own bank… It’ll be like being a communication clerk between banks. “I’ve found a job for you!” Then, he stared at my face with his dauntless
and important eyes, adding: “I mean, I invented one. It isn’t too taxing. You’ll keep track of our business at different banks, particularly at our
own bank… It’ll be like being a communication clerk between banks. David Gramling/Martina Schwalm 34 When business is slow, you can sit down and do your own thing. Write
poetry as much as you want. I talked with the president. We’ll get the hir-
ing taken care of…6 (Ali 2014: 4) When business is slow, you can sit down and do your own thing. Write
poetry as much as you want. I talked with the president. 6 “‘Sana bir iş buldum!’ dedi. Sonra, yüzüme o cesur ve manalı gözlerini dikerek ilave etti: ‘Yani bir iş
icat ettim. Yorucu bir şey değil. Bazı bankalarda ve bilhassa kendi bankamızda işlerimizi takip
edeceksin... Adeta şirketle bankalar arasında irtibat memuru gibi bir şey... Boş zamanlarında içeride
oturur, kendi işlerine bakarsın... İstediğin kadar şiir yaz... Ben müdürle konuştum, tayinini
yapacağız...’” (Ali 1998: 17) 7 “‘Bırak böyle şeyleri canım!’ diyerek pratik hayatın muvaffakiyetlerinden, edebiyat gibi boş şeylerin
mektep sıralarından sonra ancak zararlı olabileceğinden bahsetti. Kendisine cevap verilebileceğini,
münakaşa edilebileceğini asla aklına getirmeden, küçük bir çocuğa nasihat verir gibi konuşuyor ve bu
cesareti hayattaki muvaffakiyetinden aldığını tavırlarıyla göstermekten de hiç çekinmiyordu.” (Ali
1998: 15) The Purpose of the Frame Narrative We’ll get the hir-
ing taken care of…6 (Ali 2014: 4) With this triumphal gesture, the lugubrious creative-writer frame narrator is
brought back into the fold of commercial and fiscal trade and traffic, as befitting
an aspiring republic attempting to strategically incorporate artists and poets. The
frame narrator functions as the embodied idiom of inter-bank communication,
under the lenient auspices of which he may opt to exercise his freedom to write the
occasional poem. The prospective productivity of literature and poetry—the aes-
thetic and imaginative use of written language—is thus embedded in political
economy by way of this invented profession of inter-bank communicator, which
itself speculates on the unbridled optimism in the fiscal sector. The empty and
infinite promise of literature—“write poetry as much as you want”—finds its tem-
porary refuge and self-justification in the interstices of domestic capital specula-
tion. Less a clerk and more of a fiscal translator himself, the frame narrator realizes
he has bargained for more than he has gained. This result is born out in the
subsequent behavior of his benefactor, Hamdi, the normative civic subject of the
novel, who admonishes the narrator: “Cut the nonsense, man,” he then told me all about the triumphs of living
pragmatically, and insisted that good-for-nothing distractions like literature
could only hold you back once you’re done with school. Without even
considering that I might have a response of some kind, let alone a coun-
ter-argument, he just kept holding forth as if advising a child, making no
bones about how his confidence in these matters came directly from his
own successes in life.7 (Ali 2014: 3) 8 “emin olduğu için pervazsızca konuşuyordu” (Ali 1998: 15)
9 “çok alıngan, gayet ince görünüşlü ve dikkatliydi” (Ali 1998: 34)
10 “Karşımdaki masada canlı olduğundan şüphe ettirecek kadar hareketsiz oturan, tercüme yapan veya
çekmesinin gözündeki ‘Almanca romanını’ okuyan bu adamın sahiden manasız ve sıkıcı bir mahluk
olduğuna kanaat getirmiştim.” (Ali 1998: 21)
11 “Konuşurken ağzından yabancı bir kelime çıktığı, herhangi bir zaman dil bildiğinden bahsettiği
duyulmamış; elinde veya cebinde ecnebi gazete ve mecmuaları görülmemişti. Hulasa, bütün
varlıklarıyla: ‘Biz Frenkçe biliriz!’ diye haykıran insanlara benzer bir tarafı yoktu.” (Ali 1998: 19-20)
12 ”[O]nun [...] birçok tezatların bulunduğunu seziyordum.” (Ali 1998: 23) Transnational Aphasia At the opposing pole of civic subjectivity from Hamdi is the frame narrator’s new
office-mate, the translator Sir Raif, who—in contrast to the blustery, triumphant
capitalist citizen Hamdi, whose “most natural option was to talk bald-faced non- World Literature (Already) Wrote Back: Sabahattin Ali after Germany 35 sense”8 (Ali 2014: 3)— “was intense, delicate, and very careful”9 (Ali 2014: 14). Thus begins Madonna’s apparently accidental inquiry into the subjectivity of transla-
tors who, in the form of Sir Raif, offer the frame narrator an alternative model to
Hamdi’s banal commercial nationalism, a counter-model to which the narrator
gradually finds he can aspire. This interest does not come easily to the frame narra-
tor, who, in the spirit of Lawrence Venuti’s 1995 interventions on the social invisi-
bility of the translator, is predisposed to find Sir Raif and his ilk “on the whole
tedious and irrelevant: he sat there so stiffly that I began to wonder whether he
was alive or dead there at the table across from me—translating this or that, read-
ing that ‘novel in German’ hidden in his drawer.”10 (Ali 2014: 7) As he grows more curious about the translator with whom he shares his room,
the frame narrator comes to understand that the company had long failed to give
Sir Raif a raise—despite his seniority and despite institutional profligacy in other
areas—primarily because of an underlying suspicion that he, Raif, does not actually
know the languages he translates. Raif’s behavior, or lack of behavior, is prejudi-
cially understood around the office in this dismissive way, primarily because he
abstains from the social graces presumed of his upwardly mobile clerk contempo-
raries. Raif foregoes the ostensive cultural labor of (European) cosmopolitanism,
adopting an apparently ascetic disposition that rankles those around him in his
work environment, who presume such performances of European ‘culturedness’ as
the prerequisite for ‘being a translator,’ the experience of which no one in the nov-
el seems eager to understand. We never heard any foreign words come out of his mouth, we never en-
countered him talking about his language knowledge, and we never saw a
foreign newspaper or magazine in his hands. 8 “emin olduğu için pervazsızca konuşuyordu” (Ali 1998: 15) 9 “çok alıngan, gayet ince görünüşlü ve dikkatliydi” (Ali 1998: 34) 13 “Bundan sonra Raif efendinin her hali, sahiden manasız ve ehemmiyetsiz olan hareketleri bile, bana
merak vermeye başladı.” (Ali 1998: 24)
14 “Evin bütün masrafı bizim Raif efendinin cılız ücretine yüklenmekteydi. Buna rağmen, evde zavallı
ihtiyardan başka herkesin borusu ötüyordu.” (Ali 1998: 30)
15 “Bütün bu yükleri çeken Raif efendi olduğu halde, evde onun yokluğu ile varlığı müsavi gibiydi. [...]
Sanki cansız bir makine sabahleyin birtakım siparişlerle dışarı bırakılıyor, akşamüzeri kolları dolu bir
halde dönüyordu. (Ali 1998: 31) Transnational Aphasia In short, he had nothing in
common with those types who seemed to shout, “We speak European!”
with all their being.11 (Ali 2014: 6) The narrator appears to be the only person in the novel, among Raif’s coworkers
and family-members alike, who “sensed [he] was still harboring a handful of mis-
conceptions about [Raif]”12, and this leads the frame narrator to take up a studious David Gramling/Martina Schwalm 36 emulation of his office-mate. “After that, anything about Sir Raif, even his mean-
ingless and negligible actions, provoked my interest.”13 (Ali 2014: 8) What we have in this frame narrative, long before meeting the true Madonna
in her fur coat, is a scaffolding in which a citizen-national, disheartened at the
meager affective affordances of his own nation-building environment’s version of
commercial and cosmetic cosmopolitanism, seeks succor and tutelage from pre-
cisely that element in the environment (Sir Raif) that most seeks to recede from
visibility and structural importance. The translator, a vanishingly self-effacing fig-
ure who nonetheless enables the firm to keep up its pace of commercial traffic,
turns the normative social world upon itself, becoming the spiritual and critical
fulcrum of the novel and ensuring it the world-literary obscurity it would inhabit
until the hey-day of Turkish-German revisionist literary history in the 1990s, when
it would be brought back into the limelight. The Clandestine Subjectivity of the Translator To summarize so far, the frame narrative serves as more than a social or rhetorical
sponsor for the ensuing transnational love story. The frame is arguably a self-
standing critique of the relationship between national citizenship and critical trans-
lation in contexts of compulsory progressive self-fashioning such as the 1930s
Republic of Turkey, amid whose institutions Sabahattin Ali was perennially active. Let us take a further look at the way the novel symbolically encodes the translator
into the social world of Ankara around 1940. “The whole household,” notes the
frame narrator, “depended on our Sir Raif’s tiny income. The result was that eve-
ryone in the house lived high-off-the-hog, except for our miserable old man”14 (Ali
2014: 12). The political economy of a budding affluent society, as embodied in
Raif’s extended family in Ismetpaşa, swells conspicuously on the back of this one
mistreated translator. “Although Sir Raif bore all the burden, his presence or ab-
sence in the house was almost irrelevant. [...]In the mornings, he was sent off into
the street like an inanimate machine with various errands to run, and he returned
home at night with his arms full”15 (Ali 2014: 12). Through these characterizations, it becomes clear that Ali and his frame narra-
tor are not merely interested in finding out what it is that makes translators desire
to translate, nor merely in the details of their craft or vocation. Ali’s novel offers,
rather, a political-economic set of conjectures about what kind of translingual labor
goes unacknowledged in the production of commercial and political homeostasis World Literature (Already) Wrote Back: Sabahattin Ali after Germany 37 in a monolingualist national society like Turkey in the 1940s. The translator is
metonymically linked—due to age, his pleurisy, and his lack of ostentatious Euro-
peanness—to the Ottoman past: Still, the president and our Hamdi would treat him in a manner that boils
down to: “Look, we’re not throwing you out, despite your whiny, sickly
personality!” They never missed an opportunity to scold him and then sar-
castically welcome him back after a couple days’ absence, saying: “How’d
it go this time around? 16 “Buna rağmen müdürün ve bizim Hamdi’nin Raif efendiye karşı muamelelerinde: “Bak, seni şu
mızmız, hastalıklı haline rağmen atmıyoruz!” demek isteyen bir şey vardı. Bunu ikide birde yüzüne
vurmaktan da çekinmezler, birkaç gün yokluktan sonra her gelişinde adamcağızı: “Nasıl? İnşallah artık
bitti ya?” diye iğneli geçmiş olsunlarla karşılarlardı.” (Ali 1998: 20-21)
17 “Evvela alttakileri açtım; biri bomboştu, ötekinde birtakım kâğıtlar ve tercüme müsveddeleri vardı.
Üst göze anahtarı sokarken ürperdim: Raif efendinin senelerden beri oturduğu iskemlede oturduğumu 16 “Buna rağmen müdürün ve bizim Hamdi’nin Raif efendiye karşı muamelelerinde: “Bak, seni şu
mızmız, hastalıklı haline rağmen atmıyoruz!” demek isteyen bir şey vardı. Bunu ikide birde yüzüne
vurmaktan da çekinmezler, birkaç gün yokluktan sonra her gelişinde adamcağızı: “Nasıl? İnşallah artık
bitti ya?” diye iğneli geçmiş olsunlarla karşılarlardı.” (Ali 1998: 20-21)
17 “Evvela alttakileri açtım; biri bomboştu, ötekinde birtakım kâğıtlar ve tercüme müsveddeleri vardı.
Üst göze anahtarı sokarken ürperdim: Raif efendinin senelerden beri oturduğu iskemlede oturduğumu
ve onun her gün birkaç defa yaptığı hareketi tekrar ettiğimi şimdi fark etmiştim. Acele ile gözü
çektim.” (Ali 1998: 41) 17 “Evvela alttakileri açtım; biri bomboştu, ötekinde birtakım kâğıtlar ve tercüme müsveddeleri vardı.
Üst göze anahtarı sokarken ürperdim: Raif efendinin senelerden beri oturduğu iskemlede oturduğumu
ve onun her gün birkaç defa yaptığı hareketi tekrar ettiğimi şimdi fark etmiştim. Acele ile gözü
çektim.” (Ali 1998: 41) 19 “Yalnız bazı günler birdenbire vahşileşiyor, gözleri bütün ifadesini kaybediyor, küçülüyor ve
kendisine hitap edildiği zaman yavaş, fakat her türlü yakınlaşmayı meneden bir sesle cevap veriyordu.
Böyle zamanlarında tercüme yapmayı da ihmal ediyor, çok kere kalemi yanına bırakarak saatlerce
önündeki kâğıtları seyrediyordu. Onun şimdi bütün mesafelerin ve zamanın arkasına çekilmiş
olduğunu ve oraya kimseyi bırakmayacağını seziyor ve hiç sokulmak teşebbüsünde bulunmuyordum.
Yalnız içimi bir endişe kaplıyordu: Çünkü Raif efendinin hastalıklarının, garip bir tesadüfle, ekseriya
böyle günleri takip ettiğini fark etmiştim.” (Ali 1998: 34-35) The Clandestine Subjectivity of the Translator This universalizing gesture—added to the political
economy of translation and monolingualism laid out in Raif’s workplace and
household—suggests that Ali is proposing a general ethics of translingual meaning
under conditions of what must be regarded as early forms of globalization, a coun-
ter-narrative to the performative commercial cosmopolitanisms of his day. Still, the
frame narrator is insistent that the multilingual, transnational subjectivity of his
counterpart, Sir Raif, amid the prevailing cultural politics of his social and profes-
sional life, comes at great cost to him: In this gesture, we may understand the narrator and Ali as undertaking an endeav-
or to portray translators, multilinguals, and other mediators of anational or transna-
tional experience as an alternate, or oppositional, kind of citizen—unspectacular,
unassuming, and utterly capable of existing amid the worldly affordances of au-
tochthonous, suburban life. Everyone, Ali seems to suggest, could be like Raif;
everyone could be a translator. This universalizing gesture—added to the political
economy of translation and monolingualism laid out in Raif’s workplace and
household—suggests that Ali is proposing a general ethics of translingual meaning
under conditions of what must be regarded as early forms of globalization, a coun-
ter-narrative to the performative commercial cosmopolitanisms of his day. Still, the
frame narrator is insistent that the multilingual, transnational subjectivity of his
counterpart, Sir Raif, amid the prevailing cultural politics of his social and profes-
sional life, comes at great cost to him: Some days he would suddenly go wild, his eyes would shrink and lose all
expression, and he would reply to me with a voice that, though soft, re-
jected any kind of intimacy. At times like this, he also neglected his transla-
tion work, glaring at the papers in front of him for hours, his pencil lying
on the table. I felt he had retreated behind all distance and time, and that
he wouldn’t let anyone through, so I would never try to approach. But I
did have one particular concern: I noticed that Sir Raif’s periods of illness
usually followed days like this, as coincidence would have it.19 (Ali
2014: 14) It is revealed, later in Sir Raif’s own notebook, that this anxiety of multidirectional,
multilingual memory indeed brings torment to Raif. 18 “Raif efendinin saf yüzü, biraz dünyadan uzak, buna rağmen bir insana tesadüf ettikleri zaman
tebessüm etmek isteyen bakışları gözlerimin önünde canlanıyor. Halbuki o hiç de fevkalade bir adam
değildi. Hatta pek alelade, hiçbir hususiyeti olmayan, her gün etrafımızda yüzlercesini görüp de
bakmadan geçtiğimiz insanlardan biriydi.” (Ali 1998: 11) The Clandestine Subjectivity of the Translator Is it over, God willing?”16 (Ali 2014: 6) Through watching this violent bullying—and with it, the social suppression of
translators and translating despite the profits regularly derived from them—the
frame narrator acquires a new dream: to emulate, become, replace, and perpetuate
the spirit of Sir Raif, who dies at the end of the frame narrative, forbidding the
narrator from viewing and divulging his multilingual, transnational past in Germa-
ny. Sitting alone in the office he and Raif used to share, the narrator opened the lower [drawers of the desk]: the first one was completely emp-
ty, the other had some papers and scraps of translations. When I inserted
the key for the top draws, I shuddered: I realized I was sitting in Sir Raif’s
chair and doing exactly the same thing he used to do at least twice a day.17
(Ali 2014: 18) Despite this turn toward emulation in the latter stages of their relationship, the
frame narrator does not seek to characterize Raif as particularly special, saintly, or
anointed, and he grows to understand Raif’s translator identity and translation
practice without resorting to mystical tropes of talent or chosenness. The narrator
sees his “counterpart” (“karşımdaki”), an important word throughout both the
frame narrative and the embedded love story, as a worker among workers who has
chosen a livelihood that his contemporaries respond to aphasically, but one that is
hardly worthy of such bafflement. Raif was merely: a bit distant from this world, a bit puzzled, but still eager to smile when
[his eyes] encounter another person. He was no extraordinary man, not by
any means. Quite common indeed, with no particular features worth men- David Gramling/Martina Schwalm 38 tioning. Just one of the hundreds of people you see around everyday with-
out looking at them twice.18 (Ali 2014: 1) tioning. Just one of the hundreds of people you see around everyday with-
out looking at them twice.18 (Ali 2014: 1) In this gesture, we may understand the narrator and Ali as undertaking an endeav-
or to portray translators, multilinguals, and other mediators of anational or transna-
tional experience as an alternate, or oppositional, kind of citizen—unspectacular,
unassuming, and utterly capable of existing amid the worldly affordances of au-
tochthonous, suburban life. Everyone, Ali seems to suggest, could be like Raif;
everyone could be a translator. The Clandestine Subjectivity of the Translator The novel certainly permits us
to understand this anxiety primarily as that of a person mourning his long lost love
or writhing silently in a perpetual state of penitence for failing at his personal re-
sponsibilities. Certainly, Raif does feel these things, but the frame narrative allows
and indeed privileges a broader reading, one that does not come down to tropes of It is revealed, later in Sir Raif’s own notebook, that this anxiety of multidirectional,
multilingual memory indeed brings torment to Raif. The novel certainly permits us
to understand this anxiety primarily as that of a person mourning his long lost love
or writhing silently in a perpetual state of penitence for failing at his personal re-
sponsibilities. Certainly, Raif does feel these things, but the frame narrative allows
and indeed privileges a broader reading, one that does not come down to tropes of World Literature (Already) Wrote Back: Sabahattin Ali after Germany 39 love and loss alone. Indeed, the frame narrator’s characterization of Raif, his
commentary on the workplace and the homelife of his “counterpart,” and his fore-
grounding of labor, government, and bureaucracy throughout the first fifth of the
novel remind us that Raif is also a remigrant. His experience of the Republican
project differs from those of his contemporaries in the office, in that he left Anato-
lia at the height of the post-Ottoman crisis in 1923-4 and only returned, begrudg-
ingly and forlorn, once the monolingual, nationalizing Kemalist project was well
underway. Not engaged in national rebirth and redefinition in the way that Ali’s
Translation Bureau colleagues were, Raif entered Republican society with a hybrid-
ized language, literature, and aesthetic experience that had little in common with
the grand challenges of the government and its ministries. The result is a structure
of sanction and silence, according to which Raif becomes unable to attend in
(Turkish) language to his own subjectivity in Germany. It appears to have taken
him ten years to even write down this aphasic, elliptical aspiration: I want to talk about it, I want to say things, many things… To whom? Is
there any other human being wandering on this immense earth all by him-
self like I am? What can I tell of it, and to whom? I don’t recall saying a
word to anyone for ten years now. 20 “Söylemek, bir şeyler, birçok şeyler anlatmak istiyorum... Kime?... Şu koskocaman dünyada benim
kadar yapayalnız dolaşan bir insan daha var mı acaba? Kime, ne anlatabilirim? On seneden beri hiç
kimseye bir şey söylediğimi hatırlamıyorum. Boşuna yere herkesten kaçmış, boş yere bütün insanları
kendimden uzaklaştımışım; ama bundan sonra başka türlü yapabilir miyim? Artık hiçbir şeyin
değişmesine imkân yok... Lüzum da yok. Demek böyle olması icap ediyormuş. Zaten bu defteri neden
aldım?” (Ali 1998: 47-48) 21 “Içimdekileri herhangi şekilde olursa olsun dışarıya vurmak korkusu, bu manasız ve lüzumsuz
ürkeklik yazı yazmama mâniydi. Yalnız resim yapmaya devam ediyordum. Bu iş bana, içimden bir şey
vermek gibi gelmiyordu. Dışarıyı alıp bir kâğıda aksettirmekten, bir mutavassıtlıktan ibaret
görünüyordu. Nitekim işin böyle olmadığını anlayınca bundan da vazgeçtim... Hep o korku
yüzünden.” (Ali 1998: 50) 22 “Bir ecnebi dil öğreneceğimi, bu dilde kitaplar okuyacağımı, ve asıl, şimdiye kadar sadece
romanlarda rastladığım insanları işte bu ‘Avrupa’da bulacağımı tahmin ediyordum. Zaten muhitimden
uzak duruşumun, vahşiliğimin bir sebebi de kitaplarda tanıştığım ve benimsediğim insanları
muhitimde bulamayışım değil miydi?” (Ali 1998: 52) The Clandestine Subjectivity of the Translator I ran away from people for nothing, I
pushed everyone away from me for nothing. But was there any other op-
tion? It’s impossible to change it now… There’s no point anyway… So it
must have been meant to be like this. [...] Why did I buy this notebook
anyway?20 (Ali 2014: 22) With this retreating set of self-characterizations, we come to understand not only
the suppression of translation and translators afoot in the social world of this nov-
el, but furthermore the ways in which Raif finds his way into translating, somewhat
accidentally and regretfully. He “ends up” being a mediator between cultures and
languages, not because he feels a grand intercultural zeal to bring diverse people
together, and not even because he loves language and writing, perhaps despite the
people who produce them. Rather, translating is something Raif does out of fearful
and compulsive self-protection. We discover this first when Raif decides to fail out
of art school in Istanbul, precipitating a last-ditch effort at social solvency by way
of a soap-manufacturing internship with a Swedish company in Berlin: David Gramling/Martina Schwalm 40 The fear of expressing what was inside me in any fashion whatsoever,
along with this senseless and purposeless timidity of mine, proved a hin-
drance to my writing. Painting was all I continued to do. That activity
didn’t feel like I was giving away anything inside me. Taking the outside
world and reflecting it on a canvas: that just felt like being a mediator. Still,
when I understood how much this wasn’t the case, I gave it up too… All
out of fear…21 (Ali 2014: 24) The fear of expressing what was inside me in any fashion whatsoever,
along with this senseless and purposeless timidity of mine, proved a hin-
drance to my writing. Painting was all I continued to do. That activity
didn’t feel like I was giving away anything inside me. Taking the outside
world and reflecting it on a canvas: that just felt like being a mediator. Still,
when I understood how much this wasn’t the case, I gave it up too… All
out of fear…21 (Ali 2014: 24) 23 “Sokakları biraz daha geniş, çok daha temiz, insanları daha sarışın bir şehir. Fakat ortada insanı
hayretinden düşüp bayılmaya sevk edecek bir şey de yoktu. Benim hayalimdeki Avrupa'nın nasıl bir
şey olduğunu ve şimdi içinde yaşadığım şehrin buna nazaran ne noksanları bulunduğunu kendim de
bilmiyordum... Hayatta hiçbir zaman kafamızdaki kadar harikulade şeyler olmayacağını henüz idrak
etmemiştim.” (Ali 1998: 52) An Anti-Study-Abroad Novel? It must be said that the Madonna novel does not quite permit a reading in which a
dispirited young person, feeling claustrophobic in his own cultural environs, strikes
out on an adventure abroad and comes back with brand-new insights on the world. If we are audacious enough to consider this novel an early form of Turkish-
German literature—and indeed there are adequate historical connections to make
such a claim—we notice that Madonna generally foregoes the explanatory idiom of
cultural difference, and indeed casts critical light upon it. Raif, shuttled off to
Germany for commercial reasons and to take advantage of the exchange rate, isn’t
particularly interested in cultures and languages themselves, as much as he is
poised to survey, in Occidentalist fashion, the raw material that inspired the world-
literary figures of his childhood imagination: In this “Europe”, I expected to learn a foreign language and read its
books, but more importantly I’d finally encounter the people I’d only
known in novels. Perhaps my waywardness and disaffection toward my
surroundings were merely the result of not being able to meet the people
I’d so loved and known in books?22 (Ali 2014: 24) What he discovers is something much less confirmatory of his imagination and
much more compliant with the institutional proportions of international moderni-
ty. There is little for him to prospectively ‘translate’ there, in the sense of mediating
cultural diversity upon a structuralist grid from source to target culture, or from
one national public to another. His gaze upon Berlin is indifferent to its evidentiary
or statutory Germanness, and he is confronted rather with two versions of what
we might call globalization today. Firstly, Raif encounters equivalent, transposable World Literature (Already) Wrote Back: Sabahattin Ali after Germany 41 forms that he expected from his home environment, all of the infrastructural scaf-
folding necessary for a cognate nationalism: A city with wider streets—much cleaner, and with blonder people. But
there wasn’t much about it that would make a person swoon in awe. 24 “Avuç içi kadar kâğıdın üzerinde Hamdi'yi görüyordum. Beş on basit fakat fevkalade ustaca çizginin
içerisinde bütün hüviyetiyle o vardı. Başkalarının aynı benzeyişi bulacaklarını pek zannetmem, hatta
teker teker araştırılınca belki hiçbir tarafı benzemiyordu, fakat onun biraz evvel odanın ortasında nasıl
avaz avaz bağırdığını gören bir insan için yanılmaya imkan yoktu.” (Ali 1998: 22-23) 25 “Hamdi hakkında birbirine zıt bir sürü hükümler verip duruyordum. Onu bazan mazur görmeye
çalışıyor, çok kere de istihfaf ediyordum. Asıl şahsiyetiyle, bugünkü mevkiinin ona verdiği şahsiyeti
birbirine karıştırıyor, sonra bunları ayırmak istiyor ve büsbütün çıkmaza giriyordum. İşte Raif
efendinin birkaç çizgi ile ortaya koyduğu Hamdi, benim uzun zamandan beri görmek istediğim halde
bir türlü göremediğim insandı. [...] Aynı zamanda bu resim bana birdenbire Raif efendiyi de izah
etmişti.” (Ali 1998: 23) An Anti-Study-Abroad Novel? For
my part, I was still back then unaware what kind of a thing the Europe of
my dreams really was, and how much the city I was now living in lacked,
in comparison to that image… It hadn’t yet dawned on me how the mind
can conjure the most improbable projections.23 (Ali 2014: 25) This insight is important for the broader question of world-literary translation,
because it deemphasizes—for the balance of the novel—the notion that translation
is primarily a diplomatic activity, linking one linguistically mediated culture to an-
other. In Madonna, we observe a more volatile and less instrumental account of
what translation is indeed supposed to offer, and this account surfaces most
poignantly in an instance of ekphrasis, as Raif—an erstwhile art-student, erstwhile
multilingual, erstwhile lover, and now paycheck-to-paycheck translator in a posi-
tion with no recognition or advancement—renders visible the normative model of
Republican citizenship, as embodied in the rage-filled and desperate firm vice-
president, Hamdi. On this palm-sized piece of paper, I could see Hamdi. By way of five or
ten masterly crafted lines, he existed there in all his essence. I don’t think
others would notice the same likeness; maybe if you scrutinized the lines
one by one, you could’ve come to the conclusion that it didn’t look like
him at all, but for someone who’d just witnessed the man shouting at top
of his lungs in the middle of this room, it was an unmistakable likeness.24
(Ali 2014: 7) Hamdi’s repeated anger at Raif’s ostensible tardiness with translating firm docu-
ments begins to make sense if we see it not merely as the ill temperament of an
irrational tyrant—which is how the frame narrator sees it—but rather as the neces-
sary and logical result of a domestic economy dependent on transnational,
translingual traffic and daily indebted to its disciplined instrumentalization. Hamdi
is angry at Raif, not because he dislikes him, so much as because Hamdi absolutely David Gramling/Martina Schwalm 42 needs the propositional content (or what we today would call IP or ‘intellectual
property’) in order to meet his own liberal-market benchmarks. The narrator con-
tinues, contemplating not only Hamdi, but the civic position he represents in Re-
publican post-imperial commerce and self-making: I kept coming up with contradictory assessments of Hamdi. I tried to
make excuses for him, but mostly I ended up pitying him. An Anti-Study-Abroad Novel? I always con-
fused his actual personality with the personality he’d acquired through his
stature. Then I tried dissociating them, which always led me to an impasse. Here was Hamdi, manifested in a couple of lines Sir Raif drew—the per-
son I couldn’t see into, though I’d wanted to for so long. [...] What’s more,
this picture explained Sir Raif to me.25 (Ali 2014: 8) This image is the only ‘translation’ Raif produces at the Ankara office that we read-
ers are allowed to see and assess, over the shoulder of the frame narrator. The way
our attention is called to Raif’s capacity to translate critically rather than instrumen-
tally, to render visible an essence, aura, or truth that is hidden from view even in
the ‘original’ artifact, is itself the fruit of Raif’s decades-long love- and loss-affair
with Maria Puder, whom he has drawn, conjured, and fancied in his dreams since
last he was in Berlin. This obsession to translate her has ostensibly been purpose-
less, ‘pointless’ as Raif says at the end of his life, and impervious to the kind of
instrumentalization of translation that reigns supreme in his contemporary envi-
rons. Refracting this critical capacity he learned from translating Maria Puder back
onto civil society in Ankara, Raif delivers to readers of Madonna the one indication
necessary to round out an ethics of translation practice and a complex ars poetica for
translators in the era of modern, nationalized monolingualism. We hope to have shown in this article how a pre-1945 novel, coming from the
post-Imperial contexts of early Republican Turkey, already long ago “wrote back”
to the latter-day metropoles of theory-making with a critical intervention about
world-literature-in-translation, a question which continues to vex the curricular and
theoretical imagination seventy-five years hence. Ali’s novel reminds us of the
critical resources that dwell within pre-1945 literary works that, often precisely
because of their patent hybridity and translingual bearing, elided attention from
their contemporary peers and thus remain underrepresented in world-literary cur-
ricula and theorization today. We may regard Ali himself as a theorist not only of
both literary translation and its social contexts under heightened conditions of World Literature (Already) Wrote Back: Sabahattin Ali after Germany 43 nationalism, but also of the world-literary imagination beyond the confines of the
Mercatorian model (Cheah 2014). nationalism, but also of the world-literary imagination beyond the confines of the
Mercatorian model (Cheah 2014). Literature Aksoy, Nüzhet Berrin (2010): “The Relation Between Translation and Ideology as
an Instrument for the Establishment of a National Literature,” in: Meta: Journal
des Traducteurs (55/3), 438-455. Albachtan, Özlem (2013): “Intralingual Translation as ‘Modernization’ of the
Language: The Turkish Case,” in: Perspectives: Studies in Translatology (21/2), 257-
271. Ali, Sabahattin (1998 [1943]) Kürk Mantolu Madonna, İstanbul. Ali, Sabahattin (2014 [1943]): The Madonna in the Fur Coat, translated by İlker
Hepkaner and David Gramling, unpublished. Ashcroft, Bill / Griffiths, Gareth / Tifflin, Helen (eds.) (1989): The Empire Writes
Back: Theory and Practice in Post-Colonial Literatures, London. Aytürk, İlker (2004): “Turkish Linguists against the West: The Origins of Linguistic
Nationalism in Atatürk’s Turkey,” in: Middle East Studies (40/6), 1-25. Beecroft, Alexander (2008): “World Literature without a Hyphen: Towards a
Typology of Literary Systems,” in: New Left Review (54), 87-100. Bourdieu, Pierre / Wacquant, Loic J. D. (1992): An Invitation to Reflexive Sociology,
Chicago. Casanova, Pascale (1999): La République mondiale des lettres, Paris. Cassin, Barbara (ed.) (2004): Vocabulaire Européen des Philosophies: Dictionnaire des
Intraduisibles, Paris. Cheah, Pheng (2014): “World against Globe: Toward a Normative Conception of
World Literature,” in: New Literary History 45, 303-329. Damrosch, David (2005): “Death in Translation,” in: Bermann, Sandra / Wood,
Michael: Nation, Language, and the Ethics of Translation, Princeton, 180-198. Dickinson, Kristin (2013): “A Dual Critique of Modernity in Sabahattin Ali’s ‘Kürk
Mantolu Madonna’,” in: Transit: A Journal of Travel, Migration and
Multiculturalism in the German Speaking World (9/1), 1-8. Friedrich, Werner P. (1960): “On the Integrity of Our Planning,” in: Block,
Haskell: The Teaching of World Literature, Chapel Hill, 14-15. David Gramling/Martina Schwalm 44 Gramling, David / Hepkaner, İlker (2016): “Translating the Translingual Novel in
Early Turkish Republican Literature: The Case of Sabahattin Ali,” in:
Authorizing Translation: Literature, Theory and Translation, Yearbook of the
International Association for Translation and Intercultural Studies (IATIS), edited by
Michelle Woods, London. Hymes, Dell (1965): “Some North Pacific Coast Poems: a Problem in
Anthropological Philology,” in: American Anthropologist (76/2), 316–41. Leonard, Diane R. (2010): “Werner Paul Friederich: ‘Christopher Columbus of
American Comparatism,’” In: Comparative Critical Studies (7/2-3), 179-191. Noorani, Yaseen (2013): “Hard and Soft Multilingualism,” in: Critical Multilingualism
Studies (1/2), 7-28. Schwalm, Martina (2015): “Es war mir ein Anliegen, das Geheimnis nicht zu
lüften: Sieben Formen von Unübersetzbarkeit in Ilija Trojanows Der
Weltensammler,” in: Zeitschrift für interkulturelle Germanistik (6/1), 81-100. Literature Spivak, Gayatri Chakravorty / David Damrosch (2011): “Comparative Literature /
World Literature: A Discussion,” in: Damrosch, David (ed.): World Literature in
Theory, London. Szurek, Emmanuel (2015): “The Linguist and the Politician: The Türk Dil Kurumu
and the Field of Power in the 1930s-40s,” in: Aymes, Marc / Gourisse,
Benjamin / Massicard, Élise (eds.): Order and Compromise: Government Practices in
Turkey from the Late Ottoman Empire to the Early 21st Century, Amsterdam, 68-96. Venuti, Lawrence (1995): The Invisibility of the Translator, London. Venuti, Lawrence (2016): “Hijacking Translation: How Comp Lit Continues to
Suppress Translated Texts,” in: Boundary (43/2), 179-204. Woods, Michelle (ed.) (2016): Authorizing Translation, London. Yücel, Hasan Ali (1939): “Çeviri Komisyonu Raporu,” in: Birinci Türk Neşriyat
Kongresi: Raporlar, Teklifler, Müzakere Zabıtları, Ankara, 125-127. 1 From now on referred to as Madonna. Intervening in the Humanist Legacy:
Sabahattin Ali’s Kleist Translations Kristin Dickinson Sabahattin Ali’s theoretical reflections on translation and his role as German to Turkish transla-
tor for the state-sponsored Translation Bureau form a central, but largely overlooked aspect of his
literary legacy. This article explores the overlap between Ali’s positions as author and translator
vis-à-vis the World Literature in Translation publication series (1940-1966) he helped to both
shape and execute. As a case study, it examines the relationship between Ali’s final novel “Kürk
Mantolu Madonna” (1943) (Madonna in a Fur Coat) and his translation of “Die Verlobung
in St. Domingo” (1811) (The Betrothal in Santo Domingo). Through an attention to critical
narrative gaps in each of these texts, the article calls attention to the subtle manner in which Ali
was able to resist dominant paradigms of translation—such as smooth translatability and civili-
zational transfer—through his position as a state employee. As such, this article documents an
alternative history of translation in the modern Republic of Turkey that emerges from within a
state-sponsored project of culture planning. Derided at work and largely ignored at home, the main character of Sabahattin
Ali’s final novel, Kürk Mantolu Madonna1 (1943), attests to the invisibility of the
translator and the labors of translation. A German to Turkish translator at an An-
kara bank, Raif is introverted to the extreme, to the extent that colleagues even
doubt the validity of his linguistic skills. Treated as a relic of the past, Raif’s seem-
ing invisibility within Madonna stands in contrast to a heightened visibility of trans- Kristin Dickinson 46 lation at the time of the novel’s publication, in which a wide-reaching, state-
sponsored translation movement was endorsed and set into motion. With the goal
of systematically translating the classics of Ancient Greek, Latin, and contemporary
Western European literatures, a World Literature in Translation (Dünya Edebiyatın-
dan Tercümeler) series sought to engender a Turkish renaissance and specific form
of Turkish humanism, thereby forging a common cultural repertoire for citizens of
the new Republic (Tahir Gürçağlar 2003: 114). As Ali was a founding member of the translation bureau that headed this
endeavor, his own highly visible position as state employee stands in contrast to
Raif’s hidden translation work. Yet both arguably resisted the premise of smooth
translatability that underscored wide-ranging Republican humanist reforms, and
that treated modernization, nationalization, and westernization as problem-free
processes. By reading Ali’s translation of Heinrich von Kleist’s Die Verlobung in St. 2 Ali’s translation of Verlobung was included in the collection Üç Romantik Hikaye (Three Romantic
Stories) which appeared in 1943; this collection also included Ali’s translations of Adelbert von Cham-
isso’s Peter Schlemihl’s Wundersame Geschichte (translated as Peter Schlemihl’in Acayip Sergüzeşti) and E.T.A.
Hoffmann’s Doge und Dogaresse (translated as Duka ile Karısı). Intervening in the Humanist Legacy:
Sabahattin Ali’s Kleist Translations Domingo (1811) (The Betrothal in Santo Domingo) together with Madonna, this
article explores the ways in which Ali’s literary production overlapped with his
actual translation practice. Incidentally, San Domingo’da bir Nişanlanma, which first
appeared in the journal Tercüme (Translation) in 1940, coincided with the serializa-
tion of Madonna; both texts were subsequently published in book format in 1943.2 Taking the Kleistian references in Madonna into account, I ask how Verlobung,
together with Ali’s translation of it, reflects back on the structure of Ali’s final
novel. In particular, I consider the correspondence between central, unnarrated
moments of sexual union in each text. As critical “Leerstellen” (Iser 1975) or gaps,
these moments of non-narration both create epistemological uncertainty and ac-
tively engage the reader as cross-cultural interpreter. Through the use of a frame
narrative, Ali figures the narrator of Madonna as one such interpreter, who must fill
in the gaps of the main character’s life story by ‘reading’ the diary of his youth. By pointing to the contradictory nature of its literary underside, I argue that
Ali’s translation of Verlobung works together with his final novel, Madonna, to com-
plicate the Republican premise of smooth translatability, and thus also the stable
category of the ‘West’ it presumes. Whereas translation activity was one crucial
arena through which Turkey sought to emerge as an independent political entity
that identified itself as European, Ali poses the more difficult question of what
‘Western’ values themselves might be. Toward an Alternative History of Translation Secondary scholarship on the translation bureau has largely treated the systema-
tized form of publishing it supported as an instance of culture planning, defined as
the “deliberate intervention,” either by power holders or by ‘free agents,’ “into an Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 47 extant or a crystallizing repertoire” (Even-Zohar 2008: 278). In her analysis of
private publishing efforts in the 1940s and 50s, Şehnaz Tahir Gürçağlar emphasiz-
es the need to view translation efforts of this time period not simply as processes
shaped by pre-determined norms, but also as the result of individual decision-
making (Tahir Gürçağlar 2008: 24-25). In doing so, she takes a step back from the
institutional framework of the bureau to show how private publishers and
translators formed an important counterforce that “resisted the norms offered by
the dominant discourse of the planners” (Tahir Gürçağlar 2008: 31). Building on
this argument, I ask further: to what extent did voices coming from within the
bureau resist dominant translation discourses and norms? As an author who
critically engaged with the shortcomings of modernity, and the conception of mo-
dernity as a monolithic or Western discourse, Sabahattin Ali is a case in point. y
p
Sabri Gürses situates Ali within a larger, cultural search for a delineated
program of translation in the early Republic; as such, Gürses views Ali as central to
the formation of an intellectual discourse that utilized translation—understood as a
form of cultural transfer (kültür aktarımı)—as a means for constructing a national
Turkish culture (Gürses 2013: 414). This discourse was dependent on the rhetoric
of smooth translatability, which posited a stable category of Western European
ideals and values that could be easily transferred to the Turkish context. I argue, on
the contrary, that we must read Ali’s participation in the larger translation move-
ment as a counterpoint to the concepts of cultural transfer and smooth translata-
bility that emerged in the immediate wake of the translation bureau’s founding. While Ali enthusiastically supported the translation of Western European
literatures, his writing also reveals a more ambivalent view of the ‘West’ than
dominant translation discourse of the time. By pointing time and again to the
contradictions and inconsistencies of Western modernity, his work both
complicated the very possibility of translation as a form of frictionless transfer and
constituted an important countervoice to the kind of Europhilia endorsed at the
time. ‘Translating the West’ The role of literary translations vis-à-vis the development of the modern Turkish
language and the Europeanization of Turkish society were a source of intense pub-
lic debate in the early Republican period. Following the establishment of the mod-
ern Republic of Turkey in 1923, calls for a comprehensive, systematized, and gov-
ernment-funded program of translation began to gain ground throughout the
1930s; a programmatic approach to translation activity was posited as a much-
needed corrective to what intellectuals described as the inadequate and erroneous
nature of late Ottoman translation movements. The field of translation became one crucial site from which the newly founded
Republic asserted itself over and against its Ottoman predecessor. In particular,
following the adoption of the Latin alphabet in 1928, and major language reforms Kristin Dickinson 48 throughout the mid-1930s, the translation of Western European classics became a
crucial means of building a new discursive center for Turkish society. Within the
model of modernization as westernization undertaken by the Republican People’s
Party, translation activity was understood as central to Turkey’s immersion within
the history of European thought, and its successful participation in contemporary
European civilization. throughout the mid-1930s, the translation of Western European classics became a
crucial means of building a new discursive center for Turkish society. Within the
model of modernization as westernization undertaken by the Republican People’s
Party, translation activity was understood as central to Turkey’s immersion within
the history of European thought, and its successful participation in contemporary
European civilization. The rhetoric of completion and entirety utilized by seminal theorists of transla-
tion was predicated upon the smooth translatability of western European values
into the Republican context. This premise was also central to Hasan Ali Yücel’s
conception of translation as a method of transferring humanist values to Turkey
and his underscoring of a communal human spirit (insan ruhu) across East and
West (Çıkar 1997: 62). Shortly after assuming the position of Minister of Educa-
tion in December of 1938, Yücel took the first major step toward achieving this
goal: the First Turkish Publishing Convention (Birinci Türk Neşriyat Kongresi) was
convened in May of 1939 to develop a detailed and systematic plan for the
publishing industry in the years to come.3 Yücel envisioned a program of systematized translation through which a dis-
tinctly Turkish national identity could emerge that identified itself as European. 3 Committees consisting of authors, intellectuals, journalists, publishers, and educators were
established to assess and report on the state of the following seven categories: 1) printing, publishing,
and sales 2) petitions 3) copyright for literary works 4) children’s and youth literature 5) prizes and
propaganda 6) publishing program, and 7) translation.
4 Ahmet Ağaoğlu was one critical voice who argued against a form of culture planning, which he
believed would lead to a standardization of the diverse individual thoughts and feelings that make up
an aggregate culture.
5 In addition to chairman Ethem Menemencioğlu and reporter Mustafa Nihat Özön, the translation
committee consisted of the following members: Abdülhak Şinasi Hisar, Ali Kâmi Akyüz, Bedrettin
Tuncel, Burhan Belge, Cemil Bilsel, Fazıl Ahmet Aykaç, Fikret Adil, Galip Bahtiyar Göker, Halil
Nihat Boztepe, Halit Fahri Ozansoy, İzzet Melih Devrim, Nasuhi Baydar, Nurettin Artam, Nurullah
Ataç, Orhan Şaik Gökyay, Rıdvan Nafiz Ergüder, Sabahattin Rahmi Eyüboğlu, Sabahattin Ali, Sabri
Esat Siyavuşgil, Selami İzzet Sedes, Suut Kemal Yetkin, Şinasi Boran, Yusuf Şerif Kılıçer, Yaşar Nabi,
and Zühtü Uray (Kültür Bakanlığı 1997: 35). Ahmet Ağaoğlu was one critical voice who argued against a form of culture planning, which he
believed would lead to a standardization of the diverse individual thoughts and feelings that make up
an aggregate culture.
5 In addition to chairman Ethem Menemencioğlu and reporter Mustafa Nihat Özön, the translation
committee consisted of the following members: Abdülhak Şinasi Hisar, Ali Kâmi Akyüz, Bedrettin
Tuncel, Burhan Belge, Cemil Bilsel, Fazıl Ahmet Aykaç, Fikret Adil, Galip Bahtiyar Göker, Halil
Nihat Boztepe, Halit Fahri Ozansoy, İzzet Melih Devrim, Nasuhi Baydar, Nurettin Artam, Nurullah
Ataç, Orhan Şaik Gökyay, Rıdvan Nafiz Ergüder, Sabahattin Rahmi Eyüboğlu, Sabahattin Ali, Sabri
Esat Siyavuşgil, Selami İzzet Sedes, Suut Kemal Yetkin, Şinasi Boran, Yusuf Şerif Kılıçer, Yaşar Nabi,
and Zühtü Uray (Kültür Bakanlığı 1997: 35). ‘Translating the West’ His call for a comprehensive (geniş), and concrete (muayyen) program of translation
over a period of several years that could serve as the basis for such learning was
strongly supported by leading authors and intellectuals of the time; figures such as
Ahmet Hamdi Tanpınar, Yunus Kazım Köni, and Yaşar Nabi Nayır all emphasized
the need for state intervention in order to make quality, affordable literature in
translation available to an undereducated public with little disposable income.4 Sabahattin Ali served as one of 27 members on the translation committee,5
which took the first major step in realizing Yücel’s vision by producing an initial
list of 294 works to be translated into Turkish. A permanent bureau was then es-
tablished in 1940 under the direction of Nurullah Ataç. Sabahattin Ali served as
one of seven permanent board members for this bureau, as well as editor and
translator of German texts until 1944, when he was put under surveillance for Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 49 suspicious political activity.6 Describing this brief window of time as a “beautiful
period” (güzel dönem) (Togar 2011: 70) of Sabahattin Ali’s life, Melahat Togar’s rec-
ollection of his enthusiasm for the project at large is a reminder that state interven-
tion in the publishing industry initiated previously unthinkable opportunities in the
realm of translation; it set into motion the extensive World Literature in Transla-
tion series, and established a bureau in which authors came together to grapple
with the question of how to translate the great works of world literature into a
newly reformed modern Turkish language that was still struggling to establish its
own vocabulary and modes of expression. y
p
The existence of such debate suggests that supporting state intervention in the
publishing industry did not necessarily preclude one’s ability to critique the state
and its modernizing institutions from within. While Ali himself described the in-
commensurability of his identities as government employee and independent au-
thor (Ali 2008a: 424), the fact remains that they overlapped during his time at the
translation bureau. Written precisely during this time period, Madonna passed under
the radar of state censorship and continues to be read in a largely apolitical light. 6 Additional board members included Nurullah Ataç (chairman), Saffet Pala (secretary general), Sa-
bahattin Eyüboğlu, Bedrettin Tuncel, Enver Ziya Karal, and Nusret Hızır (Tahir Gürçağlar 2008: 70-71).
7 Among the authors Ali translated throughout the 1930s are: Max Kemmerich, Feodor Mihayloviç
Dostoyevski, Heinrich Heine, Friedrich Stendhal, and Gottfried Keller. ‘Translating the West’ By showing how this novel is closely tied to Ali’s translation activity for the bureau,
I argue, on the contrary, that he utilized his positions as both author and translator
to subtly assert disagreement with the modernization project at large. Among the authors Ali translated throughout the 1930s are: Max Kemmerich, Feodor Mihaylov
Dostoyevski, Heinrich Heine, Friedrich Stendhal, and Gottfried Keller. Identities in Crisis Ali was an active translator prior to his involvement with the translation bureau,7
and by the time of the first publishing convention in 1939, he had clearly estab-
lished himself as an expert on German literature and culture. While it is impossible
to determine the exact role he played in selecting works for the World Literature in
Translation series, it is only logical to assume his opinion had significant influence
with regard to the German portion of this list. The initial selections from German
literature included standard Enlightenment (Goethe, Lessing, Schiller), Romantic
(Hoffmann, Chamisso, the Grimm brothers), and Realist (Keller) authors. The
inclusion of several works by Heinrich von Kleist is not surprising, considering his
canonical status at the time of the publishing convention. Yet the placement of his
texts within a translation series meant to generate a comprehensive humanist re-
form movement in Turkey raises larger discursive questions that are also pertinent
to Ali’s involvement with the bureau. In the introduction to all texts published within the translation series, Yücel
describes literature as an embodiment of the humanist spirit; here—and in diverse
other public statements and publications—he underscores the value of translation
activity as a means of participating in humanist civilization, strengthening the Turk- Kristin Dickinson 50 ish educational system, and enriching Turkish readers’ perception of the world. With an overwhelming number of Western European classics, the World Litera-
ture in Translation series nevertheless upheld an understanding of both humanism
and civilization as intrinsic to the West. ish educational system, and enriching Turkish readers’ perception of the world. With an overwhelming number of Western European classics, the World Litera-
ture in Translation series nevertheless upheld an understanding of both humanism
and civilization as intrinsic to the West. What would it mean to translate Kleist into both Yücel’s idealistic vision and
the actual program of translation he endorsed? Although Ali describes Kleist as a
Romantic in the introductory remarks to his translation, Kleist’s literature does not
easily fit into any specific literary movement. His work offers, rather, a provocative
and inconclusive exploration of topics central to the experience of modernity, such
as the inauthenticity of the self, the psychology of national belonging, and anti-
colonial struggle. Kleist’s grueling everyday experiences in the Prussian military
(1792-1799) exposed him to the contradictions of enlightened humanism at a
young age. 9 Texts by Kleist included in the original translation list were: the complete short stories, Der zer-
brochene Krug, Hermanns Schlacht (sic), and Prinz von Homborg (Kültür Bakanlığı 1997: 278). Texts actually
translated include: Die Familie Schroffenstein and Der Prinz von Homburg (translated by Burhanetin
Batıman), Michael Kohlhaas (translated by Necip Üçok), selected short stories (translated by Melahat
Togar), Die Verlobung in St. Domingo (translated by Sabahattin Ali), Der zerbrochene Krug (translated by
Hayrullah Örs), Penthesilea, Das Käthchen von Heilbronn, and Die Hermannsschlacht (translator unknown)
(Tercüme Listesi 1959: 73). 10 The story is set in 1803, following General Jean-Jacques Dessalines’ decisive defeat of French
forces in the battle of Battle of Vertières; under orders of Napoleon the French army sought to
reintroduce slavery in Santo Domingo, even though the French National Convention had promised
the abolition of slavery in 1794. The revolt succeeded in eliminating slavery and led to the establishment of the Republic of Haiti. Helmut Schneider goes so far as to read Kleist’s entire textual production as an ongoing challeng
the project of Enlightenment Humanism (Schneider 2003). Identities in Crisis Two years following his decision to leave the service, his famous ‘Kant
crisis’ (1801) marked a crucial turning point in his career, after which he began to
doubt his earlier investment in the Enlightenment ideals of autonomy, reason, and
progress, and develop in its place a radically skeptical view of the world (Fischer
2003: 4). Further informed by his experience of the French Revolution, its violent
disruption of established power relations, and the period of political instability that
followed, Kleist’s diverse textual production reveals the tensions and paradoxes of
Enlightenment rationalism and 18th-century humanist discourse.8 Thus, whether
intentional or not, the inclusion of Kleist’s short stories and dramas within the
World Literature in Translation series contains the potential to subvert a Republi-
can investment in the ideals of humanist discourse and their presumed value for
Turkish society. Among Kleist’s texts included in this series,9 Ali’s decision to
translate Verlobung is further notable for several reasons—from the relevance of
the turbulent political context and cross-cultural modes of belonging it evokes, to
the subtle, yet unmistakable, Kleistian references within Madonna. Through its focus on the continued practice of slavery in the French colony of
Saint Dominque following the French Revolution, Kleist’s novella exposes the
contradictions of a race-based colonial order.10 Set at the tail end of the first suc-
cessful slave revolt in the Western Hemisphere (1791-1804),11 it problematizes the Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 51 categories of race, gender, and nationality within a complex matrix of colonial
power relations. Verlobung explores these issues from a German perspective prior
to the establishment of a nation-state, following the occupation of German lands
by France under Napoleon in 1806. y
p
Within this context, Todd Kontje reads Verlobung as the reflection of a crisis of
German national identity at the time of its publication in 1811 (Kontje 1999). Ra-
ther than a patriotic expression of Germanness, Verlobung reveals a deep ambiguity
regarding what it means to be German at the turn of the century. In his discussion
of different forms of “passing”12 that occur within the novella, Kontje emphasizes
in particular Kleist’s use of the Swiss protagonist, Gustav, who fights for the
French army. 12 Kontje describes “passing” as an act that “complicates identity politics by highlighting the tension
between cultural constructs and biological essence, between convention and nature. The figure who
crosses borders between fixed sexual, racial, or national identities provokes what Marjorie Garber has
termed a category crisis” (Kontje 1999: 68-69). g
y
(
j
)
13 Darülfünun means House of Knowledge. The institution was established in 1863. Identities in Crisis After hiding
his extended family in the wilderness, the character of Gustav seeks provisions and
shelter from the plantation of Congo Hoango—a leader of the slave revolt who
has killed his former master and occupied his house. Here, Gustav encounters the
mulatto Babekan and her daughter, Toni. While Kleist refers to Toni as a mestiza—
or a mixture of European and Native American descent—she is actually part
French and part African. This use of the term mestiza nevertheless emphasizes a
key difference between Congo and Toni: unlike Congo, who was taken from his
home on the African Gold Coast and sold into slavery, Toni was born on the is-
land of Haiti. Under the rule of Congo, she is nevertheless subject to a different
form of oppression; Congo forces her to lure in white travelers—who are com-
forted by the relative whiteness of her skin—in order to then trap and murder
them. As hybrid constructions that defy ‘black and white definitions’ of race, nation-
ality or ethnicity, all of the major characters in Verlobung enact processes of cultural
translation, understood as “an anti-essentialist and anti-holistic metaphor that aims
to uncover... heterogeneous discursive spaces within a society” (Bachmann-Medick
2006: 37). They point to a dynamic concept of culture as a practice of negotiating
cultural differences, while at the same time exposing deadly power dynamics at
work within the colonial system. The question of what it means to translate such racial and cultural hybridities
into the heightened nationalism of 1940s Turkey takes on new meanings within
translation rhetoric of this time period. Anxieties and concerns regarding the prob-
lem of imitation were also prominent in early Republican translation theory. Such
anxieties were expressed through the wide-spread criticism of late Ottoman trans-
lation movements as inadequate, haphazard, and incomplete. In accusing late Ot-
toman translators of merely copying the West, Republican critics participated in
the larger nation-building project by asserting a break with the Ottoman Empire,
and upholding the idea that a “complete” translation of Western literature and / or
Western culture could be realized in the Republican era (Dickinson 2013: 3). With-
in this cultural climate, İsmail Habib developed a concept of tam Avrupa (Europe in
its completeness), which designated a canon of timeless world literary texts. Identities in Crisis As the citizen of a country that was also occupied by France, Gustav
plays a double role: he is both a victim and a perpetrator of French imperialism,
and he passes for the enemy within the slave revolt. Read in this vein, Verlobung has subversive implications regarding the histori-
cally important role Germany played for key Turkish reformers of the early twenti-
eth century. 19th-century German humanism and philology provided an important
model for the Darülfünun, the first institution of higher education in the Ottoman
Empire modeled on the European university system.13 Worried that the university
was becoming too German, professor of pedagogy İsmail Hakkı Baltacıoğlu, who
later became president of the university, feared a form of foreign infiltration: he
warned against the dangers of remaining German (Alman kalmak), appearing Ger-
man (Alman görünmek), and working in German (Almanca çalışmak) (Konuk 2010:
59). )
In contrast, the leading architect of Turkish nationalism, Ziya Gökalp, under-
stood the German model as crucial for both a successful restructuring of the uni-
versity and the establishment of a Turkish national consciousness. In the 1916
article “Maarif Meselesi” (The Question of Education), for example, he argues that
a German national conscience (milli vicdan) came into being via the rejection of
French cultural dominance. Just as Germans could not discover their essential
character while striving to imitate the French, he argues, neither can a Turkish
national literature come into being without excising a lingering Persian and more
recent French influence. While Gökalp strictly warns against all forms of cultural
imitation, he suggests that Turkish literature should follow the German example, in
establishing its own national preferences and tastes (milli zevk) (Gökalp 1972: 111-
12). A story such as Verlobung thoroughly complicates the idea of what it means to
follow a ‘German’ model. While it can be read in response to the French occupa-
tion of Germany, it in no way posits an authentic German national character. On Kristin Dickinson 52 the contrary, its tragic interracial love story—populated by characters who cannot
be clearly defined along racial or national lines—engages in processes of cultural
translation that undermine its potential to represent a unified understanding of
Germanness. A brief plot summary makes this abundantly clear: Following the defeat of
French forces in Saint Dominique by General Dessalines in 1803, a Swiss family
attempts to make its way across the island to escape via the city’s port. Identities in Crisis Hilmi
Ziya Ülken proposed in turn the concept of tam tercüme (complete translation),
which perpetuated an image of the West as a fixed, monolithic entity that could be
‘translated’ at face value. Even while attempting to establish an original Turkish
identity, such discursive terminology inevitably led to an understanding of the Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 53 ‘West’ as originary, in relation to which Turkish translations could only be under-
stood as both derivative and belated. ‘West’ as originary, in relation to which Turkish translations could only be under-
stood as both derivative and belated. In this respect, Verlobung offers a fascinating case study, as a text that 1) posits
a certain hybrid or non-essential aspect of culture and 2) consciously reflects on
the potential inadequacy of its Western sources. While there is no evidence that
Kleist explicitly condemned the practice of slavery in the French colonies, Ver-
lobung does offer a critical portrayal of the slave revolt through tensions between
the mode and content of its narration. Ali hints at this aspect of the text in his
introduction to the translation. Here he argues that Verlobung seems to conform to
the popular European portrayals of the Haitian Revolution Kleist consulted while
writing the novella; these sources all viewed the slave revolt as an act of brutality
against the ‘white’ population. At the same time, he argues, Kleist does not refrain
from exposing the tyranny endemic to white colonials’ brutal treatment of the
African slave population through the action of his text (Ali 1943: 5). Within this
context, Ali’s translation of Verlobung also suggests the need to consciously reflect
on the category of the “West” within the large-scale translation movement of the
1940s. 14 Translations mine unless otherwise noted; KD.
Sie folgte ihm mit einer plötzlichen Bewegung, fiel ihm um den Hals, und mischte ihre Thränen mit
den seinigen.
Was weiter erfolgte, brauchen wir nicht zu melden, weil es jeder, der an diese Stelle kommt, von
selbst lies’t. (Kleist 1988: 43) 14 Translations mine unless otherwise noted; KD. ‘Engaging’ in World Literature It is within the complex matrix of colonial power relations and racial tensions
Kleist lays out in Verlobung that the fatal attraction between Gustav and Toni de-
velops. Ordered by her mother to lull this fugitive into a false sense of security,
Toni nevertheless sympathizes with and is drawn to Gustav; in an initial attempt to
test her loyalty, Gustav also finds himself enamored by Toni’s youthful counte-
nance and likens her to his deceased beloved. What follows is a crucial unnarrated
moment in which an implied sexual encounter marks a turning point in the story. Toni and Gustav’s physical union is marked by a textual absence that simultane-
ously points to the unspoken speech act of engagement from which the story takes
its title: She went over to him with a sudden movement, threw her arms around
his neck, and let her tears merge with his. There is no need to describe what happened next, as everyone who has
come to this point can read it for himself.14 She went over to him with a sudden movement, threw her arms around
his neck, and let her tears merge with his. There is no need to describe what happened next, as everyone who has
come to this point can read it for himself.14 Kristin Dickinson 54 This moment of non-narration leads to a series of crucial misreadings that ulti-
mately incite Gustav to accuse Toni of prostitution and betrayal; in a moment of
confusion he shoots her in the chest just below the cross pendant he had offered
her as a sign of their engagement. Upon realizing his mistake, Gustav shortly
thereafter commits suicide by shooting himself in the head. In asking what it
means to translate a moment of non-narration, I turn to Ali’s rendition of this
passage into Turkish: “There is no need to say what happened next, because
everyone who has come this far can read the subtext for themselves.” (“Bundan
sonra ne olduğunu söylemeye lüzum yok çünkü buraya kadar gelen herkes alt
tarafını kendiliğinden okur.”) (Ali 1943: 31) Whereas the idiom alt tarafı means
‘after all,’ Ali’s literal use of this phrase carries critical spatial implications. By call-
ing attention to both a literal and a literary ‘underside,’ it points to the existence of
a subtext, or textual layerings that work both with and against one another. ‘Engaging’ in World Literature As
such, Ali’s translation also offers a commentary on what it means to read the am-
biguous and contradictory nature of Kleist’s text as a whole. Verlobung is marked by
myriad textual inconsistencies; these range from semantic issues (such as missing,
misplaced, or open-ended quotation marks), to orthographic differences in charac-
ter names (Gustav is alternately referred to as August), and contradictory narrative
information. Much more than potential mistakes or flaws, such inconsistencies
point to a heightened level of semantic ambiguity; they set up situations in which
the narrative drives characters’ actions on the surface, even while such actions
undermine the very meaning of the language in which they are expressed. The non-narrated sexual union between Gustav and Toni is one critical
instance in which the language of Verlobung contradicts itself, in that it asks us to
read a moment of non-narration. Andreas Gailus analyses this aspect of the text as
an “unmooring” of language, or a paradoxical situation in which we are actually
asked “to stop reading the text...[and; KD] the semantic and typographical signs of
absence and discontinuity the text puts before us” (Gailus 2010: 34). In support of
this argument, Gailus argues that we cannot overlook Kleist’s use of outmoded
typography: the verb “lies’t” (in place of lieset, to read) contains a contractual eli-
sion that marks the very gap the passage asks us to ignore, and thus runs counter
to the sentence in which it is embedded. Wolfgang Iser more explicitly addresses the role played by the reader in such
moments through his concept of the Leerstelle, or narrative gap. Within his theory
of reception aesthetics, Iser examines the changing relationship between reader
and text as ever-increasing levels of indeterminacy have become a central feature
of literature from the 18th century to the present. The more texts foreground their
own incompleteness—by pointing to events outside the frame of narration, or by
highlighting moments of contradiction and ambiguity—the more readers are called
upon to actively engage in an interpretive process of reading as meaning making Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 55 (Iser 1975: 29-31).15 While Iser’s concept of the Leerstelle can apply to all levels of a
text, Madonna could be said to explicitly call attention to the central Leerstelle of
Verlobung through its own use of narrative elision. 15 Narrative ambiguity and epistemological uncertainty is a central aspect of Kleist’s poetics. The
concept of the Leerstelle in particular has been utilized to describe the famous dash in Kleist’s Die
Marquise von O (1808, The Marquise of O), which stands in place of the marquise’s rape by Graf F (Reif
2005: 87). David Roberts has also used this concept in his discussion of Das Erdbeben in Chili (1807,
The Earthquake in Chile) to describe the earthquake as an event that shakes the very basis of society in
St. Jago; Roberts builds here on Werner Hamacher’s discussion of the novella as “eine
Erschütterung... die auch die Logik der Repräsentation ergreift und daher den Begriff der Darstellung
als solchen in Frage stellt” (a shock that seizes the very logic of representation, thereby calling the
concept itself into question) (Roberts 2000: 45).
16 “Vücudunun titrediğini hissettim. Kesik kesik nefes alarak: ‘Tabii … Tabii!’ dedi. ‘Tabii sizi se-
viyorum […] Fakat neden şaşırıyorsunuz? Başka türlü olacağını mı zannediyorsunuz? Beni ne kadar
çok sevdiğini anlıyorum ... Ben de sizi şüphesiz o kadar çok seviyorum.’ Başımı kendisine doğru çekti
ve bütün yüzümü ateş gibi buselere boğdu. Sabahleyin uyandığım zaman onun derin ve muntazam
nefeslerini duydum ...” (Ali 2008b: 116) 15 Narrative ambiguity and epistemological uncertainty is a central aspect of Kleist’s poetics. The
concept of the Leerstelle in particular has been utilized to describe the famous dash in Kleist’s Die
Marquise von O (1808, The Marquise of O), which stands in place of the marquise’s rape by Graf F (Reif
2005: 87). David Roberts has also used this concept in his discussion of Das Erdbeben in Chili (1807,
The Earthquake in Chile) to describe the earthquake as an event that shakes the very basis of society in
St. Jago; Roberts builds here on Werner Hamacher’s discussion of the novella as “eine
Erschütterung... die auch die Logik der Repräsentation ergreift und daher den Begriff der Darstellung
als solchen in Frage stellt” (a shock that seizes the very logic of representation, thereby calling the
concept itself into question) (Roberts 2000: 45). p
q
16 “Vücudunun titrediğini hissettim. Kesik kesik nefes alarak: ‘Tabii … Tabii!’ dedi. ‘Tabii sizi se-
viyorum […] Fakat neden şaşırıyorsunuz? Başka türlü olacağını mı zannediyorsunuz? Beni ne kadar
çok sevdiğini anlıyorum ... Ben de sizi şüphesiz o kadar çok seviyorum.’ Başımı kendisine doğru çekti
ve bütün yüzümü ateş gibi buselere boğdu. Sabahleyin uyandığım zaman onun derin ve muntazam
nefeslerini duydum ...” (Ali 2008b: 116) ‘Engaging’ in World Literature A line break in the text marks an
implied sexual encounter between the main character Raif and his beloved Maria: I felt her body shivering. She breathed in gasps, saying: “Of course!... Of
course! Of course I love you. […] But why are you surprised? Did you
think there was any other way? I understand how much you love me… I,
without any doubt, love you just as much…” She pulled my head toward
her and buried my face in ardent kisses. In the morning, when I woke up, I heard her deep and regular breathing. (Ali 2014: 139)16 y
In the morning, when I woke up, I heard her deep and regular breathing. (Ali 2014: 139)16 Following a frenzied New Year’s Eve celebration, and Maria’s sudden declaration
of her love for Raif, a moment of textual silence marks both the apex and the tem-
porary breaking point of their relationship. It leads to a second “sessizlik” (Ali
2008b: 118) or silence between the lovers, in which they find themselves at a loss
for words in one another’s presence. These double silences in the text address a
problem of referentiality. Feeling suddenly estranged from Raif, Maria repeatedly
attempts, but finds herself unable, to explicitly refer to their moment of sexual
union: “I had hoped to awaken this morning to a completely different world” (“Bu
sabah uykudan, başka bir dünyaya doğar gibi uyanacağımı sanmıştım”) (Ali 2008b:
119); “We can’t talk openly as we used to... For what did we sacrifice all this?” (Ali
2014: 144) (“Artık eskisi gibi apaçık konuşamayız ... Bunları ne diye, neyin uğrunda
feda ettik?”) (Ali 2008b: 120). ) (
)
In considering the relationship of these sexual encounters to one another, it is
notable that Verlobung and Madonna employ markedly different methods of narra-
tion. In his introduction to the translation, Ali accurately describes Verlobung as a
text that hinges on the power of the event; it is in the carrying out of key events
that the complex and contradictory nature of Kleist’s characters comes to the Kristin Dickinson 56 fore.17 Madonna is, on the contrary, a text driven by descriptive detail, long conver-
sational passages, and the psychological development of its main characters. Con-
sidering the otherwise divergent nature of these texts, what does it mean for Ver-
lobung and Madonna to converge precisely on a moment of non-narration? 17 This does not imply that Kleist’s characters lack complexity or serve merely symbolic functions.
According to Ray Fleming, “Kleist allows action rather than psychological analysis to present us with
the key to understanding the complexity of his black characters and their world. One might object
that this is but another example in Western canonical literature of the marginalization of the Other as
represented by Congo Hoango, Babekan and Toni, but if this were so the black characters would,
typically, only have a symbolic role in the literary work… rather than a structurally and thematically
essential role” (Fleming 1992: 309). ‘Engaging’ in World Literature In answering this question, I argue that Madonna also contains an inverted
counterpart to the textual elision of Raif and Maria’s sexual encounter: the text of
Raif’s notebook—in which is recorded the story of his youth in Weimar, Berlin,
and which we receive word for word as the internal narrative of Madonna. At the
close of the external narrative that opens the novel—in which the narrator and
Raif slowly become friends through their positions at a local bank—Raif becomes
deathly ill. After asking the narrator to retrieve this small black notebook from his
work desk, Raif then pleads with him to throw it into the fire of his stove. Raif’s
desire to have this notebook destroyed—an act that would silence his own trans-
cultural life story—points us back to the moment of textual silence that marks his
relationship with Maria; and yet Raif’s fervent desire to have his notebook burned
ironically leads the narrator to both keep, and read Raif’s life story for himself. This
act both opens Raif’s text to all external readers of the novel, and raises the ques-
tion of how to read Raif’s notebook through the silences that mark his character. Following Iser, I argue that Madonna presents this act of reading as an expres-
sion of the text’s very openness to interpretation (Iser 1975: 249). If the Leerstelle
constitutes a fundamental Ansatzpunkt (point of departure) from which the reader
can explore a text’s potential meanings (Iser 1975: 235), then the text of Raif’s
notebook—as a counterpoint to the Leerstelle of Raif and Maria’s sexual encoun-
ter—furthermore figures the act of reading as a cross-cultural encounter: the ques-
tion of what it means to read Raif’s notebook is complicated by his own offhand
depiction of it as a German novel (Almanca bir roman). While this lie is meant to
detract a colleague’s attention as Raif hides the notebook deep within a drawer, I
argue that Madonna asks us to take this claim seriously. To read Raif’s life story as a
German novel is to fundamentally question what it means to be German. It requires
an investigation of the intersubjective and intercultural framework of the novel via
a mode of reading that engages both its surfaces and subtextual layers. g
g g
y
Ali’s translation of another key scene in Verlobung is central to my argument
here. ‘Engaging’ in World Literature As mentioned earlier, following their implied sexual encounter, Gustav gives
Toni a necklace with a cross pendant as a “Brautgeschenk” (engagement gift); Ali’s
translation of this term—which attests to an otherwise unspoken engagement—as
a “nişan hediyesi” (engagement gift), conveniently plays on the double meanings of Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 57 nişan as both ‘engagement’ and ‘target’ (Ali 1943: 31). Indeed, the place where the
cross lies on Toni’s chest marks the spot where Gustav shoots her later in the sto-
ry, after misreading her actions as a sign of betrayal. In my own play on words, I argue that for Ali, translation constitutes one
method of engaging with, rather than simply transferring the classics of world liter-
ature to Turkey. I develop this argument through recourse to Ali’s own reflections
in an article written for the journal Tercüme (Translation) in March 1941: The language of a work of art, as opposed to that of other written works,
is a living being. In the course of translation and the transfer of its words’
meanings, this life perishes, leaving only a chain of dead words behind. The real translator can give this lifeless matter a new life in the language it
has been transferred to... (Ali 2012: 104) The translator must not forget that he has undertaken a debt of con-
sciousness and a serious responsibility toward the author of the work in
question, and the public mass he wants to present the work to.18 (Ali 2012:
111) Here, Ali figures the initial act of linguistic transfer (nakil) as one that literally takes
the life of a living work of art. Real translation, he argues, cannot remain at the
level of transfer, but must instead enliven the dead words of the literary text; this is
the ethical responsibility of the translator toward both the author and the public. Here, Ali figures the initial act of linguistic transfer (nakil) as one that literally takes
the life of a living work of art. Real translation, he argues, cannot remain at the
level of transfer, but must instead enliven the dead words of the literary text; this is
the ethical responsibility of the translator toward both the author and the public. 18 “Sanat eserinin dili, diğer yazılı eserlerden farklı olarak, canlı bir mevcudiyettir... Tercüme esnasında,
mütercim kelimelerin manalarını nakil ile kanaat ettiği müddetçe, bu hayat yok olur, ortada sadece
birtakım ölü kelimeler silsilesi kalır. Asıl mütercim, bu cansız malzemeye, naklettiği dilde yeni bir hayat
vermesini bilen kimsedir...” (Ali 2012: 104) 19 I borrow the term “textual substructure” from Roswitha Burwick, who argues that the substruc-
tures of Verlobung “mirror the unsolved social, political, and racial problems” (Burwick 1992: 321) it
addresses. “Mütercimin hem eserini tercüme ettiği muharrire, hem de bu eseri arz ettiği insan kütlesine karşı
büyük bir vicdan borcu olduğunu ve ağır bir mesuliyet altına girdiğini asla unutmaması lazımdır.” (Ali
2012: 111) 18 “Sanat eserinin dili, diğer yazılı eserlerden farklı olarak, canlı bir mevcudiyettir... Tercüme esnasında,
mütercim kelimelerin manalarını nakil ile kanaat ettiği müddetçe, bu hayat yok olur, ortada sadece
birtakım ölü kelimeler silsilesi kalır. Asıl mütercim, bu cansız malzemeye, naklettiği dilde yeni bir hayat
vermesini bilen kimsedir...” (Ali 2012: 104)
“Mütercimin hem eserini tercüme ettiği muharrire, hem de bu eseri arz ettiği insan kütlesine karşı
büyük bir vicdan borcu olduğunu ve ağır bir mesuliyet altına girdiğini asla unutmaması lazımdır.” (Ali
2012: 111)
19 I borrow the term “textual substructure” from Roswitha Burwick, who argues that the substruc-
tures of Verlobung “mirror the unsolved social, political, and racial problems” (Burwick 1992: 321) it
addresses. 20 Maria expresses this idea in the following quote: “Demek ki insanlar birbirine ancak muayyen bir
hadde kadar yaklaşabiliyorlar ve ondan sonra daha fazla sokulmak için atılan her adım daha çok uzak-
laştırıyor. Seninle aramızdaki yakınlaşmanın bir hududu, bir sonu olmamasını ne kadar isterdim. Beni
asıl, bu ümidin boşa çıkması üzüyor...” (So, people can get close to a certain extent, and then, every
step taken to become closer makes them more distant. I wish very much that our convergence had
not had such a boundary, an end. What really disappoints me is this unfulfilled hope.) (Ali 2008b:
120) ‘Engaging’ in World Literature p
y
p
In Ali’s own terms, then, to engage with Verlobung in translation is to bring this
text to life in Turkish; Ali arguably does this by exploring new semantic ambigui-
ties—like those of alt tarafı and nişan—that highlight the textual surfaces and sub-
structures of Kleist’s novella.19 Just as Verlobung asks us to read a moment of non-
narration, I argue that the kind of translation Ali endorses is closely tied to the
ability to read through the double meanings and potentially contradictory layers
within a given text. As such, Ali’s translation practice enacts the kind of double
dislocation Theo Hermans describes in his concept of “thick translation,” whereby
concepts in both the source and target languages are unhinged from their apparent
meanings. Hermans builds here on Clifford Geertz’ notion of “thick description,”
which resists universalizing tendencies by actively reflecting on the interpretive and Kristin Dickinson 58 constructivist nature of the ethnographer’s observations. In recognizing that acts
of translation, interpretation, and description play out in the same discursive space,
Hermans’ concept of “thick translation” resists the imposition of categorical defi-
nitions. In working from the bottom up—rather than from the top down—“thick
translation contains within it both the acknowledgement of the impossibility of
total translation and an unwillingness to appropriate the other through translation
even as translation is taking place” (Hermans 2003: 386-387). Civilized Encounters, Impure Mixtures Yet, in
one of their first excursions together, Maria brings Raif to the Botanical Gardens
in Berlin, where she compares the strange (garip) and uprooted (sökülerek) plants it
houses to her Jewish ancestors (ecdat) (Ali 2008b: 91-92). Through this conversa-
tion, we learn that Maria’s father was a Jew born in Prague who converted to
Christianity before she was born. It seems no coincidence that Maria shortly there-
after describes her mother as “a Protestant of pure German blood” (Ali 2014: 131)
(“halis Alman kanında bir Protestan”) (Ali 2008b: 110). This reference to “pure”
German blood highlights National Socialist discourses on race, according to which
Maria would have been categorized as an interracial child. g
Maria’s racial background is arguably tied to the mixing of blood in the crucial
scene in which Raif envisions Kleist’s suicide. Here, Raif’s fantasy of his own un-
ion with Maria—in which she becomes bound to him in memory by blood—is
also the projection of an impure mixture and serves as commentary on the kinds
of hybridities and interracial couplings Kleist employs in Verlobung. Ali’s recourse to the rhetoric of purity offers a clear reference to the racial poli-
tics of National Socialism at the time of Madonna’s publication in the early 1940s. Raif’s notebook is furthermore composed in the summer of 1933, a year that
marked both the official end of the Weimar Republic and Hitler’s systematic con-
solidation of power. With regard to these historical implications, I return again to
the question of what it means for Ali to translate the key moment of non-narration
in Verlobung, which marks the implied sexual union between Toni and Gustav. In
addition to the text of Raif’s black notebook, I suggest that this moment of non-
narration finds a second inverted counterpart at the close of Madonna’s embedded
narrative through the event that finally leads Raif to document his life in Berlin: A
chance encounter with Frau von Tiedemann, the owner of the boarding house
where Raif lived in Berlin, leads him to discover the existence of his ten-year-old
daughter. Described as thin (zayıf), of pale complexion (soluk benizli), and well be-
haved and silent (huylu ve sessizdir), this girl serves as a ghostly physical testament to
Raif’s relationship with Maria, who he now learns passed away shortly after giving
birth (Ali 2008b: 152-156). Civilized Encounters, Impure Mixtures Following the implied sexual encounter in Madonna, and Maria’s expression of her
inability to love another human being, Raif wanders blindly through the streets of
Berlin until he reaches the southernmost limits of the city. In a key turning point
for the novel, he finally takes notice of his surroundings: on the shore of Berlin’s
Wannsee, he recognizes the spot where Kleist and his lover, Henriette Vogel, had
committed suicide in 1811 (Ali 2008b: 123), a date that incidentally also marks the
publication of Verlobung. This famous murder-suicide pact—in which Kleist first shot his lover and then
himself—does not serve as a clear point of reference for Raif and Maria’s relation-
ship, but rather as a bitter point of inversion. In contrast to Maria’s assertion that
two people can never really become one,20 Raif imagines the lovers Heinrich and
Henriette with a bullet through the temples and chest, respectively, their blood
streaming into a single pool at his feet: “Like their destinies, their blood had also
mixed with one another.” (“Mukadderatları gibi kanları da birbirine karışmıştı”)
(Ali 2008b: 123). In a self-deprecating fantasy, he imagines first calling Maria, then
shooting himself in the head, so that he might listen to her saying his name as he
lies, dying, in a pool of his own blood. Only in this way, “she would understand
that she would never forget me until the end of her life, and that I had bound
myself to her memory with my blood.” (Ali 2014: 151) (“ömrünün sonuna kadar
beni unutamacağını, kendimi kanla hatırasına bağladığımı anlayacaktı.”) (Ali 2008b:
125) The manner in which Kleist committed suicide is also eerily reminiscent of the
suicide in Verlobung, in which Gustav first murders Toni by shooting her in the
chest, and then kills himself out of remorse with a bullet through the head. Indeed,
Raif’s bitter fantasy also recalls the image of Toni writhing in her own pool of
blood at the close of Kleist’s novella. This intertextual reference, together with
Ali’s multiple visual references to blood, lead me to question the stakes of Raif and Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 59 Maria’s intercultural relationship in Madonna. While this novel is consistently read
as a tragic love story, scholarship has not sufficiently addressed the significance of
Raif and Maria’s specific backgrounds for the development of the narrative. Civilized Encounters, Impure Mixtures The Leerstelle that marks both Gustav and Toni’s, as well as Raif and Maria’s,
sexual encounters is translated into the silence of this small child. Whereas the love
story in Verlobung reveals a crisis of German identity prior to the establishment of
the German nation-state in the face of French imperialism, Ali gestures through
this child to the colonial power structures within which Raif’s relationship to Maria
is embedded in the 20th century. Frau von Tiedemann—who is, incidentally, Ma-
ria’s cousin—reveals that she is traveling through Ankara en route to Berlin along
the Bagdad Railway. Her Prussian husband, whom she describes as a colonial mer-
chant (müstemleke tüccarı) (Ali 2008b: 152), is now involved in the date trade in Iraq. Recalling her husband, Herr Döppke’s, previous experience with the date trade in Kristin Dickinson 60 the German colony of Cameroon, Raif notes perplexedly that Bagdad is not a
German colony. To this Frau von Tiedemann replies: “my husband specializes in
the produce of warm countries.” (Ali 2014: 187) (“kocamın sıcak memleket
mahsulleri üzerinde ihtisası var”) (Ali 2008b: 152). In conclusion, I argue that it is precisely this kind of categorical cultural essen-
tialization against which Kleist works through the hybrid identities and semantic
ambiguities in Verlobung. Ali’s translation of Verlobung, then, works together with
the intertextual references in Madonna to underscore the deep irony of a project
meant to transfer European humanist values to Turkey at a time when these very
values were being destroyed by fascist governments in Europe or put into question
via the European colonial imposition of slavery following the French Revolution,
respectively. While Ali did enthusiastically support both the translation project and
other humanist reforms instated by Hasan Ali Yücel throughout the 1940s, I argue
that both his translation practice and his fiction suggest that translating the ‘West’
involves a careful consideration of the contradictions and potentially negative as-
pects of Western civilization. As such, these texts also challenge depictions of civilization within translation
rhetoric of the early Republican period: Hilmi Ziya Ülken proposes the concept of
a universal civilization that is nevertheless situated in the West; Turkey’s entrance
into it is thus aligned with what he views as the Ottoman goal of Westernization
(garplılaşma), which he states can only be fully realized with the systematic program
of translation in the Republican era. Civilized Encounters, Impure Mixtures İsmail Habib Sevük argues along similar lines
that textual participation in European civilization is premised on the ‘entire’ trans-
lation of Western antiquity, suggesting a positive ideal of European civilization that
is complete in and of itself. Both of these authors strongly believe in the possibility
of transferring otherwise vaguely defined values of Western civilization to Turkey. By pointing to the contradictory nature of its literary underside, I argue that
Ali’s translation of Verlobung works together with his final novel, Madonna, to com-
plicate the Republican Turkish premise of smooth translatability—and thus also
the stable category of the ‘West’ it presumes—upheld by scholars such as Ülken
and Habib. Whereas translation activity was one crucial arena through which Tur-
key sought to emerge as an independent political entity that identified itself as Eu-
ropean, Ali poses the more difficult question of what ‘Western’ values themselves
might be. Intervening in the Humanist Legacy: Sabahattin Ali’s Kleist Translations 61 Works Cited Ali, Sabahattin (2014): The Madonna in the Fur Coat, translated by David Gramling
and İlker Hepkaner, unpublished. Ali, Sabahattin (2012): “İkinci Dilden Tercüme Meselesi ve Bir Misal,” in: Hikmet
Altınkaynak / Sevengül Sönmez (eds.): Markopaşa Yzıları ve Ötekiler, İstanbul,
104-111. Ali, Sabahattin (2008a): Hep Genç Kalacağım: Bütün Yapıtları Mektup, edited by
Sevengül Sönmez, İstanbul. Ali, Sabahattin (2008b [1943]): Kürk Mantolu Madonna, İstanbul. Ali, Sabahattin (1943): Üç Romantik Hikaye: Kleist, Chamisso, Hoffmann, Ankara. Bachmann-Medick, Doris (2006): “Meanings of Translation in Cultural
Anthropology,” in: Hermans, Theo (ed.), Translating Others, Manchester, 33-42. Burwick, Roswitha (1992): “Issues of Language and Communication: Kleist’s ‘Die
Verlobung in St. Domingo,’” in: The German Quarterly (65/3,4), 318-327. Çıkar, Mustafa (1997): Hasan Ali Yücel ve Türk Kültür Reformu, Ankara. Dickinson, Kristin (2013): “A Dual Critique of Modernity in Sabahattin Ali’s ‘Kürk
Mantolu Madonna’,” in: Transit: A Journal of Travel, Migration and Multiculturalism
in the German Speaking World (9/1), 1-8. Dünya Edebiyatından Tercümeler Listesi (1959), Ankara. Dünya Edebiyatından Tercümeler Listesi (1959), Ankara. Even-Zohar, Itamar (2008): “Culture Planning, Cohesion, and the Making and
Maintenance of Entities,” in: Pym, Anthony / Schlesinger, Miriam / Simeoni,
Daniel (eds): Beyond Descriptive Translation Studies: Investigations in Homage to Gideon
Toury, Amsterdam, 277-292. Fischer, Bernd (2003): “Introduction: Heinrich von Kleist’s Life and Work,” in:
Fischer, Bernd (ed.): A Companian to the Works of Heinrich von Kleist, Rochester. Fleming, Ray (1992): “Race and the Difference it Makes in Kleist’s ‘Die Verlobung
in St. Domingo,’” in: The German Quarterly (65/3, 4), 306-317. Gaillus, Andreas (2010): “Language Unmoored: On Kleist’s ‘The Betrothal in St. Domingue,’” in: The Germanic Review (85), 20-43. Gökalp, Ziya (1972): “Maarif Meselesi,” in: Milli Terbiye ve Maarif Meselesi, Ankar Gürses, Sabri (2013): “Sabahattin Ali’nin Hayatında ve Yapıtında Çeviri,” in:
Sönmez, Sevengül (ed.): Sabahattin Ali, İstanbul, 395-418. Kristin Dickinson 62 Hamacher, Werner. (1984): “Das Beben der Darstellung,” in: Wellbery, David E. (ed.): Positionen der Literaturwissenschaft. Acht Modellanalysen am Beispiel von Kleists
‘Das Erdbeben in Chili’, Munich, 149-173. Hermans, Theo (2003): “Cross-Cultural Translation Studies as Thick Translation,”
in: Bulletin of the School of Oriental and African Studies (66/3), 380-389. Iser, Wolfgang (1975): “Die Appellstruktur der Texte: Unbestimmtheit als
Wirkungsbedingung literarischer Prosa,” in: Warning, Rainer (ed.):
Rezeptionsästhetik: Theorie und Praxis, Stuttgart, 228-252. Kleist, Heinrich von (1988): “Die Verlobung in St. Domingo,” in: Reuß, Roland /
Staengle, Peter (eds.): Sämtliche Werke: Berliner Ausgabe, Basel. Kleist, Heinrich von (1943): “San Domingo’de Bir Nişanlama,” in: Üç Romantik
Hikaye, translated by Sabahattin Ali, Ankara. Kontje, Todd (1999): “Passing for German: Politics and Patriarchy in Kleist,
Körner, and Fischer,” in: German Studies Review (22/1), 67-84. Konuk, Kader (2010): East West Mimesis: Auerbach in Istanbul, Stanford. Reif, Daniella (2005): Die Ästehtik der Leerstelle: Raymond Federmans Roman ‘La
Fourrure de ma tante Rachel‘ vor dem Hintergrund des Gesamtwerks, Würzburg. Roberts, David (2000): “Kleists Kritik der Urteilskraft: Zum Erhabenen in ‘Das
Erdbeben in Chili,’ ” in: Mehigan, Timothy J. (ed.): Heinrich von Kleist und die
Aufklärung, Rochester, 46-57. Şengör, A. M Celal (2001): Hasan-Ali Yücel ve Türk Aydınlanması, Ankara. Schneider, Helmut J (2003): “The Facts of Life: Kleist’s Challenge to
Enlightenment Humanism,” in: Fischer, Bernd (ed.): A Companian to the Works
of Heinrich von Kleist, Rochester, 141-163. Tahir Gürçağlar, Şehnaz (2008): The Poetics and Politics of Translation in Turkey 1923-
1960, Amsterdam. Dünya Edebiyatından Tercümeler Listesi (1959), Ankara. Tahir Gürçağlar, Şehnaz (2003): “The Translation Bureau Revisited: Translation as
Symbol,” in: Maria Calzada Perez (ed.): Apropos of Ideology: Translation Studies on
Ideology - Ideologies in Translation Studies, 113-129. T.C. Kültür Bakanlığı (1997): Birinci Türk Neşriyat Kongresi, Ankara. Togar, Melahat (2011): “Arkadaşım Sabahattin Ali,” in: Sabahattin Ali: Anılar,
İncelemeler, Eleştiriler, İstanbul, 67-73. Ülken, Hilmi Ziya (1997): Uyanış Devirlerinde Tercümenin Rolü, İstanbul. Venuti, Lawrence (1995): The Translator’s Invisibility : A History of Translation,
London. The Political behind the Fur Coat: Sabahattin Ali’s
The Madonna in the Fur Coat and Leopold Sacher-
Masoch’s Venus in Furs in an Intertextual Context İlker Hepkaner Abstract We called the novel The Madonna in the Fur Coat, because
we believe Sabahattin Ali used Madonna in a definitive manner and this version of the title satisfies
the urgency to reflect such anchorage in the novel. The relationship between the novel’s main charac-
ters Raif and Maria develops in a specific manner. Raif first sees Maria’s self portrait, makes meaning
of it through his and an art critic’s cultural repertoire, and struggles with Maria’s elusive image for a
while before they start their friendship. Maria’s image and persona concretizes throughout the novel
for Raif, and achieves a definitive and fundamental meaning for him. We therefore think the title
“The Madonna in the Fur Coat” reflects this exponential meaning making inside the novel better
than the title “Madonna in a Fur Coat.” 3 As a follow up, another user referred to a 2012 entry, which had identified some elements of
Gogol’s short stories in Madonna and had claimed that Sabahattin Ali “summarized” Gogol’s stories
in Madonna, accessible: https://eksisozluk.com/entry/27525680 [Last accessed: 01.06.2016]. In this
article, I focus on Ali’s intertextuality with Sacher-Masoch’s Venus because I look at the intertextuality
of the novel by considering its author’s time in Germany, expertise in the German language, and
active role in the state-run modernization projects as a German teacher and translator. My focus is
not his association with Russian literature or the reasons behind the novel’s intertextuality with
Gogol’s stories. However, his fascination with Russian literature was no secret, and the impact of
Russian literature on Ali’s authorship was noticed by İbrahim Tatarlı. For a detailed survey of Ali’s
works and thorough analysis of his authorship, see Tatarlı (1979). 4 Although there is no concrete evidence in Ali’s personal documents on his engagement with Sacher-
Masoch’s novel, the parallels that have been explored by other scholars and that will be discussed in
this article point to striking similarities with Sacher-Masoch’s work. The fascination over the parallel
between Ali’s Madonna and Sacher-Masoch’s Venus is nothing new, and indeed predates discussions in
the online forum Ekşi Sözlük. In our personal conversations, comparative literature scholar Kristin
Dickinson and German literature scholar Barbara Kosta suggested that there might be an important
intertextuality between Ali and Sacher-Masoch. I followed their suggestions and explored the inter-
textuality between the two novels in my master’s thesis defended in 2013. For this please see
(Hepkaner 2013). 1 For a succinct description of Ekşi Sözlük, see Gürel / Yakın (2007). In the last nine years, the
discussion over the website has intensified but its main tenets, as explained in this article, stayed more
or less the same. The original title reads “Kürk Mantolu Madonna’nın Çalıntı Olması”, accessible at:
https://eksisozluk.com/kurk-mantolu-madonnanin-calinti-olmasi--4610794 [Last accessed: 01. 06.
2016]. All translations in this article are mine unless noted otherwise. Abstract In his final novel Kürk Mantolu Madonna (1943, The Madonna in the Fur Coat), social-
realist author Sabahattin Ali deployed multiple intertextualities with Leopold Sacher-Masoch’s
novel Venus im Pelz (1870, Venus in Furs). At a time period when Turkey’s ruling govern-
ment did not shy away from occasionally cooperating with the Nazi regime and allowing Nazi
infiltration, Ali’s references to a German-language novel carry political repercussions. In this
article, I argue that Ali intentionally deployed intertextuality in order to take a political stand
and critique his contemporary context. In particular, I show how the layered intertextualities
between Madonna and Venus relate to Ali’s political and judicial struggles at the time of Ma-
donna’s publication. I conclude the article by demonstrating the urgent importance of recognizing
the multifarious political aspects of Madonna in order to reevaluate the novel’s current bestseller
status in Turkey, and the specific marketing strategies adopted for its English publication with
Penguin Classics in 2016. “The Fact that The Madonna in the Fur Coat is Plagiarized” reads a title on the popu-
lar e-dictionary Ekşi Sözlük, which has functioned less as a dictionary than as a
cyber public space where young people share information and discuss a myriad of İlker Hepkaner 64 issues since the early 2000s.1 In 2014, one user wrote a long entry describing strik-
ing plot parallels between Sabahattin Ali’s novel Kürk Mantolu Madonna (1943) (The
Madonna in the Fur Coat, hereafter referred to as Madonna)2 and Leopold Sacher-
Masoch’s Venus im Pelz (1870) (Venus in Furs, hereafter referred to as Venus) and
concluded that Ali had plagiarized the core of his novel.3 Against such accusations of plagiarism, I argue that Ali intentionally deployed
intertextuality in order to take a political stand and critique his contemporary con-
text. Madonna’s intertexualities have been discussed by a number of scholars includ-
ing myself since 2011.4 In particular, Kristin Dickinson has shown how the novel
questions the Turkish modernization project through its engagement with both 2 An English translation of the novel by Maureen Freely and Alexander Dawe was published in May
2016 under the title of Madonna in a Fur Coat. However, here I use the unpublished translation that
David Gramling and I completed in 2011. 2 An English translation of the novel by Maureen Freely and Alexander Dawe was published in May
2016 under the title of Madonna in a Fur Coat. However, here I use the unpublished translation that
David Gramling and I completed in 2011. We called the novel The Madonna in the Fur Coat, because
we believe Sabahattin Ali used Madonna in a definitive manner and this version of the title satisfies
the urgency to reflect such anchorage in the novel. The relationship between the novel’s main charac-
ters Raif and Maria develops in a specific manner. Raif first sees Maria’s self portrait, makes meaning
of it through his and an art critic’s cultural repertoire, and struggles with Maria’s elusive image for a
while before they start their friendship. Maria’s image and persona concretizes throughout the novel
for Raif, and achieves a definitive and fundamental meaning for him. We therefore think the title
“The Madonna in the Fur Coat” reflects this exponential meaning making inside the novel better
than the title “Madonna in a Fur Coat.” Abstract Kristin Dickinson wrote on the intertextuality between two novels in her disserta-
tion (Dickinson 2015). Separate from these discussions, Onur Kemal Bazarkaya published an article
on the hypertextuality between the two novels in 2015 and their interpretation according to Derrida
and Foucault’s theories on representation and sexuality. Unlike Bazarkaya, I don’t delve into different
intertextualities of Gerard Genette’s typology in this article, rather I explore possible political under-
tones in the connections Ali establishes with other texts. For such discussion, please see Bazarkaya
(2015). The Political behind the Fur Coat 65 Weimar surface culture and revolutionary Turkey’s cultural practices.5 In this arti-
cle, I build on Dickinson’s argument by highlighting the key role intertextuality
plays within Madonna as a mode of political critique.6 Weimar surface culture and revolutionary Turkey’s cultural practices.5 In this arti-
cle, I build on Dickinson’s argument by highlighting the key role intertextuality
plays within Madonna as a mode of political critique.6 On the concept of intertextuality, I draw on literary theorist Julia Kristeva’s
definition of the term. Kristeva brought Saussure’s linguistic theory together with
Mikhail Bakhtin’s literary theories in her works in the 1960s. In her article “The
Bounded Text,” Kristeva argued that “the text is […] productivity” and in addition
to its redistributive relationship to the language, the text is also “a permutation of
texts, an intertextuality: in the space of a given text, several utterances, taken from
other texts, intersect and neutralize one another” (Kristeva 1980 [1969]: 36). In this
article, I look at what type of texts left their mark on Madonna through its author,
and how these texts’ intersection can be read as a political critique. I explore the intertextuality of Madonna on two levels. Firstly, I argue that
Sabahattin Ali incorporated intertextual references into his novel as a formal prin-
ciple. By enriching Madonna’s plot and characters these references simultaneously
situate Madonna and question its place within a Turkish society undergoing cultural
transformation. Secondly, I explore the specific intertextual relationship between
Madonna and Sacher-Masoch’s Venus, a novel that also employed intertextuality as a
formal principle. In closing, I emphasize the urgent importance of recognizing the
political nature of Ali’s intertextual aspects to Venus in order to reevaluate the nov-
el’s current bestseller status in Turkey, and the specific marketing strategies adopt-
ed for its English publication with Penguin Classics in 2016. 6 Intertextuality is not the only strategy that Ali used to criticize his contemporary context in Madonna.
David Gramling and I explored how Sabahattin Ali’s stance towards translation and the language
engineering practices in the early years of the Turkish Republic, which ‘purified’ the language by
eliminating Arabo-Farsi vocabulary in favor of European and Turkic lexicon, was reflected in Madon-
na. In this piece, we argue that Sabahattin Ali used different linguistic registers for the framing narra-
tive and the story of Raif and Maria. The framing narrative has shorter sentences, and more ‘purified’
language whereas the enclosed narrative has more complex sentence structures and definitions of
situations and emotions. Sabahattin Ali may have deployed this difference as a critique of the lan-
guage reform which affected his writing directly. For more on this topic, see Gramling / Hepkaner
(forthcoming 2017). 5 For an elaborate analysis of Sabahattin Ali’s critique in Madonna, please see Dickinson (2013). Also,
İbrahim Tatarlı argued for the political insights in the novel. For more on this, please see Tatarlı
(1979: 263). 7 There are a number of scholarly works dealing with the connection between the Turkish govern-
ment and the Nazi regime in Germany until the end of the Nazi era. I list a few here which, based on
concrete evidence, delineate this close relationship between two countries: (Konuk 2010; Baer 2013;
Guttstadt 2013; Ihrig 2014). 9 In their foreword to Venus, Lotringer and Kraus argue that such erasure of boundaries between art
and reality was on purpose. They list a number of similarities between Sacher-Masoch’s life and that
of Venus’ characters. They even argue that “[i]n Venus in Furs, reality and fantasy aren’t just inter-
twined, they’re indistinguishable.” For more on this, see Lotringer / Kraus (1989). I don’t know if
Sabahattin Ali knew exactly the autobiographical elements of Sacher-Masoch’s life in Venus and
picked this novel for this reason. Unsurprisingly, there are a number of autobiographical elements in
Madonna, and many authors have noted this in various occasions. For a thorough explanation of Ali’s
contemporaries’ take on the novel, its political undertones, and autobiographical elements, see:
“Açıklamalar” (Arsever 1985). For autobiographical elements in his other novels, see Sönmez (2013).
Despite the plethora of autobiographical elements, I argue that the conflation between reality and art
in Venus was more important for Ali’s political agenda than imbuing autobiographical elements in the
novel. Abstract In doing so, I empha-
size that Ali’s engagement with German culture in Madonna—broadly conceived
here to include the fin-de-siècle Austrian novelist Sacher Masoch—carried real politi-
cal repercussions at the time of Madonna’s publication. In 1941, when Madonna was
first serialized, and 1943, when it first appeared as a novel, Turkey’s ruling gov-
ernment did not shy away from allowing Nazi infiltration and occasionally cooper-
ating with the Nazi regime.7 While Venus seems far removed from this political İlker Hepkaner 66 context, I argue on the contrary, that Madonna’s intertextuality with Venus carries
important political overtones. context, I argue on the contrary, that Madonna’s intertextuality with Venus carries
important political overtones. Ali establishes an intricate intertextual relationship with Venus through similari-
ties on the level of plot, character, and narrative form. These multilayered intertex-
tual references to Venus in Madonna relate to Ali’s political and judicial struggles at
the time of its publication. Ali’s political adversaries considered his literary works
incriminating evidence that proved his harmfulness to the state;8 ultra-nationalists
in particular utilized Ali’s literary work to declare him a traitor of the state and
homeland. Venus, as a work in which characters constantly cross the line between
art and reality, thus proves relevant for reflecting back on Ali’s own life and literary
career.9 Indeed, Venus provided Ali an excellent canvas with which to subtly criti-
cize his political adversaries, who could not dissociate literature from reality. 8 Yapı Kredi Yayınları, the publishing house of Yapı Kredi published a volume on Ali’s struggles against
the political court cases that were opened against him in order to curb his political dissidence. Docu-
ments such as letters, court decisions, expert witness reports, and defense scripts give a succinct
picture on how court cases were used against Ali’s authorship and dissidence (Ali 2004). 10 The following scholarly works investigate this cultural revolution in a myriad of realms: (Lewis
1999; Tahir Gürçağlar 2008; Ertürk 2008; Konuk 2010).
11 In an article he penned for M. Behet Yazar’s “Edebiyatçılarımız ve Türk Edebiyatı” (Our Literary
Men and Turkish Literature), Ali explained the dangers of being locked to one’s own cultural refer-
ences in literature as follows: “A literary tradition that is stuck in its own frail and blind self instead of
encompassing all humanity and the universe has no other virtue than being subject to psychopathol-
ogy studies. Arts should contain life with all its details, and it should awaken inside people the desire,
and even the need, to live, to live as human beings, to live by running towards the better, the higher,
and the purer.” (Bütün bir beşeriyeti ve bir kainatı içine alacağı yerde kendi cılız ve âmâ benliğine
saplanan bir edebiyatın, bence, pskiopatoloji etüdlerine mevzu olmaktan başka bir meziyeti yoktur.
Sanat bütün tefeerruatiyle hayatı ihtiva etmeli, insanda yaşamak, insan gibi yaşamak, daha iyiye, daha
yükseğe, daha temize doğru koşarak yaşamak arzusunu, hatta ihtiyacını uyandırmalıdır.) For the entire
article, see Ali Laslo / Özkırımlı (1979: 241-242). 13 For an elaborate discussion of Ali’s role in the state-run cultural initiatives, see Dickinson (2013).
14 For a number of articles and interviews Ali penned on the role of arts and social realism, please see
(Ali 1998). For a concrete example in which Ali states his social realist stance: “Literature, actually
generally the arts, is the expression, generalization of an idea or emotion thought and felt by the
artists, so, it is a kind of propaganda. I have never been a partisan of purposeless arts. Arts have one 12 For the entire letter, please see: “Letter to Cemal Hakkı Bey dated November 10, 1941” in Ali
(2008: 365-367). Ali defends the literary quality of his novel in this letter, and this is one of the rea-
sons why I don’t refer to Madonna as a romance novella in this article, although some of his contem-
poraries have done so. For an elaborate discussion of Ali’s role in the state-run cultural initiatives, see Dickinson (2013). The Purpose of Intertextuality in Madonna Beyond the clear references to Venus within Madonna, I argue that intertextuality as
a formal principle is crucial to the novel’s narrative strategy, in that it complicates
the love story at its core. For example, Ali uses two short literary texts in order to
explain the novel’s main characters’, Raif and Maria’s, personalities and life trajec-
tories in the narrative. Ivan Turgenev’s Klara Milic and Jakob Wassermann’s Der
niegeküsste Mund (The Mouth that Never Was Kissed) are mentioned in the text by name
and briefly summarized. Raif mentions these stories in the novel in a way that fore-
shadows the characters’ destinies. Turgenev’s Klara Milic is projected as someone
who “falls in love with a rather simple student, but even without breathing a word
of it to anyone, she falls victim to her astonishing obsession, merely out of the
shame of loving such a simpleton” (Ali 2014: 59). This is a hint at Maria and Raif’s
own destinies and deaths in the novel. Wasserman’s story about “a teacher who
was never loved by anyone and grew old while constantly expecting love or some
human affection, though he never confessed as much” (Ali 2014: 159) is a direct
reference to the circumstances surrounding Raif’s death. The Political behind the Fur Coat 10 The following scholarly works investigate this cultural revolution in a myriad of realms: (Lewis
1999; Tahir Gürçağlar 2008; Ertürk 2008; Konuk 2010). 10 The following scholarly works investigate this cultural revolution in a myriad of realms: (Lewis
1999; Tahir Gürçağlar 2008; Ertürk 2008; Konuk 2010).
11 In an article he penned for M. Behet Yazar’s “Edebiyatçılarımız ve Türk Edebiyatı” (Our Literary
Men and Turkish Literature), Ali explained the dangers of being locked to one’s own cultural refer-
ences in literature as follows: “A literary tradition that is stuck in its own frail and blind self instead of
encompassing all humanity and the universe has no other virtue than being subject to psychopathol-
ogy studies. Arts should contain life with all its details, and it should awaken inside people the desire,
and even the need, to live, to live as human beings, to live by running towards the better, the higher,
and the purer.” (Bütün bir beşeriyeti ve bir kainatı içine alacağı yerde kendi cılız ve âmâ benliğine
saplanan bir edebiyatın, bence, pskiopatoloji etüdlerine mevzu olmaktan başka bir meziyeti yoktur.
Sanat bütün tefeerruatiyle hayatı ihtiva etmeli, insanda yaşamak, insan gibi yaşamak, daha iyiye, daha
yükseğe, daha temize doğru koşarak yaşamak arzusunu, hatta ihtiyacını uyandırmalıdır.) For the entire
article, see Ali Laslo / Özkırımlı (1979: 241-242).
12 For the entire letter, please see: “Letter to Cemal Hakkı Bey dated November 10, 1941” in Ali
(2008: 365-367). Ali defends the literary quality of his novel in this letter, and this is one of the rea-
sons why I don’t refer to Madonna as a romance novella in this article, although some of his contem-
poraries have done so.
13 For an elaborate discussion of Ali’s role in the state-run cultural initiatives, see Dickinson (2013).
14 For a number of articles and interviews Ali penned on the role of arts and social realism, please see
(Ali 1998). For a concrete example in which Ali states his social realist stance: “Literature, actually
generally the arts, is the expression, generalization of an idea or emotion thought and felt by the
artists, so, it is a kind of propaganda. I have never been a partisan of purposeless arts. Arts have one The Political behind the Fur Coat 67 By playing an integral role within the narrative, these texts establish organic
connections between Ali’s novel and other European literatures. Such narrative
sophistication is in line with the cultural revolution’s broad terms in early Republi-
can era Turkey, in which state-run cultural revolution projects aimed to connect
Turkish literature and culture with a European heritage.10 By engaging intertextu-
ality as formal principle, Ali positioned Madonna beyond the confines of contempo-
rary Turkish literature and the generic restrictions of a romance novel.11 y
g
Nazım Hikmet’s letter of May 1943 further attests to the novel’s sophistica-
tion. Praising the social realist aura of the frame narrative yet lamenting the lack of
this same social realist dimension in the inner narrative, the prominent poet calls
the story of Raif and Maria “an experience [that] was necessary not only for [Ali]
but also for Turkish literature.”Ali defended the sophistication of his novel many
times in the face of criticism by fellow intellectuals and those involved in the publi-
cation of the novel. For example, when Cemal Hakkı Bey, the owner of the
Hakikat newspaper, which had serialized the novel, refused to pay Ali’s fees be-
cause “the novel did not sell at all,” and was comparable in quality to other ro-
mance novellas of the time, Ali defended his novel’s quality with rigor, arguing that
it was not geared towards popular demand.12 Apart from adding sophistication, the novel’s intertextual form intensifies Ali’s
critique of the political and cultural conditions in Turkey at the time. Before writ-
ing Madonna, Ali was already a prominent player in the state-run cultural incentives
that connected European literatures to the Turkish literary scene through transla-
tion and education.13 Deemed a social realist author even before he published Ma-
donna, Ali often expressed his utmost belief in using the arts to change people’s
lives.14 With its intertextual form, Madonna was no different than his previous and İlker Hepkaner 68 subsequent works. The novel even represented some of the cultural contentions of
its time. In the following, I address a central encounter in the text through which
intertextuality mocks the state-run mode of cultural change. subsequent works. The novel even represented some of the cultural contentions of
its time. In the following, I address a central encounter in the text through which
intertextuality mocks the state-run mode of cultural change. 15 “Bu çehreyi veya benzerini hiçbir yerde, hiçbir zaman görmediğimi ilk andan itibaren bilmeme
rağmen, onunla aramızda bir tanışıklık varmış gibi bir hisse kapıldım. Bu soluk yüz, bu siyah kaşlar ve
onların altındaki siyah gözler; bu koyu kumral saçlar ve asıl, masumluk ile iradeyi, sonsuz bir melal ile
kuvvetli bir şahsiyeti birleştiren bu ifade, bana asla yabancı olamazdı. Ben bu kadını yedi yaşımdan
beri okuduğum kitaplardan, beş yaşımdan beri kurduğum hayal dünyalarından tanıyordum. Onda
Halit Ziya’nın Nihal’inden, Vecihi Bey’ın Mehcure’sinden, Şövalye Büridan’ın sevgilisinden ve tarih
kitaplarında okuduğum Kleopatra’dan, hatta mevlit dinlerken tasavvur ettiğim, Muhammed’in annesi
Âmine Hatun’dan birer parça vardı. O benim hayalimdeki bütün kadınların bir terkibi, bir
imtizacıydı.” (Ali 2004 [1943]: 56-57) clear and sole reason: Advancing people towards the better and more correct, and instigating the
desire of this advancement. I want the arts, and our subject here the literature, to develop in this vein.
In this regard, we need to turn away from inidividualism as much as possible and turn toward life to
acquire many things from our environs and give back plenty. The first condition for this is to allow
the author to be realist.” (“Edebiyat, hatta alelumum sanat, bence sanatkarın düşündüğünü ve
duyduğu bir fikrin ve bir hissin ortaya atılması, tamim edilmesi demektir; yani bir nevi propagandadır.
Ben hiçbir zaman sanatın maksatsız olduğuna kani olmadım. Sanatın bir tek ve sarih maksadı vardır:
İnsanları daha iyiye, daha doğruya, daha güzele yükseltmek, insanlarda bu yükselme arzusunu
uyandırmak. Sanatın, ve burada mevzuumuz edebiyat olduğuna göre edebiyatın, bu manada
gelişmesini isterim. Bu takdirde de endividüalizmden mümkün olduğu kadar hayata, muhite dönmek,
muhitten birçok şeyler almak ve muhite birçok şeyler vererek yazmak lazımdır. Bunun
yapılabilmesinin birinci şartı ise, muharrire realist olmak müsaadesinin verilmesidir.”) (Ali 1998: 19). clear and sole reason: Advancing people towards the better and more correct, and instigating the
desire of this advancement. I want the arts, and our subject here the literature, to develop in this vein.
In this regard, we need to turn away from inidividualism as much as possible and turn toward life to
acquire many things from our environs and give back plenty. The first condition for this is to allow
the author to be realist.” (“Edebiyat, hatta alelumum sanat, bence sanatkarın düşündüğünü ve
duyduğu bir fikrin ve bir hissin ortaya atılması, tamim edilmesi demektir; yani bir nevi propagandadır.
Ben hiçbir zaman sanatın maksatsız olduğuna kani olmadım. Sanatın bir tek ve sarih maksadı vardır:
İnsanları daha iyiye, daha doğruya, daha güzele yükseltmek, insanlarda bu yükselme arzusunu
uyandırmak. Sanatın, ve burada mevzuumuz edebiyat olduğuna göre edebiyatın, bu manada
gelişmesini isterim. Bu takdirde de endividüalizmden mümkün olduğu kadar hayata, muhite dönmek,
muhitten birçok şeyler almak ve muhite birçok şeyler vererek yazmak lazımdır. Bunun
yapılabilmesinin birinci şartı ise, muharrire realist olmak müsaadesinin verilmesidir.”) (Ali 1998: 19).
15 “Bu çehreyi veya benzerini hiçbir yerde, hiçbir zaman görmediğimi ilk andan itibaren bilmeme
rağmen, onunla aramızda bir tanışıklık varmış gibi bir hisse kapıldım. Bu soluk yüz, bu siyah kaşlar ve
onların altındaki siyah gözler; bu koyu kumral saçlar ve asıl, masumluk ile iradeyi, sonsuz bir melal ile
kuvvetli bir şahsiyeti birleştiren bu ifade, bana asla yabancı olamazdı. Ben bu kadını yedi yaşımdan
beri okuduğum kitaplardan, beş yaşımdan beri kurduğum hayal dünyalarından tanıyordum. Onda
Halit Ziya’nın Nihal’inden, Vecihi Bey’ın Mehcure’sinden, Şövalye Büridan’ın sevgilisinden ve tarih
kitaplarında okuduğum Kleopatra’dan, hatta mevlit dinlerken tasavvur ettiğim, Muhammed’in annesi
Âmine Hatun’dan birer parça vardı. O benim hayalimdeki bütün kadınların bir terkibi, bir
imtizacıydı.” (Ali 2004 [1943]: 56-57) The Political behind the Fur Coat y
g
In the internal narrative of the novel, Raif struggles with contending dichoto-
my of meaning-making mechanisms of the so-called ‘East’ and ‘West’ when he
tries to understand the content and qualities of Maria Puder’s classical self-portrait. Raif describes his first impression of Maria Puder’s self-portrait on his own terms: Although I knew from the first instant that I had not seen this face any-
where, any time, I felt that there was some relation between us. And yet
this pale face, this dark brown hair, that expression that reconciled inno-
cence with volition, a boundless ennui with a brazen character: the combi-
nation could be nothing but familiar to me. I knew this woman from the
books I had been reading since age seven, and from that realm of dreams
I’d constituted since age five. Pieces of her came from Halit Ziya’s Nihal,
from Vecihi Bey’s Mehcure, from Chevalier Buridan’s lover, from the
Cleopatra I’d read about in history books, and even from Amine Hatun,
Muhammed’s mother, as I envisioned her while listening to the mevlits. She was the amalgam, the combination of all the women of my waking
dreams. (Ali 2014: 61)15 Raif overcomes his amazement by making sense of this painting’s effects on him
through the books he has read. Yet after reading a newspaper article, his interpre-
tation changes. In this article, the author offers a different yet definite explanation
of the portrait: The Political behind the Fur Coat 69 [The author of the article] claimed that, given the strange equivalence of posture
and facial expression, the woman looked so much like the depiction of Mother
Mary in Andreas del Sarto’s painting ‘Madonna delle Arpie’ that it could actually
give someone chills. With this, the author half jokingly wished this “Madonna in a
Fur Coat” great success, and then moved on to another artist. (Ali 2014: 63)16 With this explanation, the German critic places the portrait in its contemporary
German context, and draws a parallel with an artwork from the Renaissance era. The German critic furthermore authoritatively names Maria’s self-portrait, Raif’s
image of Maria, and—though indirectly—the novel. 16 “[T]ablodaki kadının, duruşu ve yüzünün ifadesi bakımından, tuhaf bir tesadüf eseri olarak, Andreas
del Sarto’nun Madonna delle Arpie tablosundaki Meryemana tasvirine insanı şaşırtacak kadar çok
benzediği iddia ediliyor ve yarı şaka bir ifade ile bu ‘Kürk Mantolu Madonna’ya muvaffakiyetler
temenni edilerek başka bir ressamdan bahse geçiyordu.” (Ali 2004 [1943]: 58)
17 İ̇lker Aytürk gives an excellent example on how some intellectuals of the period coped with the
issue of contending repertoires in the field of linguistics (Aytürk 2004).
18 Nihal Atsız wrote a disturbing pamphlet in which he publicly attacked Sabahattin Ali and his book
İçimizdeki Şeytan (The Devil Within Us). Atsız’s pamphlet is called İçimizdeki Şeytanlar (The Devils Within
Us); it is a close reading of the novel in which he concludes the characters are from real life. In addi-
tion, Atsız argues that Ali is pseudo-Greek (and he means this as an insult), completely crazy, and a
megalomaniac, and Atsız openly threatens Ali by saying that “their problem will only be solved by
blood.” This pamphlet is accessible on the website Ulu Türkçü Nihal Atsız Otağı: http://www.nihal-
atsiz.com/yazi/icimizdeki-seytanlar-h-nihal-atsiz.html [Last accessed: 30.11.2016].
19 For more on this, please see Azamet Arsever’s note (Arsever, 1985). 16 “[T]ablodaki kadının, duruşu ve yüzünün ifadesi bakımından, tuhaf bir tesadüf eseri olarak, Andreas
del Sarto’nun Madonna delle Arpie tablosundaki Meryemana tasvirine insanı şaşırtacak kadar çok
benzediği iddia ediliyor ve yarı şaka bir ifade ile bu ‘Kürk Mantolu Madonna’ya muvaffakiyetler
temenni edilerek başka bir ressamdan bahse geçiyordu.” (Ali 2004 [1943]: 58) İlker Aytürk gives an excellent example on how some intellectuals of the period coped with the
issue of contending repertoires in the field of linguistics (Aytürk 2004).
18 Nihal Atsız wrote a disturbing pamphlet in which he publicly attacked Sabahattin Ali and his book
İçimizdeki Şeytan (The Devil Within Us). Atsız’s pamphlet is called İçimizdeki Şeytanlar (The Devils Within
Us); it is a close reading of the novel in which he concludes the characters are from real life. In addi-
tion, Atsız argues that Ali is pseudo-Greek (and he means this as an insult), completely crazy, and a
megalomaniac, and Atsız openly threatens Ali by saying that “their problem will only be solved by
blood.” This pamphlet is accessible on the website Ulu Türkçü Nihal Atsız Otağı: http://www.nihal-
atsiz.com/yazi/icimizdeki-seytanlar-h-nihal-atsiz.html [Last accessed: 30.11.2016].
19 For more on this, please see Azamet Arsever’s note (Arsever, 1985). For more on this, please see Azamet Arsever’s note (Arsever, 1985). ker Aytürk gives an excellent example on how some intellectuals of the period coped with the
of contending repertoires in the field of linguistics (Aytürk 2004). The Political behind the Fur Coat In the following paragraph,
convinced by the critic’s intervention, Raif admits the similarity when he looks at a
replica of ‘Madonna delle Arpie.’ Given the political dimensions of the novel, I argue that the tension between
the meaning-making mechanisms of the German critic and Raif, and the critic’s
definitive answer to Raif’s puzzlement, are politically charged intertextualities that
represent and critique the cultural revolution that Ali along with his fellow coun-
trymen experienced. Unlike Ali’s characters in İçimizdeki Şeytan (1940) (The Devil
Within Us, hereafter referred to as Şeytan)—which represents real-life ultra-
nationalist intellectuals—the characters of Madonna are not directly drawn from
Ali’s contemporary Turkey, yet the conflict between various meaning-making
mechanisms they experience is strikingly similar to the one his contemporaries
faced.17 The decision to place his characters outside of a Turkish context can be read as
a strategic move in relation to the political and social hardships that Ali underwent
as author. Right before the publication of Madonna, Nihal Atsız, the most promi-
nent ultra-nationalist intellectual of the time, considered the publication of Şeytan as
a personal attack. Indeed, the publication of Şeytan marks a pinnacle in the two
intellectuals’ disagreements,18 following which Atsız called for a nationalist outrage
towards Ali and openly threatened Ali’s life. In the aftermath of such vehement
reactions to Ali’s previous work the owner of Hakikat newspaper, Cemal Hakkı
Bey, asked Ali to write “a riveting love story not involved in politics.”19 Contrary
to the heated reaction and scrutiny Şeytan attracted, Madonna did not face any such İlker Hepkaner 70 reaction from the ultra-nationalist circles. Due to the subtlety with which Madonna
represented the cultural challenges Ali and his contemporaries faced, Madonna was
never a subject of state oppression, whereas Ali’s works before and after were
sanctioned by the state. reaction from the ultra-nationalist circles. Due to the subtlety with which Madonna
represented the cultural challenges Ali and his contemporaries faced, Madonna was
never a subject of state oppression, whereas Ali’s works before and after were
sanctioned by the state. It is no coincidence that Germany and the German cultural context were the
literary havens with which Ali chose to imbue his critique of the Turkish context. Ali’s past in Berlin and his fluency in German were certainly important reasons
behind his thematization of Weimar Berlin. However, I argue that there is more to
his critique than this. 20 “Ob im Glück oder ob in der Freiheit oder im Gefängnis, ich bin der Fahne, die heute des Deut-
schen Reiches Staatsflagge ist, treu geblieben.”
21 In Madonna, Raif describes one of the guests at his pension in Berlin: “This last guest was a trader
who’d been forced to leave behind his life in the German colony of Cameroon after the Armistice
and had found shelter back in his home country. He was now leading a rather modest life with the
money he had been able to smuggle back, and he spent most of his days at political meetings, which
were quite numerous at the time, and related his ideas about them at night. He often brought back
this or that freshly discharged and unemployed German officer he’d just met, and they would debate
for hours. Though my understanding could have been faulty, these guests of his seemed to be of the
opinion that Germany could survive only if a man with an iron will like Bismarck were to lead the
country, and that injustices needed to be corrected with a second war, for which the only adequate
preparation was immediate and rapid armament.” (Ali 2014: 55) (“Bu sonuncusu Almanya'nın Kame-
run müstemlekesinde ticaret yaparken mütarekeden sonra her şeyini bırakarak vatanına sığınmış bir
adamdı. Kurtarabildiği bir miktar parasıyla oldukça mütevazı bir hayat sürüyor, gününü, o sıralarda
Berlin'de pek bol olan siyasi toplantılara gidip akşamları intibalarını anlatmak suretiyle geçiriyordu.
Çok kere, yeni tanıştığı terhis edilmiş işsiz Alman zabitlerini de yanında getirir ve onlarla, saatlerce
münakaşa ederdi. Benim yarım yamalak anladığıma göre Almanya'nın kurtuluşunu Bismark gibi demir
iradeli bir adamın işbaşına geçmesinde ve hi. vakit geçirmeden silahlanmaya baş̧layarak ikinci bir
harple haksızlıkları düzeltmekte buluyorlardı.”) (Ali 2004 [1943]: 53) When Raif talks about the ongo-
ing political discussions in the pension, he even gives hints about an infamous gesture that was al- The Political behind the Fur Coat From Ali’s private correspondence and political engagements
in the 1940s, we know that the German / Nazi context of the time and its connec-
tions with Turkey were prevalent in Ali’s life. At a time in which Ali’s disagree-
ments with Nihal Atsız were revolving around issues of nationalism and fascism,
for example, Atsız sent Ali a postcard from Germany in 1939. Atsız signed the
postcard with the words “To you and your wife, greetings and love from Berlin.”
From Atsız’s pamphlet and Ali’s other correspondences, we know that Ali and
Atsız were heading towards a rocky chapter in their relationship by the end of the
1930s. More than Atsız’s words, the image on the card offers clues about what
Germany could have meant to Ali in the 1930s. The card bears Hubert Lanzinger's
famous painting of Hitler, ‘Der Bannerträger’ (The Standard Bearer), and the fol-
lowing words by the fascist leader: “Whether in good fortune or bad fortune,
whether in freedom or captivity, I remained faithful to my own banner, which is
today the State flag of the Germans.”20 Considering the elements in Madonna that hint at the Nazis coming to power,
and the fact that Ali wrote this novel while enlisted in the army for a second time
due to World War II, receiving Hitler’s image from his soon-to-be nemesis sug-
gests that the choice of Weimar Berlin as the setting for his novel was not arbi-
trary.21 The card and its message gain poignancy with the knowledge that Ali was The Political behind the Fur Coat 71 repeatedly tried by the state on treason charges during the 1930s. Ali’s political
dissidence, connections to socialist circles of the time, and his works’ social realist
undertones made him an easy target for such allegations.22 On the other hand,
Nihal Atsız was not prosecuted for his publications that were openly anti-Semitic
in the 1930s.23 Such inequality between two intellectuals with different takes on
nationalism and fascism must have been a matter of concern and critique for Ali. Ali’s preoccupation with the Nazis’ impact on humanity was not limited to his
personal life. After the war, he was publicly vocal about the connections between
the Nazi and Turkish governments and their enduring legacy. For example, follow-
ing his incarceration under the state of siege in 1944, he penned a letter to the
Judges of 1. The Political behind the Fur Coat İstanbul Sıkıyönetim Mahkemesi (Istanbul First Court for State of
Siege).24 In the letter, after comparing the Central Police Station in Istanbul to the
Bastille Prison of pre-revolutionary France, he named bureaucrats who built the
station after their visits to Nazi Germany. He criticized the lingering legacy of Nazi
Germany in Turkey with the following words: “[T]he methods of humanity's big-
gest enemy, German fascism, are still being used in a secret manner, and kept from
the public, in Turkey shaped by the Ataturk revolution.” 25 p
p
In this light, when contextualized within Ali’s personal and political problems
as well as the conditions and horrors of World War II, Madonna’s engagement with
issues relating to Nazi Germany arguably renders the intertextual connections be-
tween Madonna and Venus even more political. 22 I haven’t seen Ali’s response to this card, if there is any, among the published documents from his
life. During the research workshop “Transnational Perspectives on the Life and Work of Sabahattin
Ali” organized by the Hagop Kevorkian Center for Near Eastern Studies at New York University in
March 2015, Sevengül Sönmez, the editor of most of these published documents, stated that all the
publishable material from the personal archive of the author has been published by Yapı Kredi Pub-
lishing House. Some of the material was left out because they contain family secrets. I have not been
able to locate a personal archive of Nihal Atsız that could have contained the other half of this corre-
spondence, if there is any. To learn about the two intellectuals’ later legal battles, see Ali (2004). 23 For more on the topic of how Nihal Atsız and intellectuals and state officials who disseminated
German anti-Semitism with no judicial consequences, see Rifat N. Bali: “Antisemitism in Turkey in
the Single Party Period: 1923-1945” (Bali 2013). 24 “Belge 27” in Ali (2004). 25 “[İ]nsanlığın en büyük düşmanı Alman Faşizmi metodları bugün Atatürk İnkılabı Türkiye’sinde
halkın gözünden gizli olarak tatbik sahası bulmaktadır.” After this sentence, this letter continues by
documenting the conditions and timeline of Ali and others’ incarceration. Ali gives excruciating
details about the inhumane conditions in the police station before providing a complex account of
torture-ridden interrogation they had to endure in the station. His style of writing is very dry except
occasional references to how the state practices do not live up to the modern standards set by the
Kemalist revolution. ready legible for the reader of the 1940s: “Even the maid who cleaned my room in the mornings tried
to drum up political conversations with me, and she would read her newspaper whenever she had a
spare moment. She had her own ardent opinions, and while talking about them, her face would flush
red and she’d swing her fist in the air.” (Ali 2014: 56). (“Sabahları odamı düzelten hizmetçi kız bile
benimle siyasetten konuşmaya kalkar, boş zamanlarında derhal gazetesini okumaya koyulurdu. Onun
da kendine göre ateş̧li kanaatleri vardı ve bunlardan bahsederken yüzü büsbütün kızarır, yumruğunu
sıkarak havada sallardı.”) (Ali 2004 [1943]: 53) 26 There are several theories why Madonna became a bestseller 70 years after its first publication. For a
TV program on the summary of these theories, see 5N1K Kanal D Youtube Channel: Kürk Mantolu
Madonna neden bu kadar çok okunuyor / Why is The Madonna in the Fur Coat a Bestseller:
https://www.youtube.com/watch?v=4H-W-KP_N9g [Last accessed: 01.08.2016].
Interviewees on this program focus on the sincerity of the love story and the lack of such feelings in
the contemporary times for the bestselling status. In this program, Sevengül Sönmez also argues that
the fact that Sabahattin Ali was killed had an impact on people’s interest, and by reading his works
the Turkish public is actually apologizing to one of the victims of the first political murders in Repub-
lican history. Sönmez also considers the book as a milestone in Turkish literature as it brings ‘eastern’
and ‘western’ elements together. I don’t delve into the reasons behind the novel’s success in the last
decade in this article. However, I should add to the reasons above that Madonna gained its best-selling
status after Yapı Kredi Yayınları, the Publishing House of Yapı Kredi (YKY), one of the biggest banks in
Turkey, started publishing Ali’s works in 1997. YKY has also published a number of volumes on the
life and works of Sabahattin Ali. I personally believe Yapı Kredi’s effective network with book retailers
might have laid the ground for the novel’s success. In addition, in 2005, the Turkish Ministry of
Education has published a list of 100 Fundamental Works (100 Temel Eser) for primary and secondary
education. They included Sabahattin Ali’s Kuyucaklı Yusuf in this list. I believe a comprehensive study
on the politics of culture of big banks in Turkey and the impact of the government’s efforts in canon-
ization can answer Madonna’s success. I would like to thank Aslı Iğsız for her remarks on this issue,
which helped me immensely in thinking about this late success of Madonna. The Political behind the Fur Coat I argue that such references are textual strategies in which Ali aimed to allure to
the judges state-centric sensibilities. For more, please check “Belge 27” in Ali (2004). 72 İlker Hepkaner 26 There are several theories why Madonna became a bestseller 70 years after its first publication. For a
TV program on the summary of these theories, see 5N1K Kanal D Youtube Channel: Kürk Mantolu
Madonna neden bu kadar çok okunuyor / Why is The Madonna in the Fur Coat a Bestseller:
https://www.youtube.com/watch?v=4H-W-KP_N9g [Last accessed: 01.08.2016]. Key intertextualities of Madonna and Venus Venus was published in 1870, whereas Madonna was first serialized in Hakikat
newspaper in 1940-1941, and then published in book format in 1943. Madonna
gained bestseller status in the early 2010s in Turkey and has sold over 1 million
copies.26 It has since been translated into a number of languages including French,
German, Macedonian, Arabic, and English. Madonna and Venus are both novels in which the main story is embedded with-
in a frame narrative. This internal / external narrative structure is one of the inter-
textualities between the two novels. Madonna begins in 1940s Ankara with the story
of the narrator’s relationship with Raif, a translator and coworker. Despite their
cold start, Raif’s fatal illness brings the two unlikely colleagues closer. Due to their
rapprochement, the narrator reads Raif’s “novel in the German language” (Almanca
bir roman), a notebook which Raif had kept hidden from everyone at his office, just
as Raif dies. This ‘novel’ is then revealed to be Raif’s autobiographical story. Simi-
larly, Venus’ frame narrative is the tale of an unnamed narrator receiving a manu-
script from his servant, Severin, who has also kept it inside a drawer. The embed-
ded narrative of Venus tells the love story between Severin and a woman named
Wanda. This is the same framework Ali employs in Madonna, with the internal
narrative detailing a love affair between Raif and the German-Jewish woman, Ma-
ria. The two novels’ plots are also intertextually related. In the internal narrative of
Madonna, Raif narrates his stay in Berlin in 1924, where, with his father’s financial
support, he spends some time learning how to modernize his family’s soap busi-
ness in Havran, Turkey. As young Raif learns German through literature and wan-
ders around Berlin, he comes across “Kürk Mantolu Madonna” (The Madonna in
the Fur Coat), a classical self-portrait at an exhibition of modernist artists. The The Political behind the Fur Coat 73 painter of this portrait is Maria Puder, a half-Jewish artist and cabaret singer. Their
relationship begins in the midst of Raif’s admiration for Maria’s self-portrait, then
turns into a friendship, and gradually also a love affair. The rules of their relation-
ship are determined solely by Maria. In Venus, Severin and Wanda also establish
their relationship within Severin’s fascination with Wanda, and their master-slave
relationship is completely determined by Wanda. Key intertextualities of Madonna and Venus painter of this portrait is Maria Puder, a half-Jewish artist and cabaret singer. Their
relationship begins in the midst of Raif’s admiration for Maria’s self-portrait, then
turns into a friendship, and gradually also a love affair. The rules of their relation-
ship are determined solely by Maria. In Venus, Severin and Wanda also establish
their relationship within Severin’s fascination with Wanda, and their master-slave
relationship is completely determined by Wanda. p
p
Maria’s illness and Raif’s family problems ultimately tear them apart. Deflated
by the impossibility of keeping in touch with Maria upon his return to Turkey, Raif
gives up on love and life, and blames Maria for his unhappiness. This confusion
lasts until a surprise encounter in Ankara ten years later, when he discovers that
Maria had died after giving birth to their child. This shocking news forces Raif to
finally write down his story. This leads to an ending similar to that of Venus: In the
final pages of Madonna, the narrator states that he had the chance to get to know
Raif better through his ‘novel in German.’ When the narrator receives the news of
Raif’s passing, he begins reading the story anew. Within Venus, Severin and Wan-
da’s relationship also comes to an abrupt end, following which Severin returns
home to help with his family business, only to hear from Wanda years later in a
letter. The novel ends with a brief dialogue between the narrator and Severin on
the moral of the story. Venus in Madonna In Ali’s contemporary context, the intertextual relationship between Ali’s Madonna
and Sacher-Masoch’s Venus carries crucial political undertones. I argue that Ali’s
use of intertextuality as a formal principle effectively blurred the lines between the
Turkish and European cultures and literatures that preoccupied intellectuals of the
time. Given the interconnectedness of the two contexts that Ali experienced in his
life, together with select depictions in the novel that downplay any differences
between Europe and Turkey, the intertextual relationship between Madonna and
Venus that translates as shared structures and components become additional criti-
cal commentary on the cultural revolution, which aimed to transcend the lines
between the so-called separate worlds of Europe and Turkey. The Turkish context,
with its cultural and political revolution, was not independent from the develop-
ments beyond the country’s borders, and Madonna’s formal intertextuality and its
intertextual relationship with Venus reflect (upon) this. While Ali never explicitly refers to Venus—unlike other texts with which he es-
tablishes intertextualities—he does hint at a connection between Raif’s story and a
novel in German in the text. When asked, Raif Efendi refers to his notebook, in
which the story of Maria and himself is written, as “a novel in the German lan-
guage” (Almanca bir roman) although he wrote it in Turkish (19). As the text of
Raif’s diary, this notebook / novel also comprises the text of Madonna’s internal
narrative. Due to the myriad intertextual similarities I have outlined above, I pro-
pose that one way to read Raif’s notebook / novel is in reference to Venus. This 74 İlker Hepkaner argument is strengthened by the fact that Raif Efendi does not call his notebook ‘a
German novel’ (bir Alman romanı) but rather “a novel in the German language”
(Almanca bir roman), which underscores Venus’s status as an Austrian novel written
in German. argument is strengthened by the fact that Raif Efendi does not call his notebook ‘a
German novel’ (bir Alman romanı) but rather “a novel in the German language”
(Almanca bir roman), which underscores Venus’s status as an Austrian novel written
in German. In the narrative, Raif Efendi writes the novel in 1933, when Austria was a
country of its own. When Sabahattin Ali started to write the novel in 1941, the
Anschluss had already happened. 27 “Mir gegenüber an dem massiven Renaissancekamin saß Venus, aber nicht etwa eine Dame der
Halbwelt, die unter diesem Namen Krieg führte gegen das feindliche Geschlecht, gleich Mademoisel-
le Cleopatra, sondern die wahrhafte Liebesgöttin.” (Sacher-Masoch1980 [1870]: 9) Venus in Madonna In another
example, when Severin questions Wanda’s reasoning on a matter in the embedded
narrative of the novel, Wanda replies with the following: The Political behind the Fur Coat 75 From my cradle onward I was surrounded by replicas of ancient art; at ten
years of age, I read Gil Blas, at twelve La Pucelle. Where others had Hop-
o’-my-thumb, Bluebeard, Cinderella, as childhood friends, mine were Ve-
nus and Apollo, Hercules and Lackoon. (Sacher-Masoch 1989 [1870]:
76)28 From my cradle onward I was surrounded by replicas of ancient art; at ten
years of age, I read Gil Blas, at twelve La Pucelle. Where others had Hop-
o’-my-thumb, Bluebeard, Cinderella, as childhood friends, mine were Ve-
nus and Apollo, Hercules and Lackoon. (Sacher-Masoch 1989 [1870]:
76)28 From my cradle onward I was surrounded by replicas of ancient art; at ten
years of age, I read Gil Blas, at twelve La Pucelle. Where others had Hop-
o’-my-thumb, Bluebeard, Cinderella, as childhood friends, mine were Ve-
nus and Apollo, Hercules and Lackoon. (Sacher-Masoch 1989 [1870]:
76)28 Wanda marks her intelligence through the books she has read and the artworks
that surrounded her. Raif’s self-definitions are similar. Very often, he names the
books he read and their impact on him in order to explain one of his own charac-
ter traits. In the narrative, the books the characters read, as mentioned earlier, even
foreshadow the course of their fates. This interconnectedness between the charac-
ters and literary works lays the groundwork for Ali’s discussion of the blurred line
between the arts and reality. Both novels offer a commentary on the conflation of
art with reality, but when Ali imbues this into his novel, this confusion becomes a
part of his critique. Notably, both Raif and Severin are profoundly affected by the
paintings that surround them, and at times conflate art with reality.29 Ali’s treatise
on Maria Puder’s self-portrait and the portrait’s emotional impact on Raif—which
also causes the ambiguity between art and reality in Madonna—is parallel to Sacher-
Masoch’s treatment of art. For example, just as Raif confuses Maria with Madonna,
Severin often confuses Wanda with Venus, and uses the terms interchangeably: In the middle of the night there was a knock at my window; I got up,
opened it, and was startled. Without stood, “Venus in Furs,” just as she
had appeared to me the first time. 28 “Ich war von der Wiege an mit Abgüssen antiker Bildwerke umgeben, ich las mit zehn Jahren den
Gil Blas, mit zwölf die Pucelle. Wie andere in ihrer Kindheit den Däumling, Blaubart, Aschenbrödel,
nannte ich Venus und Apollo, Herkules und Laokoon meine Freunde.” (Sacher-Masoch 1980 [1870]:
28)
29 I would like to thank Kristin Dickinson for pointing out the image-making aspect common to both
novels.
30 “Mitten in der Nacht klopfte es an mein Fenster, ich stand auf, öffnete und schrak zusammen.
Draußen stand Venus im Pelz, genau so wie sie mir das erstemal erschienen war.
‘Sie haben mich mit Ihren Geschichten aufgeregt, ich wälze mich auf meinem Lager und kann nicht
schlafen’, sprach sie, ‘kommen Sie jetzt nur, mir Gesellschaft leisten.’
‘Im Augenblicke.’ 28 “Ich war von der Wiege an mit Abgüssen antiker Bildwerke umgeben, ich las mit zehn Jahren den
Gil Blas, mit zwölf die Pucelle. Wie andere in ihrer Kindheit den Däumling, Blaubart, Aschenbrödel,
nannte ich Venus und Apollo, Herkules und Laokoon meine Freunde.” (Sacher-Masoch 1980 [1870]:
28) “Mitten in der Nacht klopfte es an mein Fenster, ich stand auf, öffnete und schrak zusamme
Draußen stand Venus im Pelz, genau so wie sie mir das erstemal erschienen war. ie haben mich mit Ihren Geschichten aufgeregt, ich wälze mich auf meinem Lager und kann nic
chlafen’, sprach sie, ‘kommen Sie jetzt nur, mir Gesellschaft leisten.’ Venus in Madonna Ali never explicitly mentions Venus as a source for
Madonna, yet the political context of the time, in which the Nazi state was taking
over other countries with the aim of erasing any cultural difference to theirs, ren-
dered Venus, a novel written by an author whose country was erased from the po-
litical maps of the time, a powerful signifier of the political destruction that reflect-
ed the destabilized linguistic and cultural contexts of Europe at the time. g
p
Ali establishes intertextuality between the two novels most clearly by adopting
Venus’ plot line and using an internal / external narrative. In addition to this, the
use of intertextuality as a formal principle deepens the political aspects of the rela-
tionship between two novels. In both novels, the characters are described through
art and literature, and the male characters are unable to separate art from reality. Such adopted strategies in form help Ali to elucidate the over-arching conflation
between art and reality in Madonna, a central tenet of the critiques Ali voiced at this
time. Ali constructs Madonna as an intertextual novel in a manner similar to how Sa-
cher-Masoch constructed Venus. Both authors describe people or events through
historical or literary characters. For example, Sacher-Masoch describes Venus, who
appears in the first segment of the framing story in a dream sequence, as follows: Opposite me by the massive ‘Renaissance’ fireplace sat Venus; she was not
a causal woman of the half-world, who under this pseudonym wages war
against the enemy sex, like Mademoiselle Cleopatra, but the real, true god-
dess of love. (Sacher-Masoch 1989 [1870]: 53)27 Sacher-Masoch’s description of Venus references Cleopatra, just as Raif does in his
description of Maria Puder’s self-portrait. In reality, Venus is a painting in front of
the narrator, but in his dream, Venus is alive and speaks to him. Another striking
parallel is apparent when Sacher-Masoch mentions the books his characters read. During the dream sequence, the narrator is holding a book by Hegel, who penned
the seminal text on the master-slave dialectic, the very theme of Venus. )
I would like to thank Kristin Dickinson for pointing out the image-making aspect common to bo
ovels. g
‘Sie haben mich mit Ihren Geschichten aufgeregt, ich wälze mich auf meinem Lager und kann nicht
schlafen’, sprach sie, ‘kommen Sie jetzt nur, mir Gesellschaft leisten.’
‘Im Augenblicke.’ 28 “Ich war von der Wiege an mit Abgüssen antiker Bildwerke umgeben, ich las mit zehn Jahren den
Gil Blas, mit zwölf die Pucelle. Wie andere in ihrer Kindheit den Däumling, Blaubart, Aschenbrödel,
nannte ich Venus und Apollo, Herkules und Laokoon meine Freunde.” (Sacher-Masoch 1980 [1870]:
28)
29 I would like to thank Kristin Dickinson for pointing out the image-making aspect common to both
novels.
30 “Mitten in der Nacht klopfte es an mein Fenster, ich stand auf, öffnete und schrak zusammen.
D
ß
d V
i
P l
i
i
i d
l
hi Als ich eintrat, kauerte Wanda vor dem Kamin, in dem sie ein kleines Feuer angefacht hatte.”
(Sacher-Masoch 1980 [1870]: 44)
31 “‘Maria’ dedim. ‘Maria! Benim Kürk Mantolu Madonnam! Birdenbire ne oldu? Sana ne yaptım?
Hiçbir şey istemeyeceğimi vaat etmiştim. Sözümü tutmadım mı? Birbirimize her zamandan ziyade
yakın olmamız lazım gelen bu anda neler söylüyorsun?’” (Ali 2004: 121) 32 For an example of failing to recognize the political in the novel, please see Lichtig (2016).
33 I would like to thank Rüstem Ertuğ Altınay for pointing out the proliferation of
the novel’s images on social media. After our conversation, one of the marketing pieces for the Eng-
lish version by Freely and Dawe mentioned this saturation of images. See: Hürriyet Daily News,
2.12.2016: “‘Madonna in a Fur Coat’ makes a glorious comeback’”, accessible at:
http://www.hurriyetdailynews.com/madonna-in-a-fur-coat-makes-a-gloriouscomeback.aspx?
page-
ID=238&nID=98840&NewsCatID=386 [Last accessed: 1.06.2016]. The reason behind the abun-
dance of Madonna’s images on the social media is beyond the scope of this article. However, it cannot
be thought apart from its current bestseller status. Repoliticizing Madonna Reading Madonna’s multifarious intertextualities as mere coincidence or even pla-
giarism ignores both the developments in Ali’s personal life and the contemporary
context in which he wrote. Madonna takes place in pre-war Berlin and was pub-
lished during World War II in a cultural environment that underwent a turbulent
state-run revolution while maintaining close contact with Nazi Germany. Sabahat-
tin Ali remained a vocal dissident throughout his life, and the intertextualities in
Madonna provide us with enough evidence to include Ali’s last novel within his
body of works that voice social and political critique. Madonna was subtly vocal
about its contemporary context, and it is crucial to recognize this aspect of the
novel as it enjoys its persistent spot on the bestseller lists in Turkey. Such recognition is important for two reasons. First, it shows that criticism of
the turbulent cultural and political landscapes of Turkey was indeed possible de-
spite oppressive state policies in the 1940s. The subtlety of Ali’s persistent criticism
in this novel invites us to question the limits of the Turkish state’s oppression at
the time. It suggests a new timeline in understanding literature’s stand against the
dominant ideology of early republican Turkey. It also shows that intellectuals did
not wait until after World War II in order to question the connections of the Turk-
ish government with Nazi Germany and its ideologies. In short, Madonna is far
from merely a romance novel or a plagiarized novel. Its intertextualities crystallized
a number of discussions within the intellectual circles of its time. Secondly, recognizing the political aspects of Madonna is important for today’s
readership. It challenges and transforms the depoliticized consumption of the nov-
el, which has been the driving force behind its revalorization in contemporary
Turkish public and visual spheres.32 The last decade’s ‘Madonna-mania’ in Turkey
goes hand in hand with the novel’s bestseller status 70 years after its publication. 33
The saturation of social media with images of people reading or quoting from the
book without including or acknowledging intrinsic discussions of the novel may
assume new meanings by recognizing the political stance that Ali took with Madon-
na. Maybe this will also give new ideas to publishers who market various versions
of the book as a love story, which is precisely what Sabahattin Ali opposed. Venus in Madonna The Political behind the Fur Coat 77 Venus in Madonna “You have disturbed me with your stories; I have been tossing about
in bed, and can’t go to sleep,” she said. “Now come and stay with me.” “In a moment.” As I entered Wanda was crouching by the fireplace where she had
kindled a small fire. (Sacher-Masoch 1989 [1870]: 95)30 İlker Hepkaner 76 In Madonna, Raif also confuses Maria Puder with her self-portrait. Raif uses Maria
and “Madonna in the Fur Coat” interchangeably: Maria!” I said. “Maria! My Madonna in the Fur Coat! What happened so
suddenly? What have I done to you? I promised not to want anything fur-
ther. Haven’t I kept my promise? Look at what you are saying, at a time
when we are supposed to be closer than ever!” (Ali 2014: 109)31 Raif continues to conflate the image and its painter, and does not force himself to
dissociate one from the other. Indeed, Raif refers to Maria as “Maria, the Madonna
in the Fur Coat” many times in the novel, especially as the two characters bond
more closely. Raif continues to conflate the image and its painter, and does not force himself to
dissociate one from the other. Indeed, Raif refers to Maria as “Maria, the Madonna
in the Fur Coat” many times in the novel, especially as the two characters bond
more closely. This relation between Madonna and Venus underscores the politically charged
connotations of intertextuality between the two novels, although we don’t have
hard evidence that suggests Ali purposefully wrote Madonna based on Venus. The
conflation of art and reality was indeed the main reason why Atsız launched a pub-
lic attack on him—he considered the characterizations of ultra-nationalists in Ali’s
novel Şeytan a personal attack against himself and the movement to which he be-
longed. In a similar vein, the state punished Ali with severe measures for his liter-
ary works, conflating what was in his literary works with his behavior as a citizen. In a context where representation is taken too seriously, Ali writes an intertextual
novel rife with the conflation of art and reality. In a politically dire environment for
himself, he writes a politically subversive novel criticizing its own Turkish context,
disguised as a love story set in Weimar Germany. I thus argue that Madonna’s con-
nection with Venus points to Ali’s multilayered political critique of his contempo-
raries and the context in which he lived. 32 For an example of failing to recognize the political in the novel, please see Lichtig (2016). Repoliticizing Madonna In-
deed, Madonna is much more than a romance novel, and there is ample evidence
for us to recognize the sophisticated socio-political critique that lies both behind its
cover and ‘behind the fur coat.’ Secondly, recognizing the political aspects of Madonna is important for today’s
readership. It challenges and transforms the depoliticized consumption of the nov-
el, which has been the driving force behind its revalorization in contemporary
Turkish public and visual spheres.32 The last decade’s ‘Madonna-mania’ in Turkey
goes hand in hand with the novel’s bestseller status 70 years after its publication. 33
Th
i
f
i l
di
i h i
f
l
di
i
f
h İlker Hepkaner 78 Works Cited Ali, Sabahattin (2008): Hep Genç Kalacağım: Mektup, edited by Sevengül Sönmez,
İstanbul. Ali, Sabahattin (2004): Mahkemelerde: Belgeler, edited by Nüket Esen / Nezihe
Seyhan, İstanbul. Ali, Sabahattin (1998): Markopaşa Yazıları ve Ötekiler, edited by Hikmet Altınkaynak,
İstanbul. Ali, Sabahattin (2004 [1943]): Kürk Mantolu Madonna, İstanbul. Ali, Sabahattin (2014 [1943]): The Madonna in the Fur Coat, translated by David
Gramling and İlker Hepkaner, unpublished. Arsever, Azamet (1985): “Açıklamalar / Explanations,” in Ali, Sabahattin: Kürk
Mantolu Madonna, İstanbul. Aytürk, İlker (2004): “Turkish Linguists against the West: The Origins of Linguistic
Nationalism in Atatürk’s Turkey,” in: Middle Eastern Studies (40/6), 1-25. Atsız, Nihal: “İçimizdeki Şeytanlar,” in: Ulu Türkçü Nihal Atsız Otağı: accessible at:
http://www.nihal-atsiz.com/yazi/icimizdeki-seytanlar-h-nihal-atsiz.html [Last
accessed: 30.11.2016]. Baer, Marc D. (2013): “Turk and Jew in Berlin: the First Turkish Migration to
Germany and the Shoah,” in: Comparative Studies in Society and History (55/2),
330-355. Bali Rifat N. (2013): Antisemitism and Conspiracy Theories in Turkey, İstanbul. Bazarkaya, Onur Kemal (2015): “Sabahattin Ali’s ‘Kürk Mantolu Madonna’ als
Hypertext von Leopold von Sacher-Masochs ‘Venus im Pelz’,” in: DİYALOG. Interkulturelle Zeitschrift für Germanistik 1, 42-56. Dickinson, Kristin (2013): “Translating Surfaces: A Dual Critique of Modernity in
Sabahattin Ali’s Kürk Mantolu Madonna,” in: Transit (9/1), 1-8. Dickinson, Kristin (2015): Translation and the Experience of Modernity: A History of
German Turkish Connectivity, unpublished dissertation, University of California,
Berkeley. Ertürk, Nergis (2008): “Modernity and its Fallen Languages: Tanpinar's ‘Hasret,’
Benjamin’s Melancholy,” in: PMLA: Publications Of The Modern Language
Association Of America (123/1), 41-56. Gramling, David / Hepkaner, İlker (forthcoming 2017): “Translating
Translinguality in Early Turkish Republican Literature: The Case of Sabahattin The Political behind the Fur Coat 79 Ali,” in: Woods, Michelle (ed.): Authorizing Translation: Literature, Theory and
Translation, New York, 32-46. Ali,” in: Woods, Michelle (ed.): Authorizing Translation: Literature, Theory and
Translation, New York, 32-46. Guttstadt, Corry (2013): Turkey, the Jews, and the Holocaust, Cambridge. Gürel, Emet / Yakın, Mehmet (2007): “Ekşi Sözlük: Postmodern Elektronik
Kültür,” in: Selçuk İletişim-Selçuk Üniversitesi İletişim Fakültesi Dergisi (4/4), 203-
219. Hepkaner, İlker (2013): Real Intellectuals: Discussions of Social Realism and State
Oppression in Turkey through Sabahattin Ali and Yılmaz Güney, unpublished MA
Thesis, The University of Arizona. Ihrig, Stefan (2014): Atatürk in the Nazi Imagination, Cambridge, MA. . Konuk, Kader (2010): East-West Mimesis: Auerbach in Turkey, Stanford. Kristeva, Julia (1980 [1969]). Desire in Language: A Semiotic Approach to Literature and
Art. New York. Works Cited Lewis, Geoffrey (1999): The Turkish Language Reform: A Catastrophic Success, Oxford Lotringer, Sylvère / Kraus, Chris (1989): “Foreword,” in: Sacher-Masoch, Leopold,
(1989 [1870]): Venus in Furs: a Novel; Letters of Leopold von Sacher-Masoch and
Emilie Mataja, New York. Lichtig, Toby (2016): “‘Madonna in a Fur Coat’, by Sabahattin Ali,” in: Financial
Times, 20.05.2016: accessible at: http://www.ft.com/intl/cms/s/2/dceea832-
1b74-11e6-b286-cddde55ca122.html [Last accessed: 22.05.2016]. Sacher-Masoch, Leopold, (1980 [1870]): Venus Im Pelz, Frankfurt am Main. Sacher-Masoch, Leopold, (1989 [1870]): Venus in Furs: a Novel; Letters of Leopold von
Sacher-Masoch and Emilie Mataja, New York. önmez, Sevengül (2013): Sabahattin Ali, Ankara. Sönmez, Sevengül (2013): Sabahattin Ali, Ankara. Tahir Gürçağlar, Şehnaz (2008): The Politics and Poetics of Translation in Turkey, 1923-
1960, Amsterdam. Tatarlı, İbrahim (1979): “Sabahattin Ali: Hayatı, Kişiliği ve Yaratıcılığına Genel Bir
Bakış/ Sabahattin Ali: A General Survey of His Life, Personality, and
Creativity,” in: Ali Laslo, Filiz / Özkırımlı, Atilla (eds.): Sabahattin Ali, İstanbul,
243-274. Between Languages: Translative Acts in Sabahattin
Ali’s Comprehensive Germanistan Travelogue Zeynep Seviner Renown for his works focusing on the social reality of his time, Sabahattin Ali’s care for language
often takes a second position to his political engagement in the scholarship. He was nonetheless
concerned with intricacies of language, which he knew to be an indispensable device for creating the
literary reality he has been praised for. One can find traces of this care throughout his life: his
service as a German language teacher, involvement in the First Turkish Publishing Congress
(Birinci Neşriyat Kongresi) of 1939, as well as in Hasan Ali Yücel’s Translation Bureau
(Tercüme Bürosu). This article focuses on the translative acts in Ali’s best-selling novel Madonna in a Fur Coat
(Kürk Mantolu Madonna), and posthumously published private letter entitled “Comprehensive
Germanistan Travelogue” (“Mufassal Cermenistan Seyahatnamesi”) to comprehend his stance
toward language and national identity in the wake of the Turkish language reform. While think-
ing of translation as a means to cultural transfer across modes of intelligibility, this approach
complicates binary attributes, such as old-new and local-foreign, thereby posing Ali’s engagement
with language and interlinguality as a way to tackle Ottoman literary past as well as concepts
such as humanism and worldliness that dominated the political discourse of the time. 82 Zeynep Seviner In Madonna in a Fur Coat (Kürk Mantolu Madonna, published in 1943, henceforth
Madonna), Sabahattin Ali introduces his protagonist Raif Efendi as follows: In Madonna in a Fur Coat (Kürk Mantolu Madonna, published in 1943, henceforth
Madonna), Sabahattin Ali introduces his protagonist Raif Efendi as follows: He was not exceptional by any means. He was, rather, one of those men
without any particularity of whom we see hundreds and pass by every day
without looking. There surely is nothing worth one's curiosity in the ap-
parent and hidden parts of their lives.1 Yet, Ali would go on to tell Raif’s life story as if peeling the layers of an onion,
painstakingly revealing a life story that is nothing short of extraordinary. His early
life in Germany, his talent for painting, and his passionate love affair with Maria
Puder, all discovered through an unauthorized reading of his private journal by the
impudent yet sympathetic narrator, stand in dramatic contrast with how the old
Raif appears to his immediate entourage, and with the tiresome mediocrity of his
unappreciative family. 1 The original reads: “Halbuki o hiç de fevkalade bir adam değildi. Hatta pek alelade, hiçbir hususiyeti
olmayan, her gün etrafımızda yüzlercesini görüp de bakmadan geçtiğimiz insanlardan biriydi.
Hayatının bildiğimiz ve bilmediğimiz taraflarında insana merak verecek bir cihet olmadığı muhak-
kaktı.” (Ali 2004: 11). All quotations from the novel are my translation.
2 Venuti remarks, “[t]here is even a group of pejorative neologisms designed to criticize translations
that lack fluency, but also used, more generally, to signify badly written prose: ‘translatese,’ ‘transla-
tionese,’ ‘translatorese’.” (Venuti 1995: 4) 3 The original reads: “Fakat bunu düşünürken yalnız o adamların dışlarına bakarız; onların da birer
kafaları, bunun içinde, isteseler de istemeseler de işlemeye mahkûm birer dimağları bulunduğunu,
bunun neticesi olarak kendilerine göre bir iç âlemleri olacağını hiç aklımıza getirmeyiz. Bu âlemin
tezahürlerini dışarı vermediklerine bakıp onların manen yaşamadıklarına hükmedecek yerde, en basit
bir beşer tecessüsü ile, bu meçhul âlemi merak etsek, belki hiç ummadığımız şeyler görmemiz,
beklemediğimiz zenginliklerle karşılaşmamız mümkün olur.” (Ali 2004: 11) Between Languages: Translative Acts in Sabahattin
Ali’s Comprehensive Germanistan Travelogue “the violence that resides in the very purpose and activity of translation:
the reconstitution of the foreign text in accordance with values, beliefs and
representations that preexist it in the target language, always configured in
hierarchies of dominance and marginality, always determining the produc-
tion, circulation, and reception of texts” (Venuti 1995: 18). This concealment is by no means inconsequential. The initial impression Raif the
translator makes as a blatant failure is symbolic of this error in judgment, which
the entire rest of the novel works very hard to undo. Similarly, translation as an act
of intercultural transfer maintains the right to such recognition, as Ali hints at
through Raif’s remarkably rich life story. The narrator gently warns us, even at the
very beginning, of the deceptiveness of external appearances: “[…] we only look at
the exteriority of these men we judge and never take into consideration that they
too have a head and a mind inside of it, one that works even when they don’t want
it to, and, as a result of all this, an internal world.” And he adds, “if we were to
become curious about this world […] and look into those that it does not easily
give away, we would perhaps see things we did not hope for, to encounter a kind
of wealth we did not expect.”3 For those who move past appearances, the act of translation reveals the hidden
worlds of both languages involved. It does so through the sense of estrangement it
creates when one looks at one’s own language (or a language with which one is
familiar) rendered in another language and realizes just how strange it looks now. It
is in the safekeeping of differences while attempting interlingual transfer that trans-
lation reveals the odd in the familiar. As such, the impact of translation is not just
in transferring one language into another and thus rendering the content intelligible
in the target language, but also in the alienating effect of hearing the well-known in
the foreign. Between Languages: Translative Acts in Sabahattin
Ali’s Comprehensive Germanistan Travelogue Raif Efendi is a character (the protagonist of the novel no less) who comes in-
to prominence through his apparent ordinariness, in what he hides rather than
what he reveals. No wonder that the original title Ali had intended for his novel
was Lüzumsuz Adam (unnecessary man) (Gürses 2013: 405). Noting the tentative
title that never materialized, Sabri Gürses observes that it reflects Ali’s anxiety in
the face of “the Western world, foreign languages and the act of translation”
(Gürses 2013: 408). According to Gürses, for Ali, the fear of winding up “the un-
necessary man” would mean becoming as invisible as Raif Efendi, and remaining a
provincial young boy who would never be taken seriously and whose work would
never be translated into other languages. Building on Gürses’s observation, I argue
that it is indeed no coincidence Raif Efendi is a translator, and that this choice on
the part of the author hints at a general observation on the act of translation, just
as much as it channels a personal frustration, one that engages the intensive policy
implementations at this time surrounding issues of language, translation, and cul-
ture by the young Turkish state. Raif’s invisibility as a translator echoes Lawrence Venuti’s milestone mono-
graph, The Translator’s Invisibility: A History of Translation, where he criticizes the
transparency inflicted upon the act of translation, which equates “successful trans-
lation” with “the absence of any linguistic or stylistic peculiarities [thus] giving the
appearance that it reflects the foreign writer’s personality or intention or the essen-
tial meaning of the foreign text” (Venuti 1995: 4).2 Objecting to Norman Shapiro’s Between Languages: Translative Acts in Ali’s Comprehensive Germanistan Travelogue 83 perception of an ideal translation as “a pane of glass,” only noticed “when there
are little imperfections—scratches, bubbles,” Venuti argues that such idealized
transparency dangerously conceals “the violence that resides in the very purpose and activity of translation:
the reconstitution of the foreign text in accordance with values, beliefs and
representations that preexist it in the target language, always configured in
hierarchies of dominance and marginality, always determining the produc-
tion, circulation, and reception of texts” (Venuti 1995: 18). Between Languages: Translative Acts in Sabahattin
Ali’s Comprehensive Germanistan Travelogue To explain this phenomenon, Venuti argues that [t]he translator is the secret master of the difference of languages, a differ-
ence he is not out to abolish, but rather one he puts to use as he brings vi-
olent or subtle changes to bear on his own language, thus awakening with-
in it the presence of that which is at origin different in the original (Venuti
1995: 307). 84 Zeynep Seviner By thus unveiling the structural violence embedded in the act itself, translation
invites attentive readers to look for differences while reflecting on the reasons why
these differences exist and what they can make of them in order to understand
what they had previously thought to be their very own. Looking at Sabahattin Ali’s life through the lens of translation reveals remark-
able similarities with that of his protagonist Raif Efendi. They both spend some
time in Germany in their early youth (Ali in Potsdam and Raif Efendi in Berlin),
learn the German language, and work as translators back home. More interesting,
however, is the fact that Raif’s invisibility is matched by a lack of emphasis in the
scholarship on the ‘translational’ aspect of Ali’s work, which is instead referred to
as a strong example of the social realist movement in Turkey. Yet, Ali’s entire ca-
reer is immersed in a period of intensive linguistic engineering orchestrated by the
young Turkish nation-state of the 1920s and 30s, the most dramatic being the swift
Alphabet Reform of 1928, which sought to craft a new communicative medium
for the new republic and distance its cultural trajectory from its Ottoman predeces-
sor. Having returned from Germany in the wake of the reform, Ali took an active
part in this process. He participated in the First Turkish Publishing Congress (Bi-
rinci Neşriyat Kongresi) of 1939 and became one of the seven founding members of
the newly-founded Translation Bureau (Tercüme Bürosu), where he both translated
literary works and wrote theoretical pieces on translation for the Bureau’s periodi-
cal, Tercüme4. During his stay in Potsdam to learn the German language on a government fel-
lowship in 1928-9, Ali had already begun to reflect on how translation can be a
useful tool to grasp the centrality of language as a site of conflict between oppos-
ing interests, particularly during periods of fast-paced transformation. 4 Other members were Nurullah Ataç (chairman), Saffet Pala (secretary general), Sabahattin
Eyüboğlu, Bedrettin Tuncel, Enver Ziya Karal and Nusret Hızır (Tahir Gürçağlar 2008: 70).
5 The letter was never published until 1979 when it appeared in Sabahattin Ali (edited by Filiz Ali
Laslo and Atilla Özkırımlı). Between Languages: Translative Acts in Sabahattin
Ali’s Comprehensive Germanistan Travelogue One exam-
ple of this is a private letter he sent from Potsdam to Pertev Naili Boratav and a
number of his close friends, including Orhan Şaik Gökyay, Ekrem Reşit Rey, and
Nihal Atsız.5 Entitled Mufassal Cermenistan Seyahatnamesi (Comprehensive German-
istan Travelogue, henceforth Seyahatname), and written less than a year after the
Alphabet Reform of 1928, this letter is a strangely humorous amalgam of the tradi-
tional seyahatname (travelogue) form with its old-fashioned expressive spectrum,
and the ‘indecent’ content Ali provides, depicting an alcohol-ridden New Year’s
party in Potsdam. This provocative mismatch invites the reader to pay attention to
the connections between language / form and content. g
g
Through a close reading of this letter, I propose that Ali’s trip to Germany and
engagement with the German language and culture were vital to this reflection on
language and translation, one that ultimately sheds light both on the significance of
the act of translation (thus making it ‘visible again’) and on the linguistic reforms Between Languages: Translative Acts in Ali’s Comprehensive Germanistan Travelogue 85 currently underway in his home country, thus emphasizing the connection between
language and politics. The effect of estrangement created by translation parallels
and reinforces the physical displacement of the author, his move away from home
in order to be able to look at it with a fresh set of eyes. To use Emily Apter’s term,
remaining in a “translation zone” (Apter 2006) where more than one language
factors in the fulfillment of his quotidian needs enables him to establish different
and conflicting ways of perceiving the outside world. g
y
p
g
In making this argument, I embrace a more inclusive definition of the term
‘translation,’ one to which Walter G. Andrews draws attention when he confesses
that the more he translates, the more confused he gets on what translation really
means: “The result—quite strangely—has been that I am no longer confident that
I know what it means to translate,” he ponders, “or more accurately, where transla-
tion ends and something else begins” (Andrews 2002: 15). Translation is then best
described as a spectrum of activities which serves the general purpose of rendering
a set of ideas or information from one to another system of intelligibility. Between Languages: Translative Acts in Sabahattin
Ali’s Comprehensive Germanistan Travelogue In a simi-
lar vein, Even-Zohar defines translation as the movement of meaning between
cultural repertoires, which are by no means “generated by our genes, but need to
be made, learned and adopted by people, that is, members of the group” (Even-
Zohar 1997: 378). This looser and larger definition does justice to the centrality of
the act to everyone’s daily operations and also, as Emily Apter argues, renders
“self-knowledge foreign to itself,” and thus serves as “a way of denaturalizing citi-
zens, taking them out of the comfort zone of national space, daily ritual, and pre-
given domestic arrangements” (Apter 2006: 6). Ali’s Seyahatname, a letter of humor-
ous self-mockery addressed to a close group of friends, engages with issues of
estrangement, foreignness and denaturalization through translation, right at the
historical juncture between the Ottoman Empire and the young Turkish Republic,
and seeks to make translation visible anew during a period in which it is so central,
it cannot afford to be overlooked. Toward an Alternative History of Translation Secondary scholarship on the translation bureau has largely treated the systema-
tized form of publishing it supported as an instance of culture planning, defined as
the “deliberate intervention,” either by power holders or by ‘free agents,’ “into an
extant or a crystallizing repertoire” (Even-Zohar 2008: 278). In her analysis of
private publishing efforts in the 1940s and 50s, Şehnaz Tahir Gürçağlar emphasiz-
es the need to view translation efforts of this time period not simply as processes
shaped by pre-determined norms, but also as the result of individual decision-
making (Tahir-Gürçağlar 2008: 24-25). In doing so, she takes a step back from the
institutional framework of the bureau to show how private publishers and
translators formed an important counterforce that “resisted the norms offered by
the dominant discourse of the planners” (Tahir-Gürçağlar 2008: 31). Building on
this argument, I ask further: to what extent did voices coming from within the Zeynep Seviner 86 bureau resist dominant translation discourses and norms? As an author who
critically engaged with the shortcomings of modernity, and the conception of mo-
dernity as a monolithic or Western discourse, Sabahattin Ali is a case in point. y
p
Sabri Gürses situates Ali within a larger cultural search for a delineated
program of translation in the early Republic; as such, Gürses views Ali as central to
the formation of an intellectual discourse that utilized translation—understood as a
form of cultural transfer (kültür aktarımı)—as a means for constructing a national
Turkish culture (Gürses 2013: 414). This discourse was dependent on the rhetoric
of smooth translatability, which posited a stable category of Western European
ideals and values that could be easily transferred to the Turkish context. I argue, on
the contrary, that we must read Ali’s participation in the larger translation move-
ment as a counterpoint to the concepts of cultural transfer and smooth translata-
bility that emerged in the immediate wake of the translation bureau’s founding. While Ali enthusiastically supported the translation of Western European
literatures, his writing also reveals a more ambivalent view of the ‘West’ than
dominant translation discourse of the time. By pointing time and again to the
contradictions and inconsistencies of Western modernity, his work both
complicated the very possibility of translation as a form of frictionless transfer and
constituted an important countervoice to the kind of Europhilia endorsed at the
time. 7 The original reads: “Bu çanların akabinde mesakin, ticaretgah makûlesi yerlerin cümlesinin babları
küşade edilüp Kasaba-i kebirenin zükur u nisa bütün ta’fesi nim-üryan, eşkal-i garibede, mest-i şarab
olarak huruc eyledi. Aciz de, adab-ı Frenge kesb-i vukûf ile tezyid-i ma’lumat eylemek arzuy-i ulvisiyle
bu güruha iltihâk eyleyüp esvâk -ı bi-nihayede geşt ü güzara mübaşeret eyledim. Amma bir manzara ki
Yevm-i Kıyamet misal... Ve lakin bir duhterler dolaşur idi ki insan bu manzara-i Kıyameti Cennet
zann eylemekten kendini men’ eyleyemez...” (Ali 1979: 352) All English language citations are from
Dickinson and Seviner’s translation, which appears in this volume of Jahrbuch Türkisch-deutsche Studien
as The Comprehensive Germanistan Travelogue. 6 The original reads: “Burası da en nihayet bir şehirdi. Sokakları biraz daha geniş, çok daha temiz,
insanları daha sarışın bir şehir. Fakat ortada insanı hayretinden düşüp bayılmaya sevk edecek bir şey
de yoktu.” (Ali 2004: 53) For more on the representation of urban space in this novel, see Dickinson
(2013). An Oddity of Language: Ali’s Seyahatname An Oddity of Language: Ali’s Seyahatname In this text intended as a private letter, Ali narrates his trip from Istanbul to Pots-
dam and his first days as a visiting student there. Written in riq’a (a prominent
handwriting style for personal correspondence) the year following the Alphabet
Reform, when the utilization of the Arabic script was prohibited in all public doc-
uments and generally stigmatized, the letter plays with the notion of translation on
two complementary levels in order to reveal what its author seeks to accomplish:
first, there is the intralingual and diachronic play with language in which Ali uses
the seventeenth-century Ottoman language of the seyahatname genre to talk about a
New Year’s party in 1929, when this communicative system is quickly becoming
unintelligible to native speakers of Turkish; second, there is the interlingual and
synchronic play with language in which Ali speaks of a celebration taking place in
Germany, far outside the geographic realm of the language he is using, and of ritu-
als that are not readily intelligible to those who have not traveled west of the new
nation-state’s borders. In other words, this letter displays three important cultural-
linguistic elements; Ottoman Turkish, the main means of communication; Ger-
man, the main supplier of content (i. e. the tradition of New Year’s parties); and
modern Turkish, a language under construction at the time, and the supposed
means of communication between the writer and his readers, which is present in
its unexpected absence. Between Languages: Translative Acts in Ali’s Comprehensive Germanistan Travelogue 87 As I show below, Ali creates an estrangement effect through two major tech-
niques. First, he shows how language has a direct impact on the nature of the given
message, or how when the language itself changes, the content changes, even when
the same person talks about the same subject, inviting readers to reflect on how
their own native tongue plays an active role in their perception of the world. Sec-
ond, he treats German proper nouns as if they were made of Turkish subparts,
equating signifiers of one language with those of another (unrelated) one. An “Ottoman man” in Potsdam: Ali’s Encounter with Exotic Germany An “Ottoman man” in Potsdam: Ali’s Encounter with Exotic Germany An “Ottoman man” in Potsdam: Ali’s Encounter with Exotic Germany As mentioned earlier, by adopting a certain set of lexical preferences that are
deemed ‘old-fashioned’ and, thus‚ ‘foreign’ to the contemporary language, Ali
demonstrates how a linguistic shift to express ideas transforms those very ideas. This becomes manifest through a comparison of descriptions of the German ur-
ban landscape in Seyahatname and Madonna: while Raif Efendi’s Berlin is not so
different from any other big city, Sabahattin Ali’s Potsdam is nothing but strange
and alien. Raif Efendi observes, “[u]ltimately, this was just another city. A city with
wider streets—much cleaner, and with blonder people. But there was nothing
about it that would make a person swoon with awe.”6 This stands in stark contrast
with Ali’s description of his personal experience in Potsdam. Following the church bells, all stores opened their doors and the intoxicat-
ed inhabitants of this grand town - both men and women - went out half-
naked, sporting peculiar outfits. This humble servant soon joined the
crowd and began to wander along the long string of shops, with the noble
intention of acquainting himself with European manners and advancing
his knowledge of the subject. What a scenery it was, akin to Judgment
Day…And yet the girls walking around would make one think it were Par-
adise instead...7 Lusting after two beautiful girls he met in the crowd, and clearly enjoying the party
where young people are dancing and celebrating the new year, Ali nonetheless
chooses not to betray the stylistic framework of his letter and legitimizes his be- Lusting after two beautiful girls he met in the crowd, and clearly enjoying the party
where young people are dancing and celebrating the new year, Ali nonetheless
chooses not to betray the stylistic framework of his letter and legitimizes his be- Zeynep Seviner 88 friending the German girls in terms of a Muslim’s duty to show them ‘the right
path.’ He narrates, friending the German girls in terms of a Muslim’s duty to show them ‘the right
path.’ He narrates, [t]his spectacle offended my Islamic zeal; I wanted to help make the hearts
of these girls as pure and illuminated (enlightened) as their beautiful faces,
filling them with Islamic wisdom and moral values. 8 The original reads: “Bu manzara hamiyyet-i islamiyyeme dokunup bu duhterlerin vech-i dilberleri
gibi kalblerini de pak, münevver eylemek, hikmet-i islamiyye, faza’il-i ahlakıyye ile memlu kılmak
isteyüp irşada muvafık kimesne taharri eylemekte idim ki canib-i yesarımda andelib misal bir sedanın
‘sene-i cedide mübarek ola!’ me’alinde bir cümle sarf ettiğini guş eyledim.” (Ali 1979: 353)
9 The original reads: “Bunları hep ol bi-dinleri din-i hakka imale içün yaptığımı düşündükçe bir hayli
müsterih olurdum. […] Amma te’essürüm füzun olmadı, çünkü din-i mübin-i İslam içün bu denlü
cihadlar yapılup kahramanlıklar gösterilür, canlar erzan kılınurken bir müddet ayazda beklemekle, bir
mıkdar tekeffür etmekten ibaret olan benim gazam tabii ha’iz-i ehemmiyet olamazdı.” (Ali 1979: 355) 8 The original reads: “Bu manzara hamiyyet-i islamiyyeme dokunup bu duhterlerin vech-i dilberleri
gibi kalblerini de pak, münevver eylemek, hikmet-i islamiyye, faza’il-i ahlakıyye ile memlu kılmak
isteyüp irşada muvafık kimesne taharri eylemekte idim ki canib-i yesarımda andelib misal bir sedanın
‘sene-i cedide mübarek ola!’ me’alinde bir cümle sarf ettiğini guş eyledim.” (Ali 1979: 353)
9 The original reads: “Bunları hep ol bi-dinleri din-i hakka imale içün yaptığımı düşündükçe bir hayli
ü t ih
l
d
[
] A
t ’
ü ü
fü
l
d
ü kü di
i
übi
i İ l
i ü
b
d
lü sene-i cedide mübarek ola! me alinde bir cümle sarf ettiğini guş eyledim. (Ali 1979: 353)
9 The original reads: “Bunları hep ol bi-dinleri din-i hakka imale içün yaptığımı düşündükçe bir hayli
müsterih olurdum. […] Amma te’essürüm füzun olmadı, çünkü din-i mübin-i İslam içün bu denlü
cihadlar yapılup kahramanlıklar gösterilür, canlar erzan kılınurken bir müddet ayazda beklemekle, bir
mıkdar tekeffür etmekten ibaret olan benim gazam tabii ha’iz-i ehemmiyet olamazdı.” (Ali 1979: 355) An “Ottoman man” in Potsdam: Ali’s Encounter with Exotic Germany I was just searching for
an appropriate candidate for this mission when I heard a nightingale-like
voice call forth something that implied ‘Blessed New Year!’ on my left
side.8 One can trace this narrative style, illustrating, with humorous irony, the connection
between form and content, and between language and meaning, all the way to the
end of his little story when he ends up kissing one of the girls and setting up a date
with them for the following day in a Catholic Church, of all places. g
y
p
The deliberate mismatch between his language-discourse and his acts (there-
fore, between the narrative style and content) becomes even stronger when the Ali
walks into the church the next day. Unable to find the girls and guessing that he
has been stood up, Ali nevertheless remains in the church and fully participates in
the religious ceremony, crossing himself, kneeling down and praying to Jesus, and
singing along with the crowd. “I felt very pleased thinking that I was doing all this
to lead these blasphemers into the path of true religion,” he explains, and his joy is
not diminished even when he confirms that the girls are not there: “my gaza, which
consisted of waiting in the cold and being involved in a few blasphemous acts, was
nothing compared to that of others, who have displayed acts of heroism and given
their lives in the name of jihad and the true religion of Islam.”9 The seyahatname form adopted in this letter is certainly a central element in the
production of estrangement, in that it freezes the narrative time somewhere in or
around the seventeenth century, while the content clearly belongs to another era. It
precludes any other way of accounting for the events other than that which the
form allows, and determines the way the author grasps the world around him. An “Ottoman man” in Potsdam: Ali’s Encounter with Exotic Germany The
rules of the narrative were set at the very beginning, when Ali describes his journey
through the Balkans in a mocking, yet nostalgic tribute to the newly deceased em-
pire, the antagonist par excellence of the republic: “as the train moved forward,”
he writes, “places like Plovdiv that used to be filled with Muslims made us remi- Between Languages: Translative Acts in Ali’s Comprehensive Germanistan Travelogue 89 nisce about the old Ottoman state with longing.”10 As his current-day experiences
are translated into historical narrative, the equivalence between the absent modern
text and the present pre-modern one is deliberately distorted to make the reader
aware of the historical gap between the real time of experience and the ‘fake’ time
of narration.11 In other words, the ‘translation’ of his experiences into a historical
genre in Ottoman Turkish exposes and challenges the ‘function’ of translation,
defined as “the potentiality of the translated text to release diverse effects, begin-
ning with the communication of information and the production of a response
comparable to the one produced by the foreign text in its own culture” (Venuti
2004: 5). Of course, here, Ali aims for the opposite effect, and uses the texts to
show how he perceives and reflects events differently, merely because he operates
in a different historical register of the same language.12 g
g
g
This deliberate play with the act of translation as a means to reveal the often
concealed centrality of language in the perception of reality is in line with how
translation was perceived in Ali’s own time, the first decades of the twentieth cen-
tury. Rooted in German Romanticism, hermeneutics and existential phenomenolo-
gy, translation theorists and practitioners thought of language as constitutive of
reality and translation as reconstituting the reality of the original text in the target
language. In “The Task of the Translator” (1923), for example, Walter Benjamin
speaks of the “afterlife” (Überleben) of the foreign text in the target language, a new
life informed by how the text is perceived in the translated version, or, in other
words, as part of a different system of intelligibility (Benjamin 2004: 16). Similarly,
in the 1930s, José Ortega y Gasset argues that translation is “a literary genre apart,
with its own norms and its own ends” (Ortega y Gasset 1992: 109). 10 The original reads: “Tiren ilerüledikte Filibe ve saire gibi bir vakıtlar Müsliman ile memlu olan kebir
şehirler Devlet-i kadime-i Osmaniyyeyi yad-ı tahassürümüze getirmekte idi.” (Ali 1979: 349)
11 Venuti explains, “Equivalence has been understood as “accuracy,” “adequacy,” “correctness,”
“correspondence,” “fidelity,” or “identity”; it is a variable notion of how the translation is connected
to the foreign text. Function has been understood as the potentiality of the translated text to release
diverse effects, beginning with the communication of information and the production of a response
comparable to the one produced by the foreign text in its own culture” (Venuti 2004: 5).
12 In the subject of intralingual translation, Jakobson points out, for example, that “synonymy, as a
rule, is not complete equivalence: for example, every celibate is a bachelor, but not every bachelor is a
celibate” (Jakobson 2004: 114). This is certainly more marked in the difference between the dia-
chronic versions of the same language. p
p
y
g
(
)
12 In the subject of intralingual translation, Jakobson points out, for example, that “synonymy, as a
rule, is not complete equivalence: for example, every celibate is a bachelor, but not every bachelor is a
celibate” (Jakobson 2004: 114). This is certainly more marked in the difference between the dia-
chronic versions of the same language. The original reads: “Tiren ilerüledikte Filibe ve saire gibi bir vakıtlar Müsliman ile memlu olan keb
ehirler Devlet-i kadime-i Osmaniyyeyi yad-ı tahassürümüze getirmekte idi.” (Ali 1979: 349) Potsdam or “O Mother Mary, hide the idols!”: Ali’s Play on Words Potsdam or “O Mother Mary, hide the idols!”: Ali’s Play on Words Ali prefaces his first descriptions of Germany and German people with the defini-
tion of the word ‘Potsdam’ where he was to stay for the upcoming months; he
explains that the word is made up of three separate words (in Ottoman Turkish):
“put,” “sedd,” and “üm,” the first meaning “images and idols Christians (infidels)
worship,” the second, “to conceal, to hide,” and the third, “mother.” He con-
cludes, “All together, by means of cubism, it means: ‘O Mother Mary, cover the
idols!’ This meaningful wish is indeed granted, as the eternal God covers the
aforementioned city with a coat of white snow, concealing all statues in public
parks and gardens.”13 Taken to the extreme, this phonocentric literalism causes the
interlingual transfer to end in absurdity, again rendering the act more visible than it
would have been otherwise. Remarking on the preservation of original spelling for foreign proper nouns in
modern (latinized) Turkish, translation scholar Şehnaz Tahir Gürçağlar argues that
the alienating effect of this decision “is a strictly ‘foreignizing’ strategy, interfering
with the text’s fluency, […] inviting the reader to ponder it as a mediated work, i. e. translation” (Tahir Gürçağlar 2008: 204). Such moves indeed carried remarkable
strategic weight during this period of intense linguistic transformation, as the new
Turkish state needed to firmly (re)define what was considered ‘foreign’ in order to
understand what ‘national’ meant. During the early 1930s, right on the wake of
Ali’s return from Germany, the level of linguistic experimentation seems to have
reached an all-time high; on July 12, 1932, Mustafa Kemal founded the Society for
the Study of Turkish Language (Türk Dili Tetkik Cemiyeti; later, Türk Dil Kurumu,
henceforth TDK), an establishment that intensively engaged in creating neolo-
gisms in ensuing years in an attempt to ‘clean’ the Turkish language of foreign
(particularly Arabic, Persian and French) influence. Native speakers of the language expectedly joined in this process of re-
creation, albeit in unofficial terms, sometimes to take the initiative to judge the
effectiveness of the new words, and other times to mock some of the official deci-
sions. 13 The original reads: “Potsdam kelimesi, iştikâkıyyun -ı zamandan Hayrullah Molla Beyin tefsiri üzere
‘Put’, ‘sedd’, ‘üm’ kelimelerinden mürekkeb olup, ‘Put’, ma’lum olduğu üzere kena’is-i Küffarda
mevcud Hıristos tasaviri ile heyakil makuulesi esnamdır; ‘sedd’, kapamak, örtmek, setr eylemek;
‘Ümm’, valide, burada Meryem Ana manasınadır. Cümlesi toplu olarak, kübizm üzere, ‘Meryem
Valide, esnamı setr eyle!’ demek olur. Bu temenni’-i pür ma’na elhak müstecab olup Huda-yı lem yezel
medine-i mezkureyi bir setre-i sefid-i berf ile ruz u şeb setr eylemekte ve bağ u besatin-i miriyyede
mevcud bilcümle heyakil dahi örtülmektedir.” (Ali 1979: 351) An “Ottoman man” in Potsdam: Ali’s Encounter with Exotic Germany As a result,
translators engaged in meta-translative acts, such as translation through literalisms,
aiming to render translated text foreign to its target language, in order to empha-
size both the act itself and the qualities of the original language (Venuti 2004: 11). In fact, the second way in which Ali creates linguistic estrangement in Seyahatname
has to do with a play on literalism, defining a German proper noun in Ottoman
Turkish. Zeynep Seviner 90 Potsdam or “O Mother Mary, hide the idols!”: Ali’s Play on Words In The Turkish Language Reform: A Catastrophic Success, Geoffrey Lewis com-
ments on public engagement during reformative moments and points out how it
has, at times, been a disruptive force against the nation-building project, since peo-
ple cared more about the utility of words than their historicity: Between Languages: Translative Acts in Ali’s Comprehensive Germanistan Travelogue 91 Although people with a feeling for language may not have liked the new
words,” they soon found themselves obliged to use at any rate some of
them if they wished to communicate. But […] few knew or cared anything
about the origins of the words they used, which is why one hears bölgevî
for ‘regional’ and önemiyyet for ‘importance’, both being Öztürkçe [pure-
Turkish] words with Arabic suffixes (Lewis 2002: 144). This tendency continued well into the later years of the republic; for example, dur-
ing the 1960s, TDK had to issue a disclaimer for some of the humorous public
coinages, among which were “gök konuksal avrat” (sky guest-ish dame) for “uçak
hostesi” (air hostess), “öz ittirimli götürgeç” (self-propulsional carry-thing) for
“otomobil” (automobile), and “ulusal düttürü” (clannish ditty) for “milli marş”
(national anthem) (Lewis 2002: 160). The disclaimer suggests that those witty public suggestions were taken to be
official by some, pointing to the experimental nature of the process. Lewis com-
ments that “[t]he reason why unsophisticated people thought these were genuine
TDK products is that they found them no different in kind from some of the So-
ciety’s own creations; how can one tell that a grotesque parody is a parody when
the original is itself grotesque?” (Lewis 2002: 160) The early 1930s constitute a
peak time for this type of neological activity, in an attempt to quickly, yet effective-
ly engineer a new language that would reflect the spirit of the new republic. As
many cultural historians of the early republic point out, the discourse on the lin-
guistic reform emphasized an essential, yet dormant national core that needed to
be rediscovered as it was to be an indispensable element of the emergent nation-
state. Yet, despite the emphasis on the archaeological nature of the reform, this
linguistic project was very much a feat of engineering to create an epistemological
revolution, one to which Ali humorously draws attention as he ‘disguises’ himself
as an Ottoman man through his play with linguistic conventions in flux. Potsdam or “O Mother Mary, hide the idols!”: Ali’s Play on Words If, in the words of Walter Andrews, “it is actually impossible to translate,
transmit, or disseminate between epistemic domains until words and meanings can
be assumed to exist in the target language, which embody the ‘unthought’ assump-
tions of the source language,” Ali’s letter manages to expose the contemporary
epistemological shift by staging an awkward turn in narrative style, from 1930s
informal language to seventeenth century Ottoman travel writing (Andrews 2002:
27). Like the neologisms that did not stick, his linguistic choices sound comical
because they do not reflect the conventions of the time; yet they also create the
effect of alienation that jolts the reader into the realization of the historicity of the
moment in which they live. Zeynep Seviner 92 Turkey in the 1930s: Debates on Humanism and the Birth of Comparative
Literature Turkey in the 1930s: Debates on Humanism and the Birth of Comparative
Literature Why should anyone care about a personal letter, one might ask, especially consid-
ering that it was not in any way intended for publication to begin with? Though
unpublished until the late 1970s, Ali’s humorous Seyahatname speaks to an im-
portant issue of the era, one that fuels the foundation of the TDK in 1932 and of
Hasan Ali Yücel’s Translation Bureau (Tercüme Bürosu) in the early 1940s, the prep-
aration of various dictionaries, grammar books, attempts at vocabulary collection
from various registers of the language, and so on: the question of, ‘Who are we,
really?’ This question was central to the first few years of the new republic; in fact,
in the first issue of the magazine İnsan (Human) published in 1938, editor-in-chief
Hilmi Ziya Ülken conceived of the entire idea of a ‘Turkish Renaissance’ around
it. Ülken argued that “[t]oday, we are engaged in a Renaissance in the truest sense:
We are joining the world anew. Western methods will guide us in re-discovering our-
selves” (Tahir Gürçağlar 2008: 64). Here, as in the state policy in general, “re-discovering ourselves” implied a path
towards self-discovery (or, the discovery of the national identity) through westerni-
zation. Seemingly counter-intuitive, this stance was nonetheless in line with the
attempts to become a part of the European civilization by way of discovering a
sense of common humanity through works of art, and, thereby, to override what
they perceived as the casting of the European as the civilizational ‘Other’. Potsdam or “O Mother Mary, hide the idols!”: Ali’s Play on Words In the
words of Hasan Ali Yücel speaking in the late 1930s, “one nation’s repetition of
other nations’ literature in its own language, in other words, in its own understand-
ing means raising, reviving and recreating its intelligence and ability to understand
accordingly” (Berk 2004: 154). Therein stood the gist of the linguistic reforms that
highly emphasized the act of translation. Not surprisingly, Hilmi Ziya Ülken stated
in 1935 that “he expected translation activity to lead to westernization,” and İsmail
Habib Sevük, that “the way to become ‘fully European’ went, not through learning
foreign languages, but through translation.” To Sevük, translation was “a great
ideal, the mission of all missions, our greatest flag” (Tahir Gürçağlar 2008: 101). g
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It follows from this deliberate emphasis placed on translation, that translators
were the very agents shaping the Turkish language anew. This, of course, is not an
exclusively modern phenomenon. Writing about premodern literary traditions in
Turkish, Zehra Toska calls attention to how “[t]ranslating scientific and literary
works from such sophisticated languages as Arabic and Persian, using patterns of
expression in oral Turkish literature, was evidently difficult,” and that thus “[t]he
translators were at the same time the creators of the language of Turkish literature”
(Toska 2002: 62). A twentieth century instance of the same phenomenon is mani-
fest in the words of Ali’s contemporary and colleague Azra Erhat, who notes that
“this was a period when Turkish had not yet been established properly,” and that
she did not have any problem understanding the notes she was given in German; Between Languages: Translative Acts in Ali’s Comprehensive Germanistan Travelogue 93 the major problem to her was translating them into Turkish, with the appropriate
terms still non-existent (Berk 2004: 142). At this historical juncture, Ali’s Seyahatname becomes all the more important in
that it provokes one to reflect on where Ottoman literary language stands in rela-
tion to the process of linguistic reengineering. In an attempt to create a Kulturspra-
che that would reflect the expressional habits of the majority of its speakers and
that would thus be an essential part of this self-rediscovery, the pre-republican literary
language (in all its temporal and stylistic variations) was cast as a lingua non grata, an
artificial, imitative and inauthentic entity that had been hiding a dormant essence. 14 Some of the other scholars situated in Turkey at the time were mythographer Georges Dumézil,
Leon Trotsky, Romanist Traugott Fuchs, philosopher Hans Reichenbach, philologist Georg Rohde,
Carl Ebert, and Henri Prost (Apter 2006: 51). Potsdam or “O Mother Mary, hide the idols!”: Ali’s Play on Words As a result, most of the Ottoman literary heritage (including most recent texts) was
left ‘untranslated.’ But it also meant that the system of education had to go through
a dramatic process of ‘translation’ from the old into the new Turkish. Ali’s genera-
tion, as the young adults of the Alphabet Reform, was particularly caught in the
middle of this transition. The perils of this forced linguistic change were foreseen
by some dissident voices, such as Hüseyin Cahit Yalçın, who advocated allowing
the language to follow its own natural dynamics, or Refi’ Cevad Ulunay, who ar-
gued that eastern classics, which influenced Ottoman literary culture, should also
be translated into Turkish (Tahir Gürçağlar 2008: 58, 120). During the 1930s, the questions ‘Who are we?’ and ‘Which texts would help us
find ourselves?’ were by no means particular to the young Turkish state. As Kader
Konuk extensively elaborates in her book East West Mimesis: Auerbach in Turkey,
prominent Jewish scholars escaping the Nazi regime, including Erich Auerbach
and Leo Spitzer, took refuge in Turkey, forming sizable communities in its major
urban centers.14 Interestingly, Turkey served as a space for them where values of
western humanism could be salvaged from a war-torn Europe and constructed
anew. As Apter observes, [a] fascinating two-way collision occurred in Istanbul between a new na-
tion’s ideology dedicated to constructing a modern Turkish identity with
the latest European pedagogies, and an ideology of European culture ded-
icated to preserving ideals of Western humanism against the ravages of na-
tionalism (Apter 2006: 50). [a] fascinating two-way collision occurred in Istanbul between a new na-
tion’s ideology dedicated to constructing a modern Turkish identity with
the latest European pedagogies, and an ideology of European culture ded-
icated to preserving ideals of Western humanism against the ravages of na-
tionalism (Apter 2006: 50). Swiss pedagogue Albert Malche introduced a supplementary course on compara-
tive literary studies into the university curriculum to encourage the establishment
of humanistic thinking, Spitzer wrote an article on “Learning Turkish” in 1934,
complicating the paradigms of linguistic (and thus literary) comparison and illus-
trating “how linguistic estrangement becomes a way of negotiating the experience
of deportation, of emigration, and of the foreignness of adoptive cultures,”
(Spitzer 2011: 765) and Auerbach, a Romanist by training, wrote Mimesis: The Repre- 94 Zeynep Seviner sentation of Reality in Western Literature (1946), a founding text of comparative litera-
ture. Potsdam or “O Mother Mary, hide the idols!”: Ali’s Play on Words In fact, as many have pointed out, Auerbach and Spitzer have come to be
seen as the founding fathers of this field, in no small part due to the work they
produced and their émigré experiences in Turkey. sentation of Reality in Western Literature (1946), a founding text of comparative litera-
ture. In fact, as many have pointed out, Auerbach and Spitzer have come to be
seen as the founding fathers of this field, in no small part due to the work they
produced and their émigré experiences in Turkey. What Spitzer, Auerbach and Ali had in common was that they were located in
a ‘translation zone,’ in-between languages and (thus) epistemological spheres, al-
lowing them to question the operative principles of the world they lived in: while
Auerbach and Spitzer had to escape their native Europe to recreate, ironically, the
values of western humanism within the borders of a budding nation-state, Ali trav-
eled to Germany to receive education in a European language and culture with an
aim, at least on the part of the financing institutions, to help the nation-building
project. All parties had, albeit reluctantly at first, a chance to position themselves
in-between, to learn a different language, to become estranged from their home
cultures and to reevaluate it ‘from the outside.’ Self-discovery through comparison
lies at the heart of their experiences, as it does at the center of comparative litera-
ture. As Apter states, “[i]n naming a transnational process constitutive of its disci-
plinary nomination comparative literature breaks the isomorphic fit between the
name of a nation and the name of a language” (Apter 2006: 243). An existence in
the ‘translation zone’ is certainly more likely when one is ‘in exile,’ that is, physical-
ly separated from one’s native tongue. Conclusion: Translation’s Successes, Failures, and the Tragic Ending to
Raif’s Love Story Conclusion: Translation’s Successes, Failures, and the Tragic Ending to
Raif’s Love Story The impact of translation as a cross-epistemological act is larger than it immediate-
ly seems, incorporating the relationship not only between Turkish and western
languages but also between old and new registers of the same language, as expres-
sional possibilities radically differed among the last two as well. The act of transla-
tion entails a re-arranging of the target language to create the necessary epistemic
domains that provide a destination for ‘foreign’ notions. In Seyahatname, Ali illus-
trates that this process is not smooth by any means, and possibly requires physical
and temporal displacement on the part of the translator outside of and away from
her/himself. A number of scholars have already pointed to both the difficulty and
the potential of transcending boundaries and moving into other linguistic spaces,
among whom are Emily Apter, who argues that “[t]ranslation failure demarcates
intersubjective limits, even as it highlights that ‘eureka’ spot where consciousness
crosses over to a rough zone of equivalency,” (Apter 2006: 6) and Johann Wolf-
gang von Goethe, who states that “[t]he translator who attaches himself closely to
his original more or less abandons the originality of his nation, and so a third
comes into existence, and the taste of the multitude must first be shaped towards
it” (cited in Venuti 1995: 99). The act of translation as interlingual movement is
thus also an agent that works even, at times, against the nation and the national
language. As Benedict Anderson observes, “seen as both a historical fatality and as Between Languages: Translative Acts in Ali’s Comprehensive Germanistan Travelogue 95 a community imagined through language, the nation presents itself as simultane-
ously open and closed,” since “language is not an instrument of exclusion: in prin-
ciple, anyone can learn any language” (Anderson 1991: 134, 146). p
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Despite the central role of translation as a cognitive agent that builds commu-
nities while, at the same time, constituting the amount of intercommunal porosity
that they allow, some of the best translations are often deemed to be among the
most invisible. Nothing could be further from the truth; processes of translation
thus need to be exposed, and Ali suggests this precisely that in both Seyahatname
and Madonna. Conclusion: Translation’s Successes, Failures, and the Tragic Ending to
Raif’s Love Story In that respect, one should see these as texts of meta-translation,
illuminating how languages are, despite their kinship, historically embedded in their
particularities, and how this historicity can only be discovered in comparing them,
both synchronically, as in the case of Turkish and German, and diachronically, as
in the case of pre-republican and republican Turkish. This realization, in turn, al-
lows one to understand the real impact of the sudden changes inflicted onto lan-
guages by political authorities, without necessarily isolating these events as the only
times when the linguistic and the political converge. The fate of Raif and Maria’s
love affair in Ali’s best-selling novel arguably presents an instance of this impact:
One could speculate that one of the reasons why Raif was never informed of Ma-
ria’s sudden death and of the fact that they had a baby together was that the final
letters Maria sent to Raif never reached their recipient, as the envelopes Raif had
prepared in advance for Maria were inscribed with Raif’s address in Arabic script,
banned shortly after Raif’s return to his home country. While there is no evidence
in the storyline that would positively confirm this scenario, it nonetheless remains a
possibility that would indeed make the Alphabet Reform, in the words of Geoffrey
Lewis, “a tragic success.” 1 The early Republican period represents the single party period of the Turkish Republic and the first
four years of the multi-party system (1923–50). Works Cited Ali, Sabahattin (2004 [1943]): Kürk Mantolu Madonna, İstanbul. Ali, Sabahattin (2004 [1943]): Kürk Mantolu Madonna, İstanbul. Ali, Sabahattin (1979): “Mufassal Cermenistan Seyahatnamesi,” in: Laslo Ali, Filiz /
Özkırımlı Atilla (eds.): Sabahattin Ali, İstanbul. Anderson, Benedict (1991): Imagined Communities: Reflections on the Origin and Spread of
Nationalism, London. Andrews, Walter (2002): “Starting Over Again: Some Suggestions for Rethinking
Ottoman Divan Poetry in the Context of Translation and Transmission,” in:
Paker, Saliha (ed.): Translations: (Re)shaping of Literature and Culture, İstanbul. Apter, Emily (2006): Translation Zone: A New Comparative Literature, Princeton. Auerbach, Erich (2003 [1946]): Mimesis: The Representation of Reality in Western
Literature, Princeton. Zeynep Seviner 96 Benjamin, Walter (2004): “The Task of the Translator,” in: Venuti, Lawrence (ed.):
The Translation Studies Reader, New York. Berk, Özlem (2004): Translation and Westernization in Turkey from the 1840s to the
1980s, İstanbul. Dickinson, Kristin (2013): “Translating Surfaces: A Dual Critique of Modernity in
Sabahattin Ali’s Kürk Mantolu Madonna,” in: TRANSIT (9/1), accessible at:
http://transit.berkeley.edu/2013/dickinson-2/ [last accessed: 25.10.2016]. Even-Zohar, Itamar (1997): “The Making of Culture Repertoire and the Role of
Transfer,” in: Target, (9/2), 373-381. Gürses, Sabri (2013): “Sabahattin Ali’nin Hayatında ve Yapıtlarında Çeviri,” in:
Sönmez, Sevengül (ed.): Sabahattin Ali, Ankara. Jakobson, Roman (2004): “On Linguistic Aspects of Translation,” in Venuti,
Lawrence (ed.): The Translation Studies Reader, New York. ewis, Geoffrey (2002): The Turkish Language Reform: A Catastrophic Success, Oxford. Ortega y Gasset, José (1992): “Misery and Splendor of Translation,” in: Schulte,
Rainer / Biguenet, John (eds.): Theories of Translation: an Anthology of Essays from
Dryden to Derrida, Chicago. Spitzer, Leo (2011): “Learning Turkish,” in: PMLA (126/3), 763-779. Tahir Gürçağlar, Şehnaz (2008): Politics and Poetics of Translation in Turkey, 1923-1960,
Amsterdam. Toska, Zehra (2002): “Evaluative Approaches to Translated Ottoman Turkish
Literature in Future Research,” in: Paker, Saliha (ed.): Translations: (Re)shaping of
Literature and Culture, İstanbul. Venuti, Lawrence (1995): Translator’s Invisibility: A History of Translation, New York. Venuti, Lawrence (2004): The Translation Studies Reader, New York. Translators’ Introduction to The Comprehensive
Germanistan Travelogue Kristin Dickinson/Zeynep Seviner Kristin Dickinson/Zeynep Seviner Sabahattin Ali’s short stories and his first novel, Kuyucaklı Yusuf (1937) (Yusuf of
Kuyucak), are widely recognized as pioneering texts for the genre of social realism
in the early Turkish Republican period.1 More recent scholarship has also acknowl-
edged the central roles Ali’s final two novels, İçimizdeki Şeytan (1940) (The Devil
Inside Us) and Kürk Mantolu Madonna (1943) (Madonna in a Fur Coat), played in the
development of literary modernism in Turkey. Our decision to translate Sabahattin
Ali’s little known Mufassal Cermenistan Seyahatnamesi (1929) (The Comprehensive Ger-
manistan Travelogue) aims to shed light on another, largely under-researched aspect
of Ali’s literary career: his experimentation with and satirization of Ottoman liter-
ary forms. By making this text available to an English-speaking audience, we aim to
further complicate our understanding of Ali’s identity as a writer and his profound-
ly diverse contribution to the literary landscape of modern Turkish literature. Ali composed the Mufassal Cermenistan Seyahatnamesi as a private letter during his
stay in Potsdam, Germany between the fall of 1928 and spring of 1930, for his
close friends Pertev Naili Boratav, Orhan Şaik Gökyay, Nihal Atsız, Ekrem Reşit
Rey, and a few others who were active in the literary circles of the time. In it, Ali
narrates his multi-day journey on a train through the Balkans and Eastern Europe
into Berlin, and then Potsdam, his first impressions of these two cities, German
culture in general, and an adventurous New Year’s party he attended while in Kristin Dickinson/Zeynep Seviner 98 Potsdam. Originally composed in 1929, the letter remained unpublished until it
appeared in 1979 in a collected volume of his oeuvre, entitled Sabahattin Ali and
edited by Filiz Ali Laslo and Atilla Özkırımlı. Despite its publication in transliterat-
ed form, no scholarship to date has examined the significance of this text within
the larger scope of Ali’s life and work. Potsdam. Originally composed in 1929, the letter remained unpublished until it
appeared in 1979 in a collected volume of his oeuvre, entitled Sabahattin Ali and
edited by Filiz Ali Laslo and Atilla Özkırımlı. Despite its publication in transliterat-
ed form, no scholarship to date has examined the significance of this text within
the larger scope of Ali’s life and work. Translators’ Introduction to The Comprehensive
Germanistan Travelogue As a text steeped in the intricacies of language, Ali’s Mufassal Cermenistan Seya-
hatnamesi poses two major challenges for translation: (1) on the level of genre and
its linguistic conventions, and (2) due to Ali’s subversion of these conventions
through word play. Firstly, the seyahatname, or book of travels, is a premodern liter-
ary form within the Perso-Ottoman literary tradition, making it not only a curious
choice for Ali’s purposes, but also difficult to render into English in both its his-
torical and geographic specificity. Examples of the seyahatname form can be found
throughout the Middle Ages within the Islamic world, yet the form crystallized in
the late seventeenth century in Evliya Çelebi’s (d. 1682) ten-volume work where he
narrates his impressions of Anatolia, parts of Mesopotamia, Transylvania, Central
Asia, North Africa and Eastern Europe.2 Even though Evliya’s text is a later ex-
ample of the form and reflects the sensibilities of its time through a mixed use of
the colloquial Turkish and ornate style (characterized by Perso-Arabic vocabulary
and grammar), it has become the ur-seyahatname, the first (and often only) text that
comes to mind at the mention of the form, following its 1848 publication by the
Bulaq Press in Cairo. Because of its take-all canonicity, Ali must have tried to mimic Evliya’s text in
particular. Indeed, Ali’s distortion of facts for comic effect is one key way in which
he plays off of the style of Evliya’s work, which employs inventive fiction, hearsay
and exaggeration to appeal to his readers. The irony in Cermenistan Seyahatnamesi,
which Ali constructs through a mismatch between form and content, thus depends
in large part on the reader’s recognition of Evliya’s text as much as of the seyahat-
name form in general, including the praise to God in the beginning, and the utiliza-
tion of antiquated vocabulary and syntax. Because this reliance on the reader’s
particular cultural knowledge is impossible to replicate in English, we decided
against translating the formalities of the Ottoman into another, also historically
specific, form of old English. At the same time, we have chosen to maintain historic place names, such as
Asitane, Der-i Sa’adet, and Der-i ‘Aliyye, which mean ‘The Imperial Threshold,’ ‘The
Gate of Prosperity,’ and ‘The Sublime Porte,’ respectively. 2 Parts of Evliya’s Seyahatname were translated into various languages, including English. See, for
example, the most recent translation by Robert Dankoff and Sooyong Kim, entitled An Ottoman
Traveller: Selections from the Book of Travels of Evliya Çelebi, (2010), London. Translators’ Introduction to The Comprehensive
Germanistan Travelogue While these descriptive
terms for the capital of the Ottoman Empire were not yet antiquated in 1929, they
did serve as markers of the past following the establishment of the modern Repub-
lic of Turkey in 1923 and the establishment of Ankara as the new capital of the Translators’ Introduction 99 nation state. Rather than provide descriptive translations, we have chosen to main-
tain these original place names precisely because they are not immediately recog-
nizable to an English-speaking reader. As such, they resonate as a site of linguistic
difference within the modern language of the English translation. nation state. Rather than provide descriptive translations, we have chosen to main-
tain these original place names precisely because they are not immediately recog-
nizable to an English-speaking reader. As such, they resonate as a site of linguistic
difference within the modern language of the English translation. Ali also employs antiquated place names to refer to Germany throughout his
letter. Frengistan (Frangistan), for example, was used in the Ottoman Empire as late
as the 17th century to refer to western or Latin Europe. Literally meaning ‘the land
of the Franks,’ it did not denote a specific geographic area, but was rather used to
describe any land perceived as Christian. Ali’s decision to refer to present-day
Germany as Frengistan is central to the humor of his pseudo-historical travelogue,
which describes the ‘modern’ celebration of New Year’s Eve in Potsdam, Germa-
ny through recourse to outdated vocabulary and a genre rooted in the Ottoman
literary past. Ali’s titular neologism, Cermenistan, offers a playful counterpart to the actual word
Frengistan. Derived from Cermen, meaning Germanic, and the Persian suffix -stan,
meaning ‘land’, Ali describes the present-day inhabitants of Potsdam through refer-
ence to the Germanic peoples who inhabited northern Europe from approximately
the 3rd through the 9th century. This fictionalized name pokes fun at the essential-
ization of contemporary cultural identities through recourse to medieval historical
predecessors. Our decision to translate this term through the neologism ‘German-
istan’ maintains the jarring usage of the -stan suffix to describe a Western European
country. At the same time, our translation sacrifices the ironic historicity of the
word Cermen. Rather than utilizing an older French word such as Germain, or the
English term Germania, we have chosen to maintain a phonetic similarity to Ali’s
original title. Translators’ Introduction to The Comprehensive
Germanistan Travelogue In doing so, we engage in a form of phonetic translation that Ali him-
self later employs in his letter. p
The most notable instance of phonetic translation in the Cermenistan Seyahat-
namesi is Ali’s phonetic reconstruction of the place name Potsdam as Putseddüm: According to Hayrullah Molla Bey, a contemporary etymologist, the word
Potsdam consists of the words put, sedd, and üm. Put as is known, is a
shrine found in the churches of infidels in the form of Christian portraits
and statues; sedd means to close, cover or conceal; üm, or mother, here
means the Mother Mary. All together, by means of cubism, it means: “O
Mother Mary, cover the idols!” This meaningful wish is indeed granted, as
the eternal Allah covers the aforementioned city with a coat of white
snow, concealing all statues in public parks and gardens. To a certain extent, our English rendition of this passage replicates the processes
of translation at work in the original. By maintaining the original Ottoman words
put, sedd, and üm, we have nevertheless transformed instances of intralingual (Ot-
toman-Ottoman) translation into interlingual (Ottoman-English) ones. As a result,
the translation of this passage downplays Ali’s emphasis on Turkish as a language Kristin Dickinson/Zeynep Seviner 100 in transition amidst large-scale linguistic and cultural reforms. The medium of Eng-
lish nevertheless calls attention to the interlingual translation of Potsdam into a
collection of Ottoman words. In this way, the English reader finds herself ‘learn-
ing’ new Ottoman / Turkish words within a text that also thematizes Ali’s own
learning of German. g
In a second instance of phonetic translation, Ali describes a group of young
Germans engaged in a humorous ‘dance’ (raks), which he terms “pat bi-naz.” This
word is a phonetic play on patinaj, the Ottoman term for ice skating, which is itself
derived from the French word patinage. In Ali’s comic rendition of this word, ‘pat’
serves as an onomatopoeic sound which expresses a thud or fall to the ground. Through use of the Persian preposition ‘bi,’ which is commonly used with a noun
to produce a negative prepositional phrase, the term ‘bi-naz’ literally means with-
out conceit. Together, the phrase “pat bi-naz” offers a comic description of inex-
perienced but unassuming ice skaters. More than an instance of witty word play, Ali’s neologism adds to the ironic
tone of his letter. 3 For an insightful discussion of ice skating in the late Ottoman Empire, see chapter VII of Palmira
Brummett’s Image and Imperialism in the Ottoman Revolutionary Press, 1908-1911, (2002), Albany, NY. Translators’ Introduction to The Comprehensive
Germanistan Travelogue As a European form of entertainment, ice skating had become a
sign of modernity and progress in turn-of-the-century Istanbul, in that it provided
a new form of public social interaction between men and women. At the same
time, the relative in/ability to skate served as a metaphor for one’s adaptability to
the ‘modern’ era.3 Ali’s depiction of clumsy German skaters in 1929 turns this
rhetoric on its head, raising the larger question of whether ‘modern’ values are
necessarily engendered by Western Europe. Our translation of “pat bi-naz” as “ease-skidding” maintains a phonetic play
on words with ice-skating, and expresses a sense of clumsiness through the pho-
netic similarities of skate/ skid. This form of English-English wordplay neverthe-
less loses the additional reference to patinaj as a French loan word, which under-
scores the importation of ice skating to the late Ottoman Empire from Western
Europe. Together with instances of word play such as “pat bi-naz” and “Cermenistan,”
the historic specificity of Ali’s travelogue has made its translation a difficult but
also rewarding process. In this brief introduction, we have tried to outline the ma-
jor issues at stake in translating Ali’s Cermenistan Seyahatnamesi into English. For a
more in-depth discussion of the text, please see Zeynep Seviner’s article in this
special issue, “Between Languages: Translative Acts in Sabahattin Ali’s Comprehen-
sive Germanistan Travelogue,” which further situates the linguistic playfulness of Ali’s
letter within its specific historical context. Despite the specificities of Ali’s original text that do not carry over into Eng-
lish, the very act of translation serves to metaphorically extend the interlingual and
intercultural play Ali had begun in the wake of significant linguistic and cultural
transformations in Turkey. As such we hope that this translation will both bring 101 Translators’ Introduction new attention to, and help scholars to ‘unearth’ a hitherto unexplored dimension
of Ali’s work, namely his literary engagement with Ottoman forms and conven-
tions. new attention to, and help scholars to ‘unearth’ a hitherto unexplored dimension
of Ali’s work, namely his literary engagement with Ottoman forms and conven-
tions. The Comprehensive Germanistan Travelogue Sabahattin Ali Translated by Kristin Dickinson and Zeynep Seviner Translated by Kristin Dickinson and Zeynep Seviner In the name of Allah, the Compassionate, the Merciful y
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y
1 Literally meaning ‘threshold,’ Asitane is a word that was used to refer to the capital city of the Otto-
man Empire (present-day Istanbul). Printed by courtesy of Filiz Ali via ONK Agency Ltd. Printed by courtesy of Filiz Ali via ONK Agency Ltd.
1 Literally meaning ‘threshold,’ Asitane is a word that was used to refer to the capital city of the Otto-
man Empire (present-day Istanbul). The Reason for the Composition of the Travelogue Following boundless gratitude to Allah the everlasting, the worship of whom is a
necessity, the reason for the composition of this lengthy travelogue is to be pro-
claimed. That is to say, in the beginning of year 1346 Hijri and 1928 Gregorian,
this flawed writer of foolish lines, Sabahaddin Ali, was busy educating young minds
in Yozgad, the city of cities. With the sudden arrival of the season of wandering
also called vacation, he took on his back his saddle-bag and embarked on the road
toward Asitane.1 Since his weak shoulders did not allow him to carry a large load,
he had left most of his weight in that location; as it was particularly weighty, he had
donated his debt to the creditors. 104 Sabahattin Ali On the road, I first stopped in the sweet town of Engüri, and while conversing
with old friends and loved ones, I happened to tell an esteemed acquaintance
among the high officials of the Ministry of Education that I did not intend to re-
turn to Yozgad, as I would likely be beaten or even killed by the creditors. When
that person asked: “What do you intend to do?” I responded, “Allah knows, but I
desire to set out for Moscow and look for my destiny there.” Then that person
said, “Oh, for goodness sake! This is not reasonable. You are an intelligent person. There are good deeds this state and nation desires you to do. Never consider such
frivolity. But if going to the land of infidels is your ultimate desire, it is our duty to
help you realize it with ease.” His words, at times reprimanding, at others compli-
menting, convinced this humble servant to delay the realization of his desire for a
while longer. Yet, unlike other friends, he kept his promise; within a month he
announced my participation in an examination available in Der-i Aliyye2. In a letter
most kind he urged me to make every effort to avoid embarrassment in this regard,
even though, he added, the result of the examination would surely be to my ad-
vantage. As for me, as already known by my friends and acquaintances, I took the
examination and following a positive outcome, began to wait and hope for some
money to compensate for travel expenses. 2 Der-i Aliyye, meaning Sublime Porte, is a metonym for the central government of the Ottoman
Empire. The Pain of Departure and Incidents of the Road Even though I am not among the lucky ones blessed with a love of the homeland,
as we departed the Sirkevi station waving our handkerchiefs and wiping our eyes,
tears of emotion rolled down my trifling eyes for this country that I would not be
able to see for six, seven years, maybe even for eternity. Just as we traversed Yeni-
kapı onto the Gardens of Langa—which I perceived even though it was not visible
in the dark—my cries of separation became excessive and I was sent back to my
car with the helping hands of friends. I decided to tolerate my fate in silence, re-
calling that it is useless to divert tears of emotion. I desired to lay down, thinking
that sleep would have a calming effect, but some kind travel companions protested
as there would not be enough space left for them. So we began to nap ‘bitter al-
mond’ style,3 sitting up in our seats. The expression şekerleme kestirmek, ‘to take a
sweet snooze,’ would not be appropriate here. There’s no use in elaborating any
further; in short, until we passed through Edirne and reached the Bulgarian border,
I remained on the wooden couch, paralyzed by the cold weather and lack of sleep. Once we transferred to Bulgarian locomotives at the Bulgarian stations, however, a
stove-like invention that was new to us, but old to these lands was added to our
cars; these so-called radiators brought us a bit of warmth. The land of the Bulgarians was no different than ours; one could tell that it had
once belonged to us, looking at the signs of destruction that were still apparent. In
fact, Bulgarians are no better than us in their vulgarity, and different only in their
working hard like a miller’s horse. As the train moved forward, grand places like Plovdiv that used to be filled
with Muslims made us reminisce about the old Ottoman state with longing. We
arrived in the city of Sofia at nightfall, and upon touring the city we were all agape
with wonder. We were doubly astonished by the monuments built to bolster the
only nascent national pride of a three-and-a-half-day-old State of Bulgaria. The
smallest of these monuments was so much more graceful and majestic than even
the most pleasurable of monuments in our homeland. In the morning of the following day we saw the grandiose city of Belgrade. The Reason for the Composition of the Travelogue Although the wait was far too long, a
generous amount—three hundred republican Liras to be exact—poured into my
pocket like rain of mercy. No need to elaborate, as my friends know all of this
already; in short, I finished all preparations, and we gathered in Sirkevi, the grand
train station, in order to set out for the land of decadent infidels. It is not possible
to write about the state of friends who were present there to see me off, since this
humble servant, overcome with an excess of sadness, was not in a position to see
what was around him; I was rushing to attend to four different corners at a time,
and thinking “I certainly hope I don’t leave without seeing each and every one of
my friends.” y
We then exchanged kisses of farewell and shed emotional tears with friends
present there, whom I love more than my own precious life. Meanwhile, I prom-
ised to send them detailed news from Frangistan. This promise, which I made
without considering my own insufficiencies, left a knot in my heart during my first
month and a half in the land of infidels, torturing me with words like, “You un-
grateful schmuck! How could you so quickly throw friends in a corner of oblivi-
on? What happened to the news from here? What has become of the long letters
you were going to send?” At last a letter of the same reproving opinion arrived
from Pertev Hodja, which encouraged me to compose this travelogue. Despite my
excessive ineptitude, which would certainly lead to an outcome as inscrutable as
the affairs of the Ministry of Education, I was convinced that no apology would be
necessary for sending it to friends who have surely perceived my various mistakes
with a forgiving gaze thus far. The Comprehensive Germanistan Travelogue 105 This letter was composed for my dear brother Pertev Naili Mullah, his partners
Orhan Şa’ik, Nihal, Münir, Ekrem Reşid, Ziya, and Tahsin mullahs, and our dear
sister Mehpare. And so that the reading is not overly taxing, I have made every
effort to avail myself of simple language. With a final request for the reader to
forgive the hastily composed nature of this work, which even lacks connections
between sentences, let us embark on the writing of the travelogue. May Allah grant
it benevolent success. Amen! 3 This is a literal translation of the phrase acıbadem. The Pain of Departure and Incidents of the Road But
during the previous night a small incident occurred that is appropriate to tell here:
At one point, with sleeplessness piercing my soul, I became the ridicule of my Sabahattin Ali 106 companions as I began to unwittingly sway right in the spot where I was standing. Suddenly a thought occurred to my humble mind and I began to take all the pieces
of luggage from the porte-bagage above us and place them on the floor. I then went
up to that place and managed to get some tranquil sleep. But right in the middle of
my sweet sleep, as I was on the cusp of a second dream, my right leg—which was
hanging downward because it didn’t fit on the luggage rack—began to swing up
and down like a suction pump. After a good deal of time, when I opened my eyes
and righted myself, I saw that a Serbian train official was reprimanding me to get
down. Even though I snapped at him and went back to sleep, he kept on mum-
bling in a language I did not understand. Finally, when he started to yell, “Billet! Billet!” I gave him my ticket thinking he wanted to punch it. But he just shoved it in
his pocket and walked away. As the heart of this humble servant was overcome
with apprehension, I thought: “Woe is me! What shall I do if that man doesn’t
return my ticket?” And sitting up in a panic, my sleep was spoiled. Indeed, the
fellow held onto my ticket until the following morning. As we passed the border
from Serbia to Hungary, he gave it to the Hungarian officers and they returned it
to me with the utmost kindness. At the border to Hungary, upon fully comprehending that we were in Europe,
we began to admire the signs of civilization. Around noon, we entered the Great
Hungarian Plain. Though my pen cannot do justice to the description, let it suffice
to say that our train pursued the straightest of routes, traveling for six hours on
end without encountering the smallest rise or fall in elevation, or taking the slight-
est of swerves to the right or the left. Without exaggerating in the slightest, I can
say that we were all astonished by the flatness of this landscape. Toward the end,
as we began to pray and exclaim: “My God! 4 The original name of this ‘dance’ is “pat bi-naz,” which is a phonetic play on patinaj, the Ottoman
term for ice skating (derived from the French patinage). ‘Pat’ is an onomatopoeic sound which ex-
presses a thud or fall to the ground, ‘bi-naz’ literally means without conceit. The Pain of Departure and Incidents of the Road Let us turn just a finger’s width to the
right or the left!” we finally pulled into a beautiful station with one fantastic turn. Upon arriving in Pest, we toured the city as usual. This was also a fantastic place
with particularly magnificent bridges. Upon realizing that this city was once under
our administration, one cannot help but feel the pang of a memory deep inside. Early the following morning we passed through Prague, the city of cities; in the
afternoon we arrived in Dresden, the first train station after the German border. Here we had left the gothic style of architecture behind; red-walled buildings with
slated roofs came into view, followed by uniform rows of pine and beech trees, all
resembling one another... then came the surrounding Elbe River with its orderly
wharf... And in the evening we arrived in the city of light they call Berlin. Because I have already recounted a bit about the situation in Berlin in my pre-
vious letters, please allow me to give an abbreviated account here: it would be ap-
propriate to say that if one gathered all the large open squares and wide streets in
one place that would be the city of Berlin. And its orderly buildings must certainly
also be mentioned—above all the department store called Wertheim. Looking at it
from one side, you can’t see the other; it is an object bigger than a neighborhood,
and, according to reliable rumors, 5,000 employees are housed within... May Allah
protect it! The Comprehensive Germanistan Travelogue 107 After staying here for 15 days I had spent everything at my disposal. As I was
left empty handed, the embassy sent me to Potsdam out of pity. And they didn’t
forget to admonish me, saying “Quick, quick! Pick up the German language like a
parrot!” I have been busy with German language courses for a month now. May
Allah grant me success! On the Qualities of Potsdam According to Hayrullah Molla Bey, a contemporary etymologist, the word Potsdam
consists of the words put, sedd, and üm. Put, as is known, is a shrine found in the
churches of infidels in the form of Christian portraits and statues; sedd means to
close, cover or conceal; üm, or mother, here means the Mother Mary. All together,
by means of cubism, it means: ‘O Mother Mary, cover the idols!’ This meaningful
wish is indeed granted, as the eternal Allah covers the aforementioned city with a
coat of white snow, concealing all statues in public parks and gardens. And yet the
intensity of the cold air fails to reassure us that we are safe from solidifying into
blocks of ice one day, just like these statues. The lakes and rivers surrounding this
town are all frozen, and on their frozen surface—Allah forbid! —half-naked Ger-
man boys and girls were performing a dance they called ‘ease skidding.’ In line with
the name of the dance, they would skid to the ground with a thud, only to
reemerge with ease and hasten toward an unknown direction.4 The most curious
part was that some among our cohort were also eager to learn this dance, and they
made fools of themselves by constantly skidding to the ground from lack of expe-
rience. The temperature hovered between fifteen and seventeen degrees below
zero, though at times it reached a high of five or six degrees below zero out of
undue generosity on its part. In this kind of weather, we abandon ourselves to the
reality of the cold road to school, reaching an extreme degree of self-renunciation
for the sake of acquiring knowledge. From time to time there are changes in the monotonous passing of our sor-
rowful lives, one of the most important of which was what they called ‘Weih-
nacht’—the celebration of Christmas and the New Year. Let us now conclude this
travelogue by recounting the night of New Year’s Eve. The Events of New Year’s Eve The Events of New Year’s Eve That night I had gone to the embassy in Berlin to discuss an important affair. Around nine o’clock, as I was leaving to return to Potsdam, I was startled to see all
of the coffeehouses and taverns more crowded than usual. Upon asking a local at
the train station, who responded, “It is in preparation for the New Year’s celebra- Sabahattin Ali 108 tion at midnight,” I returned nonetheless to the town of Potsdam with the utmost
of haste, as I would have nothing to do with such affairs. The distance between the
city of Berlin and the town of Potsdam is, however, rather long, and as I was walk-
ing through the market in Potsdam, headed with the utmost decorum towards my
house, I began to hear all the bells from the churches and monasteries of the infi-
dels breaking into a clamor. And what a clamor! ... No words could describe it. One would need a His Master’s Voice gramophone to do it justice. Following the
church bells, all stores opened their doors and the intoxicated inhabitants of this
grand town—both men and women—went out half-naked, sporting peculiar out-
fits. This humble servant soon joined the crowd and began to wander along the
long string of shops, with the noble intention of acquainting himself with Euro-
pean manners and advancing his knowledge of the subject. What a scenery it was,
akin to Judgment Day... And yet the girls walking around would make one think it
were Paradise instead... Fireworks, sparklers, and firecrackers of all sorts painted
spirals on the snowy skies, then fell onto the snow-covered ground where they
continued to burn for a good while, reflecting their red and green glows onto the
people around, and making the space seem like a fantasy world. g
Even those girls of the town whose stature, behavior, fancy outfits, and pretty
faces suggested they were from well-to-do families walked around drunk, bewil-
dered, and full of laughter, exposing their naked arms to slowly falling snowflakes. The young men who ran into these girls ignored their protests and gave them
quick New Year’s kisses. Yet in their drunkenness, the girls barely noticed, and
their legs trembled as they fell into yet another’s arms. 5 Der-i Sa’adet is another descriptive name meaning ‘Gate of Prosperity,’ which was used to refer to
the capital of the Ottoman Empire. The Events of New Year’s Eve And if one of them yelled
too much, the police officers on duty would tell her to calm down, and would turn
their backs if they complained about the boys. This spectacle offended my Islamic zeal; I wanted to help make the hearts of
these girls as pure and enlightened as their beautiful faces, filling them with Islamic
wisdom and moral values. I was just searching for an appropriate candidate for this
mission when I heard a nightingale-like voice call forth something that implied
“Blessed New Year!” on my left side. When I turned to look, I saw a houri with
ruby-red lips and hair the color of amber, smiling with eyes as bright as the radi-
ance of this light-filled night. She welcomed my attentive gaze, and, smiling anew,
extended her delicate hand to wish me a blessed new year. She had a girlfriend with
her. We exchanged a fraternal handshake and continued walking together. During
our walk, I gave Allah— may His Name be exalted—my gratitude for sending me
such a priceless beauty in order to guide me to the right path; I thought to myself
that her heart must surely match her face, which looked very much like this hum-
ble servant’s cruel lover, his unloving beloved in Der-i Sa’adet,5 a fact that made
me feel strange inside. The Comprehensive Germanistan Travelogue 109 Convinced that silence would not be appropriate, I began to talk. Our conver-
sation was worth listening to; I knew no more than a few sentences that I had
memorized, as for her, she did not speak any language other than German, which
she now spoke in a funny way because she was intoxicated… In any case, we made
do with body language, some words from French and so on. Just to make conver-
sation, this humble servant uttered the sentence “Was ist das?” meaning “What is
this?” in that Frankish tongue. They laughed, saying “Isn’t this the New Year?”
Just then, I saw her wobble from the alcohol; I immediately grabbed her arm and
made her lean towards me, in order to show the infidels the altruism of the Turks. She then joined her girlfriend and we began to walk and talk. The Events of New Year’s Eve She uttered long
sentences, but failing to grasp her words, I said: “I don’t understand!” She re-
sponded: “Are you a foreigner?” “Yes, I am.” “Are you Italian?” “No, I am Turk-
ish.” A military officer walking by us right then heard us speak and suddenly gave
me a hug. He wouldn’t let me go, and he uttered this sentence in Frankish: “Wir
kriegen zusammen!” (The officer actually used this verb in the past tense. But as I
haven’t learned that in depth, I wrote it like this. Let brother Orhan know it is not
a mistake.) And then he kissed me on the cheeks. But this was nothing—when the
pure girls whom I had with me for the purpose of enlightening and putting on the
right path also received hugs and noisy kisses, I involuntarily began to recite the
Islamic confession of faith. After walking around for a while longer, I judged the
time of enlightenment to have come and decided to talk about the harms of alco-
hol consumption. I said: p
“You have drunk too much alcohol,” (because I didn’t know any names in
Frankish for other intoxicating drinks. I should have learned these words before
anything else but I didn’t have the time.) She answered: p
“You have drunk too much alcohol,” (because I didn’t know any names in
Frankish for other intoxicating drinks. I should have learned these words before
anything else but I didn’t have the time.) She answered: anything else but I didn’t have the time.) She answered: y
g
“No. We just drank a little bit.” “But drinking alcohol is not good for you.” “But drinking alcohol is not good for you.” They both smiled and leaned further onto my arms. In the end, this ravishing
blond who had first talked to me proceeded: p
“Look, we aren’t tipsy, and we aren’t unconscious... How can you tell we are
drunk?” “Look, we aren’t tipsy, and we aren’t unconscious... How can you tell we are
drunk?” I said: “Oh my charming beauty. I knew it because of the smell coming from
your lovely mouth.” I said: “Oh my charming beauty. I knew it because of the smell coming from
your lovely mouth.” y
y
“It’s not true,” she responded, and in order to prove my claim, I said: “Would
you blow toward my face?” She turned to me and exhaled rapidly. The Events of New Year’s Eve However, be-
cause my sense of smell was reduced by the intense cold, I said, “Please, do come
closer.” She approached me further, until the distance between us was reduced to a
space of four fingers and exhaled anew. I failed to sense anything, and asked her to
move towards me a little more. However, the damned libertine, in her cunning
ways, just came too close and made this humble servant commit a sin as he never
had before. As if this were not enough, she kissed my cheeks, now red with shame,
and broke out in immodest laughter. She then sidled up to me and whispered into
my ear: “This new year is rather lovely, isn’t it?” In a voice coarsened with shame “It’s not true,” she responded, and in order to prove my claim, I said: “Would
you blow toward my face?” She turned to me and exhaled rapidly. However, be-
cause my sense of smell was reduced by the intense cold, I said, “Please, do come
closer.” She approached me further, until the distance between us was reduced to a
space of four fingers and exhaled anew. I failed to sense anything, and asked her to
move towards me a little more. However, the damned libertine, in her cunning
ways, just came too close and made this humble servant commit a sin as he never
had before. As if this were not enough, she kissed my cheeks, now red with shame,
and broke out in immodest laughter. She then sidled up to me and whispered into
my ear: “This new year is rather lovely, isn’t it?” In a voice coarsened with shame Sabahattin Ali 110 this humble servant responded, “Yes, oh, yes, it is very lovely!” After wandering
around a little more, we went back home as it was getting late. Upon parting, I
asked them when we were to meet again, out of my still unfulfilled duty of enlight-
ening them. They promised they would be in the grand Catholic Church in the
outskirts of the city tomorrow. I went back to my house and immersed myself in
sweet sleep... In the evening of the next day, I turned to go to that blasphemous place of
worship, but despite waiting under the snow, in fifteen degrees below zero, I never
encountered them. Thinking they might be inside the church, I decided to enter. 6 Gaza is a term used to denote a holy war waged on behalf of Islam. Unlike jihad, gaza is still part of
non-religious contemporary Turkish-language usage. 15 Kanun-ı Sani (January) 1929 The Events of New Year’s Eve Not aware of Christian customs, I followed the example of an elderly person pass-
ing in front of me. I put my hand in the holy water and made the sign of the cross
along with him. I went down on my knees and prayed to Jesus. I felt very pleased
thinking that I was doing all this to lead these blasphemers into the path of true
religion. Yet, despite being tortured with all kinds of prayers and quietly participat-
ing in the songs of Christianity, somehow I never caught a glance of those women. When it was time to leave I quickly went to the door and scrutinized each person
as they left, but I still could not find them. Then I finally sensed that the wretched
girls had caught an inkling of my benevolent intentions and had not accepted my
invitation, or that, in layman’s terms, I had been ‘ditched.’ But my grief was not
excessive, because my gaza,6 which consisted of waiting in the cold and being in-
volved in a few blasphemous acts, was nothing compared to that of others who
showcased acts of heroism and gave their lives away in the name of jihad and the
true religion of Islam. Eternal Allah, what happiness it would bring if you were to
forget a portion of my sins for this gaza! As no other event worthy of recording has since caught my eye in the town of
Potsdam, this travelogue thus comes to an end. g
Allah has spoken the truth. Warning: Only Pertev Naili Mullah shall decide who can read this worthless
letter. And because the backs of the pages have been left blank, the prohibition of
drawing pictures there is also entrusted upon him. By the time this humble servant
realized that he had wasted paper, it was too late. It has just come to my attention that this travelogue ends on page thirteen. But
since our Effendis are not Christians, they would not think it inauspicious. Cermenistan Seyahatnamesi Onk Ajans aracılığıyla Filiz Ali'nin izniyle yayımlanmıştır. Mufassal Cermenistân Seyâhatnâmesi Sabahattin Ali Bismillâhi’r Rahmâni’r-Rahîm Sebeb-i tahrir-i Seyâhatnâme Hudâ-yı dâ’im ül vucûd ve vâcib üs-sücûda hamd-i bî nihâyeden sonra bu
seyâhatnâme-i mutavvelenin sebeb-i tahririnin beyânına şürû’ edildi. Şöyle ki: Hicrî
1346 ve mîlâdî 1928 senesi evâ’ilinde Yozgad şehr-i şehîrinden, ta’lîm ü terbiyye-i
sıbyân ile meşgûl iken, ta’til ta’bir olunan mevsim-i âvâregî nâgihân hulûl
edivermekle bu sutûr-ı bi-mâ’nânın muharrir-i pür kusûru Sabahaddîn Alî dahi ol
medineden heybesini sırtlayup cânib-i Âsitâneye revân oldu. Dûş-ı za’îfi hamûle-i
kesîre nakline müsâ’id olmadığından bir aded sagîr çantadan gayrı ağırlığını ol
mahalde terk eylemiş, ve bilhassa, fazla ağır olduğundan dolayı, borçlarını da
alacaklılara bağışlamıştı. ğ
Esnâ-yı râhda birinci konak olarak Engüri belde-i şîrînine konup ehibbâ ve
eviddâ-yı kadîme ile sohbet edilegelmekte iken erkân-ı ma’âriften bir yâr-ı kesîr ül-
i’tibâra, bir dahi Yozgad’a dönmek niyyetinde olmadığımı, dâyinler tarafından darb
edilmek, hattâ fevt olmak bile muhtemel olduğunu söylemiş bulundum. Ol zât:
“Ne eylemek niyyetindesin?” deyü su’âl eyledikte: “Hudâ bilir, ammâ Moskov
diyârına azm edüp kısmetimi orada aramak arzusundayım,” deyü cevâb ettim. Ol Sabahattin Ali 112 zât ise: “Bre amân! Bu akıl kârı değildir. Sen zekâvetmend bir kimesnesin. Dahi bu
Devlet ve milletin senden dilediği maslahatlar vardır. Sakın ola ki böyle bir hiffette
bulunasın. Eğer Küffâr diyârına gitmekliğin mutlakâ ârzûn ise kolayına bakmak
borcumuzdur,” deyü bu âcizi gâh itâb, gâh taltif ile bu ârzûsunu bir muddet te’hîre
sevk eyledi. Ammâ sair ehibbâ misillü sözünde durmamazlık eylemeyüp bir mâh
mürûrunda Der-i aliyyede küşâd edilen bir imtihana duhûlümü, netîce-i imtihânın
lehimde olması muhakkak ise de ol tarafta bâdi-i mahcûbiyyet olmamak içün bir
mikdar sa’y ü ikdâm eylememizi bir keremnâme ile bildirdi. Biz dahi, yârâna,
ehibbâya mâ’lûm olduğu vechile imtihâna duhûl, ve netîce-i imtihan lehimizde
zuhûr eylemekle yol masârifi için intizâr-ı dînâr ü pul eyledik. Gerçi bu intizâr
füzûn oldu ise de netîcede üç yüz lirâ-yı cumhuri gibi bir meblâğ-ı mebzûl, bârân-ı
rahmet misillü cebimize nüzûl eyledi. Ehibbâ cümlesini bildikleri için tatvîli bî-
mâ’nâdır, hulâsa cümle tedârükümüz tamâm eyleyüp bir ahşam Sirkevi nâmındaki
kebîr şimendifer konağında Küffâr-ı hâkisâr diyârına hareket içün cem’ olduk. Ol
mahalde teşyî’imiz içün hâzır bulunan ehibbâ ahvâline dâ’ir yazmak mümkin olmaz,
çünki âciz ol esnâda kemâl-i te’essüründen etrafı görebilecek halde olmayup yalnız
telâş ile dört cânibe şitâb ile, “olmaya ki bir ehbâbı görmeden hareket eylerim”
deyü düşünürdüm. İmdi orada hâzır bulunan cân-ı azîzimden ziyade sevdiğim yârân ile vedâ’
bûseleri te’âti eyleyüp dümû-ı te’essür rizân eyledik. Ammâ bu esnâda kendülerine
mufassal Frengistân havâdisi dahi vereceğimi va’d eyledim. Sebeb-i tahrir-i Seyâhatnâme Kıllet-i bidâ’ama
bakmayarak etmiş bulunduğum bu va’d Frengistânda müddet-i ikâmetim olan bir
buçuk aydan beri derûnumda bir ukde gibi durup: “Bre kadir nâ-şinâs! Ehibbâyı
böyle çabuk mu kûşe-i nisyâna atacak idin? Kanı onlara bura havâdisi? Kanı onlara
mutavvel mektûblar?” deyü beni ta’zib eylerdi. En sonra ol cânibden Pertev
Hocanın mektûbu vürûd eyleyüp bu minvâldeki tahti’aları ol dahi tekrar eylemekle
bu Seyahâtnâmenin tahririne şürû’ edildi. Kemâl-i aczimizden her ne kadar eyü bir
eser meydana çıkamayup Ma’ârif Vekâleti umûru kadar müşevveş bir nesne olacağı
vâreste-i iştibâh ise de bilcümle hatâyâmıza nazar-ı afv ile baktıkları müsbet olan
ehibbâya irsâlinde bir mahzûr olmadığı neticesi zihnime mülâyim geldi. Bu ceride, birâder-i cânım Pertev Nailî Molla ile şürekâsı olan Orhan Şâ’ik,
Nihâl, Münir, Ekrem Reşîd, Ziyâ, Tahsin mollalar ve Mehpâre isimlü ablamız için
tahrir edilmiştir. Ve kırâ’ati mûcib-i sudâ’ olmamak içün mümkin mertebe lisân-ı
sâde isti’mâline gayret olunmuştur. Bir çırpıda çıktığı içün cümleleri arasındaki
irtibât bile bir hayli noksan olan bu eser içün mütâli’ înden bir daha taleb-i afv
eyleyerek Seyahâtnâmenin tahririne mübaşeret kılındı. Hudâ muvaffak-ı bi’l-hayr
eyleye, âmîn! Iztırâb-ı hareket ve vuku’ât-ı râh Sirkevi konağından mendiller sallayup gözleri silerek ayrıldığımızda her ne kadar
vatan muhabbeti ile mühesassis olan bahtiyârândan değil isek de belki altı yedi sene,
belki de ilel’ebed göremiyeceğimiz bu memleket içün çeşm-i hakîrimizden bilâ Mufassal Cermenistân Seyâhatnâmesi 113 ihtiyâr dümû’-i te’essür rizân oldu. Hele Yenikapu’yu geçüp Langa Bostanlarına
gelindikte – karanlıkta görünmezdi, lâkin ben hissederdim – girye-i hicrânımız
füzûn olarak ehibbâ dest-i i’ânetinde vagonumuza irsâl edildik. Eşk-i te’essürün
isâlesinin bî-fâ’ide olduğu hâtıra hutûr etmekle sükûnetle kaderimize tahammüle
karar verdik. Bir mıkdâr uyumak bâ’is-i teskin olur deyü uzanmak arzu edildikte
rüfekaa-yı kirâm mâni’ olmağıla – çünkü kendilerine yer kalmıyordu –
oturduğumuz yerde acıbâdem kestirmeğe başladık. ‘Şekerleme kestirmek’ ta’biri
burada münasebet almazdı. Tatvîl-i kelâm fâ’ideyi hâvi değildir, hulâsa ertesi gün
Edirne’yi geçüp Bulgar hududuna gelinceye kadar uykusuzluktan ve soğuktan
meflûc bir hâlde tahta kanepelerde kaldım. Ancak Bulgar istasyonlarında Bulgar
lokomotifleri geldikten sonra vagonumuza kalorifer dedikleri – bize göre nev’îcâd
buralara göre atîk – soba bozması ilsâk edilüp bir mıkdar tesahhun mümkin oldu. Bulgar memâliki bizim arâzîden farksız olup bir vakıtlar yedimizde olduğu hâlâ
izâle edilemiyen âsâr-ı tahrîbden belli idi. Zâten Bulgarlar dahi kabalıkta pek bizden
aşağı olmayup yalnız dolap beygiri biri çalışmaları fazladır. Tiren ilerüledikte Filibe ve saire gibi bir vakıtlar Müslimân ile memlû olan kebîr
şehirler Devlet-i kadime-i Osmâniyyeyi yâd-ı tahassürümüze getirmekte idi. Ahşam
üzeri Sofya’ya muvasalat olunup şehir, berâ-yi müşâhede devrân edildikte cümlemiz
engüşt ber-dehân-ı hayret olduk. Üç buçuk günlük Bulgar Devletinin gayr-ı mevcûd
olan mefâhir-i milliyyesi içün rekz eylediği âbidât bilhassa hayretimizi dübâlâ
eylerdi. Bu âbidâtın en küçüğü, en zevksizi bizim memlekette rekz edilen âbidâttan
kat be-kat lâtif ve heybetlü idi. Ertesi gün sabahleyin Belgrad şehr-i mu’azzamı göründü. Lâkin bu gecenin
ufak bir vak’ası vardır ki hikâyeti münâsibdir: Uykusuzluk gayrı tâ cânımıza tâk
etmekle bî-ihtiyâr olduğumuz yerde sallanur, rüfekânın mûcib-i istihzâsı olurduk. Nâgihân zihn-i hakîrânemize bir fikir hutûr eylemekle fevkımızdaki ‘porte-
bagage’dan bütün bavulları zemîne ilkâ‘ ederek ol mahalle çıkup bir hâb-ı asûdeye
kavuştuk. Tam hâb-ı lâtîfin evâsıtında, rü’yâyı sânînin mebâdisinde idim ki,
‘portbagaj’ istî’âb eylemediği içün, aşağıya doğru sallanan sağ bacağım, emme
basma tulumba kolu gibi inüp çıkmağa başladı. Bir hayli müddet mürûrunda
gözümü açup doğrulduğumda Sırbiyyeli bir tiren me’mûru olup aşağı inmemi ihtâr
eylemekte bulunduğunu gördüm. Kendüsini tersleyüp tekrar yattım ise de ol bî-
ebed hâlâ bilmediğim bir lisana söylenür dururdu. En nihayet “bilyet, bilyet!” diye
bağırınca zımbalayacak zanniyle biletimi verdim. Lâkin o cebine attığı gibi yürüdü,
gitti. Âcizin yüreğini bir evhâm istilâ edüp: “Vay! Iztırâb-ı hareket ve vuku’ât-ı râh Bu adam bizim bileti vermezse ne
eylerim?” deyü kemâl-i telâş ile doğrulup uyku çeşmime harâm oldu. Hakîkaten
herif bileti i’âde eylemeyüp beni tâ be-sabâh arkasında gezdirdi. Sırb hududundan
Macar hududuna geçerken Macar me’mûrlarına verüp onlar da kemâl-i
nezâketlerinden bana i’âde eylediler. Macar hududunda Avrupada olduğumuzu tamamiyle idrâk edüp etrafımızdaki
âsâr-ı umrâna hayran olurduk. Öğleye doğru büyük Macaristan Ovasına girdik. Ta’rîf içün kalem âcizdir, şu kadar söyleyeyim ki tirenimiz en ufak bir yokuş ve inişe
tesâdüf etmeden, sağa sola en küçük bir inhirâf bile yapmadan tam altı sa’at Sabahattin Ali 114 mütemâdiyyen hatt-ı müstakim istikâmetinde koştu. Zerre kadar hilâf olmayup bu
düzlük dahi cümlemizin ta’accübünü bâdi olmuştur. Sonlarına doğru: “Aman yâ
Rabbi! ya sağa, ya sola bir parmak mıkdârı sapalım!” deyü du’âya başlayup en
nihayet enfes bir büküntü ile güzel bir istasyona geldik. Peşte’ye muvâsalat olunup
bermu’tâd şehir dolaşıldı. Burası da hârikul’âde bir yer olup köprüleri bilhassa
mu’azzam şeylerdi. İnsan buraların bir zamanlar yine idâremizde olduğunu tasavvur
eyledikçe derununda derin bir hâtıranın sızladığını hisseyliyor. ğ
Ertesi sabah erkenden Prag şehr-i şehîrine, ve öğleden sonra da Alman
hududunda ilk istasyon olan Dresden’e geldik. Burada gayrı gotik tarzda binalar
başlayup kırmızı duvarlı, arduvaz çatılı evler, hep birbirine benzeyen muntazam
çam ve kayın ağaçları… Etrafı muntazam rıhtımlı Elbe nehri göründü… Ve ahşam
üzeri Berlin dedikleri belde-i nûra dâhil olundu. Evvelki mektuplarımda Berlin ahvâlini bir mıkdâr hikâyet eylediğimiz içün
ihtisâr ile bu şehri ta’rîf edecek olur isek: geniş caddelerle büyük meydanları bir yere
topla, Berlin şehri meydana gelir, demek münâsib olur; mu’azzam binaları da tabii
ilâve eylemelidir… Hele bir mağazası var ki nâmı Wertheim’dır, bir ucundan
bakıldıkta öbür ucu görülmez, bir mahalleden kebir bir nesnedir; rivâyât-ı
mevsûkaya göre derûnunda 5000 müstahdem mevcûd imiş… Hudâ muhâfaza
eyleye! On beş gün mıkdar burada kalup elimizdeki avucumuzdakini bitirince makâm-ı
Sefâret bizi merhameten Potsdam şehrine i’zâm eyleyüp: “Çabuk, papağan misâli
lisân-ı Alamanı elde edesiz!” deyü tenbîhâtı da nisyân eylemedi. İmdi bir aydır
burada ta’allüm-i zebân-ı Cermân ile meşgûlüz. Hudâ muvaffak eyleye! Der vasf-ı medîne-i Potsdam Potsdam kelimesi, iştikâkıyyûn-ı zamândan Hayrullah Molla Beyin tefsîri üzere
‘Put’, ‘sedd’, ‘ümm’ kelimelerinden mürekkeb olup, ‘Put’, ma’lûm olduğu üzere
kenâ’is-î Küffârda mevcûd Hıristos tasâvîri ile heyâkil makûlesi esnâmdır; ‘sedd’,
kapamak, örtmek, setr eylemek; ‘Ümm’, vâlide, burada Meryem Ana mânasınadır. Cümlesi toplu olarak, kübizm üzere, ‘Meryem Vâlide, esnâmı setr eyle!’ demek olur. Bu temennî-i pür ma’nâ elhak müstecâb olup Hudâ-yı lem yezel medîne-i
mezkûreyi bir setre-i sefid-i berf ile rûz u şeb setr eylemekte ve bûğ u besâtîn-i
mîriyyede mevcûd bilcümle heyâkil dahi örtülmektedir. Ammâ bunun neticesi olan
şiddet-i burûdetten bizlerin de bir gün işbu heyâkil misillü incimâd edüp kas katı
kesilmeyeceğimiz mü’emmen değildir. Kasaba-i mezkûrenin etrâf ü cevânibini ihâta
eden enhâr ile göller tamamiyle buz tutup üzerinde – Hudâ hızf eyleye! – genç
Alman kızları ile oğlanları nîm-‘uryan ‘pat bî-nâz’ dedikleri raksı icrâ
eylemektedirler. Ve raksın ismine muvâfık olarak ‘pat’ diye yere yuvarlandıkları
halde ‘nâz’ etmeyerek kalkup tekrar bir istikâmet-i mechûleye şitâb etmektedirler. Garîbi şu ki: bu acâ’ib raksa bizim rüfekâdan da râgıb olanlar bulunmakta, ve
mübtedîliklerinden mütemâdiyyen yuvarlanarak etrâfı kendilerine hande-nisâr
eylemektedirler. Derece-i harâret ’ale-l’ekser that es-sıfır on yedi ile on beş arasında Mufassal Cermenistân Seyâhatnâmesi 115 raksân ise de ba’zen kemâl-i kereminden taht es-sıfır beşe, altıya çıktığı da vâki’dir. Biz ise bu havalarda dahi mektebimizin râh-ı bürûdet iktinâhına kendimizi terk
eyleyerek tahsîl-i ilm içün ferâgat-i nefsin mertebe-i kusvâsına varmaktayız. raksân ise de ba’zen kemâl-i kereminden taht es-sıfır beşe, altıya çıktığı da vâki’dir. Biz ise bu havalarda dahi mektebimizin râh-ı bürûdet iktinâhına kendimizi terk
eyleyerek tahsîl-i ilm içün ferâgat-i nefsin mertebe-i kusvâsına varmaktayız. g
Hayât-ı pür ıztırâbımız hayli yeknasak mürûr eylemekte ise de ara sıra
değişiklikler olup bunların en mühimmi ‘Weihnacht’ dedikleri Noel yortusu ile
yılbaşı idi. Yılbaşı gecesini burada hikâyye eyleyerek bu seyâhatnâmeye de nihâyet
verelim. Vukunât-ı leyle-i sene-i cedîde Ol gece bir mes’ele-i mühimmeyi müzâkere içün Berlin’e, sefâret cânibine revân
olmuştum. Sâ’at zevâli dokuz raddelerinde oradan çıkup Potsdam’a avdet
eylemekte idim ki bilcümle kahvehânelerin, harâbâtların haddinden fazla memlû
olduğunu görüp tahayyür eylerdim. İstasyonda bir âşinâya sorup: “Nısf ül-leylde
vâkı’ olacak sene-i cedîde â’id hazırlıktır,” cevabını alınca zinhâr böyle işlerle alâkam
olmadığından kemâl-i isti’câl ile kariyye-i Potsdam’a avdet eyledim. Lâkin, medîne-i
Berlin ile kariyye-i Potsdam arasındaki mesafe vâfir mıkdarda uzun olduğundan
tam Çârşû-yı Potsdam’da, kemâl-i te’eddüble hâneme revân olurken bilcümle deyr
ü kenâ’is-i Küffârın nâkuusları velvele-endâz olmağa başladı. Ammâ ne velvele!.. ta’rîfi kalem ile edilmez, bir adet ‘Sahibinin sesi’ gramofon lâzımdır. Bu çanların
akabinde mesâkin, ticaretgâh makûlesi yerlerin cümlesinin bâbları küşâde edilüp
Kasaba-i kebîrenin zükûr u nisâ bütün tâ’fesi nîm-üryân, eşkâl-i garîbede, mest-i
şarâb olarak hurûc eyledi. Âciz de, âdâb-i Frenge kesb-i vukûf ile tezyîd-i ma’lûmat
eylemek ârzûy-i ulvîsiyle bu gürûha iltihâk eyleyüp esvâak-ı bî-nihâyede geşt ü
güzâra mübâşeret eyledim. Ammâ bir manzara idi ki Yevm-i Kıyâmet misâl… Ve
lâkin bir duhterler dolaşur idi ki insan bu manzara-i Kıyâmeti Cennet zann
eylemekten kendini men’ eyleyemez… Havâî fişenkler, mâhtâblar, gûnâ-gûn el’âb-ı
nâriyye semâ’yı berf-efşânda halezûnlar resm eyleyüp zemîne, karlara sukuut
eylemekte, orada da bir müddet yanarak civârında dolaşan kesânı kırmızı, yeşil
renklerde gösterüp pür hayâl bir âlem-i dîger manzarası vermekte idiler. Asîl ül-â’ile oldukları vaz u etvârlarından, kisve-i kibârânelerinden, çehre-i
melîhlerinden ayân olan duhterân-ı belde dahi mest ü bî-hûş, pür-kahkaha, üryân
kollarını âheste âheste rizân olan kara verüp dolaşmakta idiler. Şehir delikanlıları,
bunları, her karşılaştıkça sene-i cedîde şerefine, feryâdlarına bakmayarak birer kerre
bûs eyleyüp bırakurlardı. Ammâ onlar şiddet-i mestîden pek farkına varmazlar,
lerzân bacaklariyle bir az sonra diğer birinin âğûşuna düşerlerdi. Ba’zıları fazla
feryâd eyledikleri zaman ortalıkta dolaşan polisler sükût eylemesini emr eyleyüp o,
kendisini yakalayan ferzendlerden şikâyet edecek oldukça arkalarını çevirirlerdi. Bu manzara hamiyyet-i islâmiyyeme dokunup bu duhterlerin vech-i dilberleri
gibi kalblerini de pâk, münevver eylemek, hikmet-i islâmiyye, fazâ’il-i ahlâkıyye ile
memlû kılmak isteyüp irşâda muvâfık kimesne taharrî eylemekte idim ki cânib-i
yesârımda andelîb misâl bir sedânın “sene-i cedîde mübârek ola!” me’âlinde bir
cümle sarf ettiğini gûş eyledim. Dönüp baktığımda kehrübâ misâl kumral saçları, Sabahattin Ali 116 la’l misâl lebleri, bu leyl-i pür-nûrun envârından daha ışıldak gözleriyle etrâfa
hande-nisâr olan bir hûrî olduğunu gördüm. Ol benim bu dikkatli nazarım altında
tekrar tebessüm eyleyüp sene-i cedîdemi bir dahi tebrik ile dest-i nâzikini uzattı. Yanında bir refika-i melîhası dahi var idi. Onunla da bir musâfaha-i biraderâneden
sonra birlikte yürümeğe devâm eyledik. Vukunât-ı leyle-i sene-i cedîde Ben esnâ-yı râhda Allâhü te’âlânın irşâd
içün bana böyle bir hüsn-i bî-bahâ göndermesinde hamd eyliyor, elbette kalbi dahi
çehresi gibidir, diyordum. Çehresi ise âcizin Der-i sa’adette mevcud olan yâr-ı
cefâkârına, ma’şuka-i nâ-âşıkasına hayli müşâbih olup bu hâl dahi derûnumu bir
tuhaf eyler idi. y
Sükûtun münâsib olmayacağını teyakkun ile kelâma mübâşeret eyledim. Lâkin
mükâlememiz istimâ’a şâyân olup ben ezberlediğim bir kaç cümleden mâ’adâ
bilmezdim, ol dahi Almancadan gayrı lisâna âşinâ bulunmazdı, bu lisânı dahi sâ’ika-i
mestî ile bir hoş telâffuz eylerdi… Biz gayrı, lisân-ı hâl, kelimât-ı Franseviyye ve
sâire ile idare eyledik. Âciz, laf olsun deyü, ilk def’a Frenkçesi “was ist das?” olan:
“Bu nedir?” cümlesini savurduğumda “Bu yeni sene değil mi ya?..” deyü güldüler…
Ol esnâda, kendisinin kesret-i küûlden sallandığını görüp, hemen kolunu alup
kendime ittikâ ettirdim, tâ ki Küffâra dîğer-endîşî-i Türkîyi ayân kılam. Ol dahi,
arkadaşı ile yürümeğe başlayup, hem yürür, hem konuşurduk. O bir çok mutavvel
lâflar etti ise de ben anlamayup şöyle dedim: “Anlamıyorum ki bunları!” Cevâb etti:
“Siz ecnebî misiniz?” “Evet, ecnebîyim.” “İtalyan mısınız?” “Hayır, Türküm.”
Bunu yanımızdan geçerken duyan bir zâbit hemân boynuma sarıldı. Beni
bırakmayup “Biz beraber harb eyledik” me’âlinde olan şu cümle-i Frengiyi vird
eylerdi: “Wir kriegen zusammen!” (Zâbitin söylediği bu fiilin mâzîsi idi. Ammâ ben
Almancayı daha o kadar derin öğrenemedim. Böyle kayd ediyorum. Orhan Ağabey
yanlış demesin.) Ve dahi yanaklarımı bûs ederdi. Bu bir şey değildi, ve lâkin, sırf
irşâd ve ikâz içün yanımda gezdirdiğim iki duhter-i pâki de der-âgûş ile birer bûse-i
pür-velvele aldığında gayr-ı ihtiyârî kelime-i şahâdet getirdim. Bir mıkdâr daha
yürüdükte artık zamân-ı irşâdın geldiğini anlayup evvelâ küûlun mazarratından bahs
eylemeğe niyyet edüp dedim: “Siz çok alkol nûş etmişsiz.” (Çünkü alkolden gayrı müskirâtın Frenkçe
isimlerini bilmezdim. Her şeyden evvel öğrenmek icâb eylerdi, ammâ vakıt müsâ’id
olmadı.) Ol cevâb edüp: olmadı.) Ol cevâb edüp: “Yok! Bir az almışız,” dedi. “Fakat alkol içmek eyü değildir.” “Fakat alkol içmek eyü değildir.”
İ İkisi birden bir hande edüp koluma daha yaslandılar. Nihayet, sarı saçlı olan ve
benimle evvelâ görüşen âfet-i devrân etti: g
“Biz sallanmazız, bî-hûş değiliz… İçtiğimize kandan hükm eylersin?” g
“Biz sallanmazız, bî-hûş değiliz… İçtiğimize kandan hükm eylersin?” Biz sallanmazız, bî-hûş değiliz… İçtiğimize kandan hükm eylersin? Dedim: “Aman letâfetlüm! Fem-i lâtifinizden çıkan râyihadan bildim.”
h h d
ld
d d
d
bâ
dd Dedim: “Aman letâfetlüm! Fem-i lâtifinizden çıkan râyihadan bildim.” “Sahih değildir,” dediğinde, isbât-ı müdde’a içün: “Hoh der misiniz?” dedim. Ol bana dönüp “Hoh!” dedi. Vukunât-ı leyle-i sene-i cedîde Lâkin şiddet-i
burûdetten şâmmem dumûra uğradığından: “Az takarrüb eyleyesiz,” dedim. Ol
yaklaşup, aramızda dört parmak mesâfe kaldığında bir dahi “Hoh!” dedi ise de ben
yine hisseylemeyüp az daha sokulmasını rica ettim. Lâkin fâcire-i mel’ûne bunu sû’- “Hoh der misiniz?” dedim. Ol bana dönüp “Hoh!” dedi. Lâkin şiddet-i
burûdetten şâmmem dumûra uğradığından: “Az takarrüb eyleyesiz,” dedim. Ol
yaklaşup, aramızda dört parmak mesâfe kaldığında bir dahi “Hoh!” dedi ise de ben
yine hisseylemeyüp az daha sokulmasını rica ettim. Lâkin fâcire-i mel’ûne bunu sû’- Mufassal Cermenistân Seyâhatnâmesi 117 i tefsîr edüp haddinden fazla takarrüb eylediğinde fakîre müddet-i hayâtında
işlememiş olduğu günâhı işletti. Ba-dehu, kâfî değilmiş gibi, hicâbından al al olan
ruhlarımı bûs edüp, bilâ te’eddüb bir kahkaha dahi attı. Arkasından hafif hafif
kulağıma sokulup: “Yeni sene pek güzel, değil mi?” deyü su’al eyledi. Fakîr ise
kemâl-i te’essürümden kısık kısık: “Pek güzel, ah!…. Pek güzel!” deyü cevâb ettim. Bir müddet dahi gezindikte gayrı vakıt ilerleyüp hanelerine ayrıldılar. Esnâ-yı
müfârakatta, bu ahşam ikmâl edilemeyen vazife-i irşâdı itmâm içün bir dahi nerede
buluşacağımızı su’âl eyledim. Onlar dahi şehrin kenârındaki kebîr Katolik
Kilisesinde ertesi ahşam hâzır olacaklarını va’d eylediler. Ben hâneme gidüp bir
hâb-ı lâtife müstağrak oldum... Ertesi gün ahşam üzeri ol ibadetgâh-ı pür dalâle gidüp kar altında taht es-sıfır
on beş derecede beklediğim halde onlara müsâdif olamadım. Belki dâhil-i
deyrdedirler, deyü içerü girmek istedim. Âdâb u erkân-ı Nasârâya vâkıf
olmadığımdan önümden giden bir ihtiyâra teba’iyyet eyledim. Anınla berâber mâ-i
mukaddese parmağım daldırup istavroz ihrâc eyledim. Diz üstü gelüp Hıristos’a
du’a eyledim. Bunları hep ol bî-dînleri dîn-i hakka imâle içün yaptığımı düşündükçe
bir hayli müsterîh olurdum. Lâkin dâhil-i deyrde envâ’-i ibâdât ile mu’azzeb olup
taganiyyât-ı hıristianiyyede pes perdeden iştirâk eylediğim halde bir dürlü ol
hatunlar müsâdif-i nazarım olmazdı. Hengâm-i hurûcda herkesten evvel kapunun
yanında gidüp çıkanları birer birer tedkik eyledimse de tesâdüf eylemek mümkin
olmadı. Ol zaman, mel’ûnelerin niyyet-i hayr-hâhânemi istişmâm eyleyerek da’vete
icâbet eylemediklerini, ta’bir-âmiyanesiyle ‘atladığımı’ hissettim.. Ammâ te’essürüm
füzûn olmadı, çünki dîn-i mübîn-i İslâm içün bu denlü cihâdlar yapılup
kahramanlıklar gösterilür, canlar erzân kılınurken bir müddet ayazda beklemekle,
bir mıkdâr tekeffür etmekten ibâret olan benim gazâm tabii hâ’iz-i ehemmiyet
olamazdı. Hudâ’yı lem-yezel günahlarımdan bir kısmını bu gazâ içün nisyân eylerse
ne mutlu! Bu vukuu’âttan berü Potsdam kariyyesinde şâyân-ı kayd vak’a görülmediğinden
Seyahâtnâme’miz böyle resîde-i hadd-i hitâm oldu. İhtar: Bu cerîde-i bî-i’tibârın kimlere kırâ’at ettirilebileceği yalnız Pertev Nailî
Mollanın re’yine terk edilmiştir. 15 Kânûn-ı Sânî 1929 Vukunât-ı leyle-i sene-i cedîde Ve dahi bu sahifelerin arkası boş olduğundan ol
mahalle not yazılup resim yapılmasının men’i de yine kendisine mevdû’dur. Âciz,
bu işin sonradan farkına varup kâğıd ziyân eylediğini anlamışsa da iş işten geçmişti. Bu Seyahâtnâme’nin on üç sahifede hitâm bulduğu şimdi müsâdif-i nazarımız
oldu. Lâkin Efendilerimiz Nasâradan olmadıklarından teşe’üm buyurmazlar. 15 Kânûn-ı Sânî 1929 1 Citations from Şenocak’s Deutschein are indicated by ‘D’ and the page number. Deutschsein: Zafer Şenocak’s Poetic and
Enlightened Vision of a Cosmopolitan German
Identity Vera Stegmann Zafer Şenocak’s recent essayistic publication, Deutschsein: Eine Aufklärungsschrift, appeared
in 2011, in the year of the 50th anniversary of Turkish immigration to Germany, a year that
also coincided with the ‘Integrationsdebatte’ in Germany. As part of the book’s subtitle, the term
‘Aufklärung,’ marginally present in Şenocak’s earlier essay collection Das Land hinter den
Buchstaben (2006), becomes a central metaphor in Deutschsein. The Enlightenment serves
as a guiding idea in his discussions on contemporary German identity in a nation of immigrants. Şenocak’s view of the Enlightenment is a moderate and tempered one, informed by the values of
Alexander von Humboldt, but also by Horkheimer /Adorno’s Dialektik der Aufklärung. Şenocak sees dangers in radical rationalism and Enlightenment fundamentalism, both in the
German and Turkish context, and he couples his view of the Enlightenment with Islamic mysti-
cism and with certain elements of German Romanticism. His vision of an enlightened German
identity is an aesthetic one, defined by language, poetry, and music. Finding a language, even a
broken German language that helps citizens come to terms with a traumatic national past, is an
essential step toward creating an open, welcoming society. Musical metaphors also abound in
Şenocak’s book, i.e. when he proposes to replace ‘tonal’ traditional societies with ‘atonal’ multicul-
tural ones. Poetry and music inform Şenocak’s highly humanistic concept of a cosmopolitan view of
Deutschsein. Vera Stegmann 120 The Turkish-German essayist and poet Zafer Şenocak is a Berlin-based author
who has become a leading voice in the discussions on multiculturalism, and na-
tional and cultural identity in Germany. Born in Ankara in 1961, Zafer Şenocak
spent his early childhood in Ankara and Istanbul, before his family moved to
Germany in 1970. He lived first in Munich and has lived in Berlin since 1989; he
has frequently acted as a mediator between Turkish and German culture. As a
widely published poet, essayist, novelist, journalist and editor, Şenocak has won
numerous literary awards in Germany, such as the Prize of the Literarische Colloqui-
um in Berlin in 1988; he assumed the position of “poet in residence” at the Univer-
sität Duisburg-Essen in 2007; and, most recently, in 2016, he received the Ham-
burger Gastprofessur für Interkulturelle Poetik at the Universität Hamburg, where an
international conference was devoted to his works in July 2016. Deutschsein: Zafer Şenocak’s Poetic and
Enlightened Vision of a Cosmopolitan German
Identity He has also written
feuilleton articles for German newspapers and journals across the entire political
spectrum, including taz, Tagesspiegel, Die Welt, Frankfurter Allgemeine Zeitung, Tagesspie-
gel, and Die Zeit. Şenocak considers himself politically neither left-wing nor right-
wing, but rather a “discerning free spirit” (Gropp 2012). Şenocak is well known in
American academic circles, since he has frequently visited the United States, as
Max-Kade Distinguished Visiting Professor (M.I.T. 1997), and as writer-in-residence at
several universities and colleges in the U.S. (Dartmouth College 1999, Oberlin
College 2000, UC Berkeley 2003, Washington University 2013, Dickinson College
2013, among others). Among Şenocak’s many book publications are his essay col-
lections Atlas des tropischen Deutschland (1992), translated into English by Leslie A. Adelson (2000), and Das Land hinter den Buchstaben: Deutschland und der Islam im Um-
bruch (Prosa, 2006); the novels Gefährliche Verwandtschaft (1998), Der Errottomane: Ein
Findelbuch (1999) and In deinen Worten: Mutmaßungen über den Glauben meines Vaters
(2016); and the poetry collections Übergang (2005) and Türsprachen – Door Languages,
published in 2008 in a bilingual German-English edition with translations by Eliz-
abeth Oehlkers-Wright. Şenocak’s recent publication Deutschsein: Eine Aufklärungsschrift, an essayistic
treatise on contemporary German identity, was published by edition Körber-Stiftung in
2011, the year of the 50th anniversary of Turkish migration to Germany. This Turk-
ish-German anniversary coincided with the so-called “Integrationsdebatte” (inte-
gration debate), provoked by the August 2010 publication of Thilo Sarrazin’s
Deutschland schafft sich ab, which dominated journalistic discourses in Germany dur-
ing the years 2010-2012. Although Şenocak never mentions Sarrazin by name,
subtle references to the ‘integration debate’ occur frequently in his book (D 49,
1441). Deutschsein almost appears as a response, or a counter-model, to Sarrazin’s
thesis that Germany is abolishing itself, as Sarrazin’s title translates into English. Karin Yeşilada calls Deutschsein a “great, much respected essay” (Yeşilada 2012:
162). Her own ground-breaking book on Turkish-German poetry, Poesie der Dritten
Sprache, mentions Şenocak’s essay only briefly and in reference to his lyrical work. Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 121 But indeed, Deutschsein has been debated frequently in the many public readings
given by the author, and the book was reviewed widely in German journals and
newspapers: Alan Posener published an interview with Şenocak in Die Welt (2011),
Jörg Lau reviewed Deutschsein for Die Zeit (2011), Christiane Florin discussed his
essay in detail for Deutschlandfunk (2011), as did Monika Stranakova for C.H. Deutschsein: Zafer Şenocak’s Poetic and
Enlightened Vision of a Cosmopolitan German
Identity Beck’s
forum on “Literaturkritik” (2011) and Lewis Gropp for the Goethe Institut forum
on “Migration und Integration” (2012), to mention a few examples. The Turkish-
German poet Nevfel Cumart placed a review of Şenocak’s book on his website. In
academic literature, Deutschsein has been discussed primarily by two critics: David
Coury’s article on “Enlightenment Fundamentalism” (2013) offers an overview of
recent European political and cultural discussions on the Enlightenment and on
Enlightenment fundamentalism, in which he contextualizes Şenocak’s Deutschsein
and Navid Kermani’s Wer ist Wir? (2012). Saniye Uysal Ünalan’s book Interkulturelle
Begegnungsräume (2013) is situated in the field of intercultural literary studies and
focuses on Turkish-German constructions of identity in Şenocak’s Deutschsein and
his novel Der Pavillon 2009), as well as Feridun Zaimoglu’s play Schwarze Jungfrauen
(2006) and his novel Liebesbrand (2008). In her analysis of Deutschsein, Uysal Ünalan
explores the connection that Şenocak draws between the past and the present in
Germany: Şenocak shows how the country’s broken relationship to its Nazi past
affects its perception of contemporary minorities, how debates on migration and
integration become debates on national identity in Germany. Uysal Ünalan also
analyzes how Şenocak’s espousal of the values of tolerance and civilization is in-
debted to both the European Enlightenment and to Islamic mysticism (Uysal Üna-
lan 2013: 75-85). Language and literature strongly influence Şenocak’s highly humanistic view of
a cosmopolitan form of Deutschsein. I will explore how the ‘Aufklärung,’ the con-
cept and the historical movement of the Enlightenment, serves as a guiding idea
and a metaphor throughout his book. Following Karin Yeşilada’s thesis that Zafer
Şenocak is, “above all, a poet” (Yeşilada 2012: 162), I will then consider the im-
portance of poetry and music in an essayistic work like Deutschsein. An Enlightenment Tempered by its Dialectic The title Deutschsein: Eine Aufklärungsschrift (Being German: An Enlightenment Es-
say), already offers a guide through Şenocak’s main arguments. Like a musical leit-
motif, the word ‘Aufklärung’ weaves itself through his writing, and different inter-
pretations of the literature and philosophy of the Enlightenment appear through-
out Şenocak’s book. Enlightenment thought becomes a model for a more open
and inclusive German society that integrates immigrant citizens, especially from
Muslim countries. Şenocak proposes that each generation needs to learn and create
its own form of Enlightenment, since the Enlightenment is not part of our genetic
composition, our “Erbmasse” (D 46), an inherited condition that we can take for
granted. He notes that frequently there is a gap, even an abyss between the En- 122 Vera Stegmann lightenment as a formula or etiquette, an empty convention or code of behavior,
and its genuine, heartfelt application in society in our engagements with other reli-
gions or ethnicities (D 46). lightenment as a formula or etiquette, an empty convention or code of behavior,
and its genuine, heartfelt application in society in our engagements with other reli-
gions or ethnicities (D 46). g
(
)
In looking at the history of the German Enlightenment, Şenocak observes that
Islam and the Enlightenment actually share a common history in Germany: Especially in regard to the German Enlightenment and Islam, there is a
long and intensive history of contact. Oriental Studies as an academic sub-
ject is above all a German creation. It not only led to an exotizing of a
world that was perceived as foreign, but also it actually created a close vi-
cinity.2 ‘Orientalistik,’ 18th century German-language academic research on Asian cultures,
began during the period of the Enlightenment. For Şenocak, “Orientalistik” did
not only create a sense of exoticism in perceiving Asian and Muslim cultures, as
Orientalism, the style of rather colonialist and stereotypical Western depictions of
the Middle East, seems to suggest. While he acknowledges the existence of orien-
talist representations of the East, he emphasizes that the German discipline of
‘Orientalistik’ did create a genuine understanding of different Eastern cultures in
Germany. In this context, Şenocak refers to Goethe, Lessing, and Friedrich Rück-
ert, poet and translator who researched, spoke, and translated works from 44 ori-
ental languages into German. 2 “Dabei gibt es gerade in Bezug auf die deutsche Aufklärung und den Islam eine lange und intensive
Berührungsgeschichte. Die Orientalistik als akademisches Fach ist vor allem eine deutsche Kreation.
Sie hat nicht nur zur Exotisierung einer als fremd wahrgenommenen Welt geführt, sondern auch viel
Nachbarschaft geschaffen.” (D 106) All subsequent translations from Şenocak’s Deutschsein and other
German language texts in this article are mine. 3 “Die Moderne spaltete Deutschland tief. Das dunkle Gemüt auf der einen, die technologischen
Errungenschaften auf der anderen Seite. Die Aufklärung mündete nicht in einen Umsturz der Herr-
schaftsverhältnisse, wie beispielsweise in Frankreich oder in den USA, sondern in eine Spaltung des
Bewusstseins.” (D 60) An Enlightenment Tempered by its Dialectic Şenocak also writes about the Muslim angel images
in Rilke’s poetry (D 106, also 33), which he does not explain in detail, although he
is probably referring to the Duineser Elegien. Germans carry more of the Orient in
themselves than they are aware, Şenocak summarizes (“Die Deutschen haben
mehr Orient in sich, als ihnen bewusst ist,” D 55). In order to achieve a truly enlightened society, Şenocak proposes to replace the
German concept of ‘Kultur’—especially in its occasional degradation as ‘Leitkul-
tur’—with the more universal idea of ‘Zivilisation’ (D 61). It is interesting to note
that in his distinction between culture and civilization, a complex subject, Şenocak
relies on an originally conservative thinker, Thomas Mann, who later became pro-
foundly democratic and anti-fascist. Şenocak’s role model is Mann in his later
phase, after he distanced himself from his early nationalistic writings, such as Geist
und Kunst and Betrachtungen eines Unpolitischen. In these early writings Mann had fa-
vored culture over civilization and defined the distinction between culture and
civilization as that between mind and politics (“Geist und Politik”), or between
soul and society; and he had characterized his brother Heinrich Mann critically and Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 123 pejoratively as a “Zivilisationsliterat,” a writer who dedicates his literature to revo-
lutionary political causes. Later, Thomas Mann revised his views and came to es-
pouse a more tolerant, democratic, and universal notion of civilization. It is debat-
able and not proven that this change of mind occurred already in 1922 during his
Berlin speech “Von deutscher Republik,” as Şenocak claims (D 69); but it hap-
pened at the latest during his exile years in the United States. Unlike the more na-
tionalist concept of culture, as Mann had defined it earlier, the idea of civilization
incorporates pluralism, diversity, and cosmopolitanism. Thomas Mann’s path from
a German nationalist novelist to a German “Weltbürger” in the spirit of Alexander
von Humboldt (D 69, 104), to a citizen of the world whose identity is no longer
based on national culture but rather founded on the principles of civilization, is a
path that Şenocak recommends to many Germans. This shift to the more universal
and enlightened idea of civilization is one he finds missing in contemporary Ger-
man debates on integration, where the term “Kultur” once again seems to predom-
inate (D 61). 4 “Ein Blick auf den anderen, der nicht durch das Prisma der Selbstkritik gebrochen ist, wird immer
verstellt bleiben. […] Wir erleben heute einen ständigen Affront gegenüber unserer eigenen Geistes-
geschichte, in deren Zentrum die Aufklärung steht, wenn wir die Fähigkeit zur Selbstkritik einbüßen
und uns nur noch durch die Ausgrenzung des anderen definieren. Die Unvereinbarkeit von Islam
und Moderne, von Demokratie und muslimischer Kultur, ist ein Lehrsatz sowohl der islamischen
Fundamentalisten als auch der westlichen Islamgegner, die sich auf die christlichen Wurzeln ihrer
Kultur berufen und dabei die Geschichte der Aufklärung verschweigen.” (D 107-108) An Enlightenment Tempered by its Dialectic (
)
He perceives that, historically, the Enlightenment had a different effect on
French or American society than on the German one: Modernity divided Germany deeply. The dark soul on the one hand, the
technological achievements on the other. The Enlightenment did not re-
sult in an upheaval of power relations, as in France or the United States,
but in a splitting of the mind.3 While France and the United States had their revolutions, the Enlightenment in
Germany produced a split consciousness, torn between the awareness of rapid
technological innovations and a mystical, dark soul that distrusted modernity,
looked inward, and emphasized tradition, especially during 19th century German
Romanticism. However, he evaluates the present situation differently: Şenocak
believes that now, in the 21st century, Germany has an advantage over other Euro-
pean nations like France, the Netherlands, or Great Britain, whose societies are
overshadowed by their long colonial history. Compared to England and France,
which Şenocak seems to know well, the integration of immigrants in Germany
seems to be in a splendid state (“nahezu glänzend” D 39). While this could be an
idealistic assessment, the absence of a colonial burden in Germany may genuinely
enable the possibility of eye-to-eye, egalitarian conversations “auf Augenhöhe”
between Germans and Turks (D 165). 124 Vera Stegmann Such conversations that are looking for an intellectual and emotional exchange
with ‘the Other’ (“den anderen”) should begin with a mindset that allows self-
criticism: A glance at the other that is not broken through the prism of self-criticism
will always be distorted. [...] Today we witness a continous affront toward
our own intellectual history, in which the Enlightenment is centrally locat-
ed, when we lose the capacity of self-criticism and define ourselves only by
our exclusion of the other. The incompatibility of Islam and modernity, of
democracy and Muslim culture, is a dictum of both Islamic fundamental-
ists as well as Western opponents of Islam, who invoke the Christian roots
of their culture and thereby conceal the history of the Enlightenment.4 Communication with another individual or cultural community that isn’t filtered
through the lens of self-criticism will not produce mutual understanding. 5 “Die Denkschulen bis in die 1980er Jahre, die Frankfurter Schule von Max Horkheimer und Theo-
dor Adorno, Philosophen wie Michel Foucault und Emmanuel Lévinas stehen mit ihrem Werk für
diese Wahrnehmung des komplexen Menschen und untergraben ein cartesianisches Verständnis
menschlicher und gesellschaftlicher Existenz.” (D 43) An Enlightenment Tempered by its Dialectic Şenocak
observes a creeping fundamentalization of the sense of identity (“schleichende
Fundamentalisierung des Selbstverständnisses,” D 44) not only on the part of cer-
tain Islamic fundamentalists, but also on the part of Western radical opponents of
Islam, who believe that Islam is incompatible with Western European values. These “Enlightenment fundamentalists” (Coury 2013: 140-143), or ‘vulgar rational-
ists,’ to use a term by Navid Kermani (“Triumph des Vulgärrationalismus,” 2012),
are as similarly narrow-minded and intolerant as Islamic fundamentalists them-
selves. In his appeal for a more open and enlightened German identity, Şenocak also
considers philosophers that were critical of, or skeptical toward, Enlightenment
discourse. The catastrophes of the twentieth century have taught us that humans
are not exclusively rational beings: The schools of thought up to the 1980s, the Frankfurt School by Max
Horkheimer and Theodor Adorno, philosophers like Michel Foucault and
Emmanuel Lévinas represent in their work a perception of the complex
human being and undermine a cartesian understanding of human and so-
cial existence.5 Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 125 While philosophers like Horkheimer, Adorno, Foucault, and Lévinas held a com-
plex view of the enlightened human and understood the dialectics and dichotomies
of enlightenment discourse, Şenocak observes an increased tendency toward sim-
plification in public conversations since the 1980s (D 43). He notes that there has
been a regression since Frankfurt School thinking, a “Rückfall hinter die Dialektik
der Aufklärung der Frankfurter Schule” (D 49). The Dialectic of Enlightenment, first
published in 1944 and again in a revised version in 1947, shaped twentieth-century
discussions on the Enlightenment, but it seems to be no longer present in public
discourses. Some conservative pundits in the integration debate assert the sup-
posed superiority of Western European civilization because it underwent the his-
torical period of the Enlightenment, and believe that Muslims who have not inter-
nalized such an Enlightenment period cannot participate in a modern, enlightened
Western European society (D 46). These same critics, however, ignore the argu-
ments of the Frankfurt School that Enlightenment is also a dialectical process and
could not stop fascism and anti-Semitism in Europe, for example. 6 “Die Dialektik der Aufklärung […] mahnt auch heute daran, dass Vernunft schnell in Vernunft-
blind-heit umschlagen kann, wenn sie meint, das komplexe Wesen Mensch ganz und gar erklären zu
können, dass Modernisierung und Rationalisierung vor dem Bösen nicht gefeit sind, wenn sie wert-
neutral und instrumentell gedacht werden.” (D 178) An Enlightenment Tempered by its Dialectic The same ra-
tionalist world view could lead first to the creation of democracy and moderniza-
tion, and later also to the brutal destruction of war and genocide in the twentieth
century: The Dialectic of the Enlightenment [...] still reminds us today that rational-
ity can change quickly to blindness toward rationality when it believes it
can fully explain the complex human being, that modernization and ra-
tionalization are not protected from evil if they are considered neutral and
purely instrumental.6 In this context, Şenocak looks at the society of his origin, and he notes that Turkey
also underwent a historical period that can be viewed as an equivalent of the Euro-
pean Enlightenment. It is possible to see the radical transformations and moderni-
zations that Mustafa Kemal Atatürk created with the formation of the Turkish
Republic in 1923, after the collapse of the Ottoman Empire, as a form of Enlight-
enment. Şenocak believes that Kemalism, one of the great cultural revolutions of
human history (“eine der großen Kulturrevolutionen der Menschheitsgeschichte”
D 144), can easily be compared to the achievements of the European Enlighten-
ment. At the same time, Kemalism is characterized by its own dialectic, and this
rational process of Turkish modernization also coincided with nationalism and
violence in Turkey: In this context, Şenocak looks at the society of his origin, and he notes that Turkey
also underwent a historical period that can be viewed as an equivalent of the Euro-
pean Enlightenment. It is possible to see the radical transformations and moderni-
zations that Mustafa Kemal Atatürk created with the formation of the Turkish
Republic in 1923, after the collapse of the Ottoman Empire, as a form of Enlight-
enment. Şenocak believes that Kemalism, one of the great cultural revolutions of
human history (“eine der großen Kulturrevolutionen der Menschheitsgeschichte”
D 144), can easily be compared to the achievements of the European Enlighten-
ment. At the same time, Kemalism is characterized by its own dialectic, and this
rational process of Turkish modernization also coincided with nationalism and
violence in Turkey: 126 Vera Stegmann Modernization gave Turkey not only the literature of Schiller and Goethe,
the music by Beethoven and Bach and the ideas of the Enlightenment. 7 “Die Modernisierung schenkte der Türkei nicht nur die Lektüre von Schiller und Goethe, die Musik
von Beethoven und Bach und die Ideen der Aufklärung, sie bescherte ihr auch einen Nationalismus,
der zur Vertreibung und Ermordung von Hunderttausenden führte.” (D 178) An Enlightenment Tempered by its Dialectic Modernization also regaled Turkey with a nationalism that led to the dis-
persal and murder of hundreds of thousands of human beings.7 He specifically refers to Greeks, Armenians, and Kurds who suddenly had a non-
Turkish identity with the formation of the Turkish Republic (D 178-179), and his
statement on the murder of hundreds of thousands represents an indirect allusion
to the killings of Armenians (D 178). In the last chapter of Deutschsein, subtitled
“Vergessene deutsch-türkische Verwandtschaften” (Forgotten German-Turkish
Kinships), Şenocak draws subtle connections between Turkish and German histo-
ry. He is clearly looking at Kemalism as a form of Enlightenment and emphasizes
the great cultural achievements of Turkish modernity: the liberation of women and
their much more equal status today, for example, which allows women to assume
leading positions at universities, in journalism, or in business in Turkey, or new
freedoms in art and science (D 144, 176). At the same time, Şenocak believes that a
similar dialectical and complex approach to the Enlightenment that Adorno and
Horkheimer used to explain twentieth century German history and the trauma of
the Holocaust could be applied to modern Turkish history, since Atatürk’s innova-
tions went along with nationalism that threatened to exclude minorities in Turkey
(D 178-179). The Enlightenment that Şenocak invokes is thus tempered by its dialectic. Ra-
tionality cannot be cold and merely functional; it needs to acknowledge the histo-
ries and stories of each country. Understanding history is essential for a nation’s
sense of identity, Şenocak reminds us already in Das Land hinter den Buchstaben; but
we may not always learn from it, since the first war of humanity would have oth-
erwise been the last one (Şenocak 2006: 144). Regarding German literature,
Şenocak mentions in Deutschsein, besides Horkheimer and Adorno’s Dialektik der
Aufklärung, particularly Jewish authors, such as Franz Kafka, the Kabbalah re-
searcher and Jewish mystic Gershom Scholem, and the contemporary writer Bar-
bara Honigmann, whose stories shaped his creative writings (D 180). In the Turk-
ish context, he foregrounds the influence that Anatolian mysticism exerted on his
work and on the country’s intellectual and spiritual history, counterbalancing Ke-
malist rationalism. In one of his closing paragraphs, Şenocak describes the Islamic
tradition with which he identifies: I am part of this tradition of a Turkish Islam that is naturally enlightened
and European. 8 “Ich stehe in dieser Tradition eines türkischen Islam, der wie selbstverständlich aufgeklärt und
europäisch ist. Dieser Islam hat es aber in Deutschland nicht einfach, weil viele Menschen, die aus
der Türkei nach Deutschland kamen, ihn nicht verinnerlicht haben und weiter Traditionen anhängen,
die nicht durch die Aufklärung gefiltert worden sind. Das Vermächtnis meiner Herkunft fordert aber
auch jene Abendländer heraus, die ihr geistiges Erbe in eine kulturgeografische Landkarte mit klar
gezogenen Grenzen einsperren wollen.” (D 190) An Enlightenment Tempered by its Dialectic This Islam is not easily achieved in Germany, because
many people who came from Turkey to Germany did not internalize it Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 127 and continue to follow traditions that are not filtered through the Enlight-
enment. The legacy of my provenance, however, also challenges those
Westerners who wish to imprison their spiritual heritage in a cultural geo-
graphic map with clearly drawn borders.8 While strongly shaped by the Enlightenment, Şenocak equally argues against En-
lightenment fundamentalists who believe that the Western European norms of the
Enlightenment should be universal and everybody living in Europe should live
according to them. He rejects the exclusionary argument that the Enlightenment is
a uniquely European phenomenon. In arguing instead in favor of an enlightened
Islam, he criticizes both the fundamentalist Westerners who consider Europe and
Islam incompatible, as well as some Turkish fundamentalists who strictly adhere to
conservative traditions that have not been filtered through the Enlightenment. Şenocak’s Islam might be characterized as a tolerant and moderate form of Euro-
Islam. Language and Poetry While Deutschsein is a collection of essays and not a novel, drama, or a volume of
poetry, Şenocak’s approach is highly literary and poetic. Yeşilada emphasizes that
Şenocak is at heart a poet and analyzes his poetry with profound insight in Poesie der
Dritten Sprache. She observes that Şenocak is perceived primarily as an intellectual,
rather than a poet or literary author (Yeşilada 2012: 163), and she has identified a
‘blind spot’ in the critical literature: While his essays are widely reviewed and dis-
cussed, the reception of his large body of poetry is virtually absent in both journal-
istic as well as academic literature (“Blinder Fleck im Feuilleton,” Yeşilada 2012:
250). This is true not only in Germany, but also in the United States, where
Şenocak has a large readership among academics at universities. In the following, I
will not look directly at his own poetry, but rather point out the lyrical and literary
dimension of Şenocak’s concepts of an enlightened German identity and particu-
larly the role that language, poetry, and music play in it. y
g
g
p
y
p y
The emphasis on language is evident throughout his book, beginning with the
dedication to his father, who taught him that roots are multilingual (“dass Wurzeln
mehrsprachig sind,” D 5). This emphasis on multilingualism opens the path for a
pluralistic, tolerant, and open-minded worldview. Language and poetry also form
the subjects of several individual chapter titles: “Die Sprache öffnen” (Opening up Vera Stegmann 128 the Language), “Gebrochen Deutsch” (Broken German), “Dichter ohne Lieder”
(Poets without Songs), and “Mein Erbe spricht auch Deutsch” (My Heritage also
Speaks German, D 7-8). the Language), “Gebrochen Deutsch” (Broken German), “Dichter ohne Lieder”
(Poets without Songs), and “Mein Erbe spricht auch Deutsch” (My Heritage also
Speaks German, D 7-8). the Language), “Gebrochen Deutsch” (Broken German), “Dichter ohne Lieder”
(Poets without Songs), and “Mein Erbe spricht auch Deutsch” (My Heritage also
Speaks German, D 7-8). p
,
)
The first chapter, “Die Sprache öffnen: Wann bietet eine Fremdsprache Ge-
borgenheit?” (Opening the Language: When Does a Foreign Language Offer Shel-
ter?) opens with a citation by the poet Paul Celan: “ins Offene, dorthin, wo Spra-
che auch zur Begegnung führen kann” (into the open, where language can also lead
to an encounter, D 9). Celan’s words guide Şenocak’s discussions of his early
childhood in Bavaria. 9 “Sprache fließt, berührt und erzeugt Lust. Nichts ist von dieser Lust spürbar, wenn in Deutschland
über Integration und Sprachdefizite gesprochen wird. Es herrscht die kühle Atmosphäre eines La-
bors.” (D 18) Language and Poetry For him, Germany was at first not a country, but a foreign
language (“kein Land, sondern eine fremde Sprache,” D 11), a thought that echoes
the title of his earlier book Das Land hinter den Buchstaben. This new language of-
fered a sensual experience for the young author, for whom words have a taste:
‘Speaking is impossible without tasting the words,’ he states in numerous variations
in this chapter, or: ‘Languages speak to the senses. They sound, they taste.’ (“Spre-
chen geht nicht, ohne Wörter zu schmecken,” “Sprachen sprechen die Sinne an. Sie klingen, sie schmecken.” D 11, 15). Growing up bilingual and learning the dis-
tinction between “Muttersprache” and “Landessprache” (mother tongue, national
language, D 16) have been foundational and emotional experiences for the author. But words need to be connected to their roots, to an understanding of people and
their environment, otherwise they are denigrated to materialism and money – a
different form of currency (“eine andere Art von Währung,” D 18). Language is
for Şenocak the bridge between thinking and feeling (“die Brücke zwischen dem
Denken und dem Empfinden,” D 21). As language mediates between thinking and
feeling, it can cause joy and pleasure bordering on the erotic: Language flows, touches, and creates desire. None of this desire is percep-
tible in the discussions on integration and linguistic deficits in Germany. The cool atmosphere of a laboratory prevails.9 Language flows, touches, and creates desire. None of this desire is percep-
tible in the discussions on integration and linguistic deficits in Germany. The cool atmosphere of a laboratory prevails.9 This statement directly critiques the tone and language of the integration debates in
Germany, which have lost any creative potential. Şenocak believes that immigrants
in Germany will learn German more joyfully if they feel that their native language
is also accepted, if they don’t see an insurmountable clash between their native
tongue and the nation’s official language: two languages that do not reject one
another, but rather move toward each other in order to be able to translate (“zwei
Sprachen, die einander nicht abweisen, sondern sich aufeinander zubewegen, um
übersetzen zu können,” D 20). If, in our contemporary times of increasingly fluid
identities, a nation-state tries to impose its language too forcefully, then language,
the maternal tongue, becomes one of several possible places for withdrawal – Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 129 “Rückzugsorte” (D 22). Language and Poetry Language, a primary means of communication, can also
become a place to which immigrants can retreat and isolate themselves. p
g
The second chapter, entitled “Gebrochen Deutsch: Die Sondersprache der
Deutschen” (Broken German: The Distinctive Language of Germans, D 23-25),
begins with a geographic reference to a part of West Berlin, the short space be-
tween Rathenauplatz and Adenauerplatz.10 Kurfürstendamm, the avenue connect-
ing these two spaces, is no longer an elegant promenade in this area and symboliz-
es instead Germany’s broken identity: The physical walk from Rathenauplatz,
commemorating the murder of the German-Jewish foreign minister in 1922, to
Adenauerplatz, commemorating the first German chancellor after World War II
and the rebuilding of German democracy, also encompasses a temporal space, the
time of the downfall of the Weimar Republic, of National Socialism, and the de-
struction of war. Şenocak describes Walther Rathenau’s vision of a secular Judaism
that embraced German nationalism as a form of Enlightenment, but it collapsed
with the advance of fascism. In his view, this failure contributes to “Gebrochen
Deutsch” – the language that even a contemporary immigrant to Germany has to
learn to speak – since a broken identity is reflected in language (D 29-30). Under-
standing this broken German, the inner language of the land (“innere Landesspra-
che,” D 29), primarily results in understanding its taboos, the unspoken historical
scenes and memories that distinguish standard spoken German from the emotive
aspects of “Gebrochen Deutsch,” the hidden national language. “Can a national
feeling openly admit its fragility, its brittleness, without having to give itself up? In
which language is such a confession formulated?”11 Şenocak suggests that Ger-
mans should thematize the unresolved contradictions and the breaks and fissures
in their national identity more openly (D 32). A Germany that would freely discuss
its ‘broken’ language and identity might ultimately have a more positive self-image
and become a more open-minded, tolerant, and welcoming place for immigrants. A ‘broken German’ would be more compatible with the structure of a civilization
than any fantasy of a homogeneous national community, he states (“‘Gebrochen
Deutsch’ könnte sich mit Zivilisationsentwürfen besser vertragen als jede Fantasie
von einer homogenen Volksgemeinschaft,” D 68). A recognition and understand-
ing of a ‘broken’ history and past could become a point of departure for an identity
based on a worldly civilization, rather than a homogeneous culture. 10 Interestingly, Das Land hinter den Buchstaben also contains a chapter on Adenauerplatz, although the
tenor of the argument is slightly different here, focusing on German self-criticism despite Adenauer’s
achievements and on German lack of patriotism that ultimately inhibits a natural relationship to
Turkish immigrants (Şenocak 2006: 163-167). 11 “Kann sich ein Nationalgefühl offen zu seiner Brüchigkeit bekennen, ohne sich selbst aufgeben zu
müssen? In welcher Sprache wird ein solches Bekenntnis formuliert?” (D 27) Language and Poetry In evoking these two Berlin locations, Rathenauplatz and Adenauerplatz, as
symbolic spaces for a broken and divided identity, Şenocak could have added one
further highly emblematic geographical detail: On Adenauerplatz, one finds not 130 Vera Stegmann only a bronze statue of Adenauer, built in 2005, but also an earlier memorial stone
commemorating Mete Ekşi, a young Berliner, the son of Turkish immigrants, who
was brutally slain in his attempt to arbitrate a fight between Berlin youths of differ-
ent ethnicities. The red granite memorial stone was placed there in 1996, predating
Adenauer’s statue by nine years. It contains the following inscription: Mete Ekşi, born in 1972, died on November 13, 1991 from severe injuries
he sustained at this place during a violent confrontation between Berlin
youths of different backgrounds. Mutual respect and a commitment to
nonviolence could have protected his life.12 The sudden and tragic death that Mete Ekşi suffered also led to the establishment
of the “Mete-Ekşi Prize” for educational projects that promote peaceful intercul-
tural understanding among children and youths in Berlin. While Şenocak does not
mention the events surrounding Mete Ekşi in his discussion of Adenauerplatz, they
could have added persuasively to his description of a “Gebrochen Deutsch”: The
image of Adenauer’s statue and Ekşi’s memorial stone existing side by side proves
that Konrad Adenauer, the first West German chancellor who achieved West
Germany’s westernization – its integration into Western Europe and NATO –
could not solve Germany’s historical and present fractures and fissures that have
resulted in a fragile identity and a broken language. g
y
g
g
Another chapter, “Dichter ohne Lieder: Ein Exkurs in die deutsche
Nachkriegslyrik” (Poets without Songs: An Excursion into Postwar German Poet-
ry) explains Şenocak’s influences and his formation as a poet. Poetic language of
the early 1970s, when he began to write poetry, was characterized by a radical cri-
tique of language and a distrust of lyrical language, above all of the metaphorical
language of poetry (“Misstrauen gegenüber der lyrischen Sprache, vor allem
gegenüber der Metaphernsprache der Dichtung,” D 74-75). As a reaction against
the linguistic abuses of the Nazis, postwar German poetic language became con-
sciously unemotional. 12 “Mete Ekşi, geb. 1972, starb am 13. November 1991 an den schweren Verletzungen, die er an
diesem Ort bei einer gewalttätigen Auseinandersetzung zwischen Berliner Jugendlichen unterschiedli-
cher Herkunft erlitt. Gegenseitiger Respekt und der Wille zur Gewaltfreiheit hätten sein Leben schüt-
zen können.” (Bannas 2014: 4) Language and Poetry Şenocak could not identify with this dry poetic language and
reminds the reader of the famous clash between Paul Celan and members of
Gruppe 47 who critiqued the pathos in his reading of his poem “Todesfuge,” which
they considered excessive (D 81-82). Şenocak clearly sides with Paul Celan, the
Jewish poet from Czernowitz, who is an important poetic role model for Şenocak,
along with Ingeborg Bachmann, Rainer Maria Rilke, and Stefan George. But he
emphasizes that not just German poets, but also Turkish poets, such as Nâzım
Hikmet or the Anatolian Sufi Yunus Emre, influenced his German language poetry
by liberating his language emotionally. For him, the Turkish language allows a di- Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 131 rect, unbroken communication with emotions; in his youth, Şenocak perceived
Turkish as a language that confronted emotions directly, in an unfractured way,
and communicated with them (“Einer Sprache, die den Emotionen ungebrochen
gegenübertrat, die mit ihnen kommunizierte,” D 79). Şenocak had left his mother
country, “Mutterland Türkei,” but his mirror country, “Spiegelland Türkei,” the
country that mirrored, reflected, and contrasted with conditions in Germany, re-
mained an important influence in the development of his literary language (D 79). p
p
g
g
Yeşilada analyzes the importance of myth and a mythological approach to lan-
guage in Şenocak’s poetry, and she defines the words that comprise Şenocak’s
concept of myth, which in German and English all start with the letter ‘m’: meta-
phor, mystery, and modernity – “Mythos,” “Metapher,” “Mysterium,” “Moderne”
(Yeşilada 2012: 179). Myths and mysteries that explore our dark, irrational origins
can also be found in fairy tales – “Märchen,” to mention another m-word in Ger-
man. In Deutschsein Şenocak recommends that Germans and Turks look at the
language of childhood and the language of fairy tales as a point of departure for
understanding each other: Cultural encounters are always fertile when they are childhood encounters. No wonder that the language of fairy tales imposes itself here. But still to-
day, the Arabian Nights and Grimms’ Fairy Tales remain a hidden treasure
in the mediation of culture.13 Şenocak points to the novel Grimms Wörter, Günter Grass’s declaration of love to
the German language, and reminds the reader that fairy tales can be an important
contribution in achieving integration. 13 “Kulturbegegnungen werden immer dann fruchtbar, wenn sie Kindheitsbegegnungen sind. Kein
Wunder, wenn sich die Sprache der Märchen hier aufdrängt. Doch Tausendundeine Nacht und Grimms
Märchen sind bislang eher ein verborgener Schatz in der Kulturvermittlung.” (D 57) Language and Poetry In the library of a Turkish relative of
Şenocak’s, the Arabian tales were placed next to Luther’s translation of the Bible,
and Goethe’s Leiden des jungen Werther stood near the Koran (D 182-183). Besides
children’s literature and religious texts, German Romantic literature also plays an
important role in an essay that he subtitles “Aufklärungsschrift” (Enlightenment
Essay). Deutschsein abounds with references to German language writers, but also to
Turkish and Persian poets, such as Nâzım Hikmet, Asaf Hâlet, Behçet Necatigil,
Yunus Emre, Hafez, and Rumi. In addition to Muslim writers, he cites Jewish au-
thors: Besides Franz Kafka, Gershom Scholem, and Barbara Honigmann, Paul
Celan is one of his major influences. Şenocak’s understanding of an enlightened
German identity is a very humanistic and poetic one, shaped less by contemporary
politics than by poets and thinkers—for example, Heinrich von Kleist, Gotthold
Ephraim Lessing, Friedrich Schiller, Johann Wolfgang von Goethe, Immanuel
Kant, and Alexander von Humboldt. Şenocak evokes the international language of Vera Stegmann 132 world poetry, a subtle allusion to Goethe’s concept of world literature (“interna-
tionale Sprache der Weltdichtung,” D 80). This world literature is cosmopolitan,
and its influence reaches far beyond the original national borders of its creation. Şenocak also champions Humboldt’s concepts of a ‘Weltbürger,’ a citizen of the
world. He suggests that Germans should internalize Humboldt’s values and calls
for “ein Alexander von Humboldt für das Innere” (D 104). Such “Welterfahrung”
(experience of the world) and “Weltaneignung” (appropriation of the world, D
105), which are readily available in today’s globalized and interconnected world and
can also lead to isolation and alienation, need to be internalized through language
and literature. The foundation of identity and integration for Şenocak remains language; and
the ability of a society to integrate its minorities is closely linked to its facility in
coming to terms with its own national identity and history. However self-critical it
needs to be, a love of self is essential for loving and understanding the other. 14 “Die Frage der Integration ist also auf Gedeih und Verderb mit der Frage nach dem Deutschsein
verknüpft, mit der Krise des deutschen Nationalgefühls, mit einer Frage, die sehr lange nicht mehr
gestellt worden ist, weil sie zu sehr schmerzte, weil sie zu viele Albträume hervorrief, weil die Sprache
versagte.” (D 55) Language and Poetry For
Şenocak, the failure of integration is above all a failure of communication and of
language: The question of integration is existentially linked with the question of the
meaning of being German, with the crisis of the German national identity,
with a question that hasn’t been asked for a very long time, because it was
too painful, because it caused nightmares, because the language failed.14 Creating a language, and, as Şenocak analyzed in his earlier chapter on “Gebrochen
Deutsch,” coming to terms with and understanding a broken German language, is
essential for a sense of identity, which, in turn, will allow for greater generosity and
tolerance to minorities. Music Besides language and literature, music plays a large role in Deutschsein. Musical met-
aphors abound in Şenocak’s book. “Die atonale Welt” (The atonal world, D 36) is
the title of an entire chapter, and the image of atonality as a metaphor for a hetero-
geneous, mixed society weaves itself through the book. He observes that many
contemporaries perceive mixed societies as helplessly and with as much irritation
as they would regard an atonal composition. (“Viele Zeitgenossen stehen vor ge-
mischten Gesellschaften so ratlos und irritiert wie vor einer atonalen Kompositi-
on,” D 36). Atonal works, “atonale Werke,” are not palatable for a broader audi-
ence (D 36); modern day-to-day existence is characterized by too much atonality in Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 133 everyday life (“zu viel Atonalität im Alltag,” D 41). The average citizen longs for
music, but not of the atonal kind – “Musik, bitte weniger atonal” (D 54). We live in
atonal times, and our contemporary democracy challenges us to achieve living
together in atonal times (“ein Zusammenleben in atonalen Zeiten,” D 71). People
feel confronted with “Dissonanz und Atonalität” in contemporary complex socie-
ties (D 59). Dissonance and atonality may be perceived similarly, but have a differ-
ent meaning in music. While dissonance primarily refers to a tension, clash, and
lack of harmony among musical notes, atonality, the structure of twentieth century
and contemporary music that lacks a key or a tonal center, is indeed a beautiful
metaphor for multiculturalism in society. In contrast, tonality, the classical system
of European music that centers around a key and a hierarchy of musical relations,
could symbolize Leitkultur. y
A related musical metaphor is polyphony. Şenocak asserts that immigration has
produced a polyphonic sound in Germany. He criticizes the rhythm of assimilating
or equalizing (“Rhythmus des Angleichens”) in post-unification Germany, which
did not lead to the desired harmony (“ersehnten Harmonie”) and he recommends
an acceptance of a polyphonic sound (“Akzeptieren eines polyphonen Klangs”)
that immigrants brought to Germany (D 58). Şenocak also notes an absence of nuances and overtones (“Zwischentöne”) in
the polarized atmosphere of the integration debate (D 49). Such intermediate
tones, however, should be defining features of an immigration society. “Zwischentöne” as a metaphor for a hybrid society already appeared in his earlier
book Das Land hinter den Buchstaben (Şenocak 2006: 66). 15 “Jedes Wort ist nicht nur aus Buchstaben gemacht. Es hat immer auch eine Melodie. Vielleicht sind
die heiligen Schriften durch eine besondere Musik erkennbar. Der Koran ist es auf jeden Fall. Noch
heute ist die Sprachmusik dieses Textes ergreifend, der Rhythmus unverkennbar.” (D 174) Music Just as “Zwischentöne”
form part of a more complex, subtle piece of music, he now proposes a more nu-
anced, sensitive, differentiated language (“sensiblen, differenzierten Sprache”) to
replace the gladiator’s language (“Gladiatorensprache”) of some recent talk show
debates (D 44). Music and literature are innately intertwined: Each word is not only composed of letters. It also always has a melody. Maybe the holy scriptures are recognizable by a special music. The Koran
definitely is. Still today, the linguistic music of this text is profoundly mov-
ing, the rhythm unmistakable.15 For Şenocak, poetry and religious texts are intensely musical. In discussing the
Koran, Şenocak emphasizes less its laws than its unique music – its aesthetic, poet-
ic, and musical beauty. When he entitles his chapter on twentieth century German
postwar poetry as “Dichter ohne Lieder” (poets without songs), he critiques these
poets’ deliberately prosaic approach to language (D 75). Poetry without song is
Şenocak’s metaphor for a poetry without emotion, without sensuality, without a 134 Vera Stegmann lyrical language. His own poetry, on the contrary, began as a “Hörerlebnis,” the
acoustic experience and sensual pleasure of listening to new languages and trans-
forming sounds to words (D 80). Şenocak’s poetry was shaped in his early years by
recitations of the Koran which he describes as a song (“Gesang”) and by the mys-
tical songs (“mystischen Gesängen”) of the medieval Anatolian Sufi Yunus Emre
(D 79-80). In Şenocak’s view, it isn’t an accident that music became the original German
language (“nicht zufällig wurde die Musik zur deutschen Ursprache,” D 70). This is
a reference to German Romanticism, which saw poetry as the closest kin to the
original art form of music. German cultural identity was created out of the spirit of
music – “aus dem Geiste der Musik,” as Şenocak states with a subtle reference to
Friedrich Nietzsche’s Geburt der Tragödie aus dem Geiste der Musik (D 71). Nietzsche
was intensely preoccupied with Richard Wagner throughout his life; he initially
admired Wagner fervently in his early work Geburt der Tragödie and then turned
against him as a fierce critic in his later writings Der Fall Wagner and Nietzsche contra
Wagner. In evoking Nietzsche, and especially by only alluding to Nietzsche’s early
supportive work on Wagner, Şenocak reminds the reader of the importance of
Richard Wagner’s music for German national identity. Music Wagner is largely responsi-
ble for the fact that Germany became the country of transfigured Romanticism
and Dionysian music (“das Land der verklärten Romantik und einer rauschhaften
Musik,” D 60). The orgiastic sensuality of Wagner’s music and its emphasis on
mythology certainly shaped German Romanticism. One might debate, however,
whether Wagner’s music still is a refuge of the cultural self-image of Germans, as
Şenocak states (“ein Refugium des kulturellen Selbstverständnisses der
Deutschen,” D 72). Wagnerian music definitely forms an important part of the
German tradition. But Wagner himself is also a controversial figure because of his
antisemitic writings, for example; and younger contemporary Germans may choose
other musical role models as well. At the same time, Şenocak himself seems profoundly shaped by a Romantic
world view, which he claims was more influential in Germany than elsewhere (D
170). When he defines the origins of German culture as “dunkel und musisch,” he
admits that the thought of describing any culture as dark and musical at its core is
highly attractive for him (D 70). The frequent reoccurrence of words like roots or
home (“Wurzeln” or “Heimat,” i.e. D 56, 70) could also be traced back to Roman-
ticism, as could his emphasis on myth and fairy tales, on emotionality and an inter-
nalized soul. Şenocak rejects a social contract based purely on the cold ink of ra-
tional reason (“mit der kalten Tinte der rationalen Vernunft,” D 171). In the last
paragraph of his book, he indirectly refers to the German folk song “Die Gedank-
en sind frei” (The Thoughts Are Free) which originated in the Middle Ages, but
was also included in Achim von Arnim and Clemens Brentano’s Romantic poetry
collection Des Knaben Wunderhorn (D 190). As Şenocak explained to Alan Posener
in his interview with Die Welt, his focus of studies at the University of Munich was
German Idealism, a philosophical movement in the late eighteenth and early nine- Deutschsein: Şenocak’s Enlightened Vision of a Cosmopolitan German Identity 135 teenth century that began as a reaction to Kant and was associated with Romanti-
cism and the revolutionary politics of the Enlightenment. While Romanticism is
traditionally perceived as a reaction against the Enlightenment, Şenocak’s “Aufklä-
rungsschrift” Deutschsein is not orthodox in its Enlightenment discourse and incor-
porates some Romantic elements. Turkish sources of literary inspiration for Şenocak’s writings include Ottoman
divan poetry and Sufi mysticism. 16 “Die anatolische Mystik, diese spezielle Form einer musisch und sozial inspirierten Gläubigkeit, die
ihre Wurzeln sowohl in koranischen Glaubenssätzen als auch in schamanistischen Naturbetrachtun-
gen findet, bildete das Fundament eines modernen, weltoffenen, aufnahmebereiten Islam.” (D 177) Music In Deutschsein, he primarily refers to Anatolian
mysticism, which he also describes as musical: Anatolian mysticism, this special form of a musically and socially inspired
faith, which has its roots in both Koranic doctrines as well as shamanistic
nature contemplations, formed the foundation for a modern,
cosmopolitan, and receptive Islam.16 He sees no contradiction between Anatolian mysticism and a modern, open, and
tolerant Islam – an Anatolian atonality, one could call it, applying Şenocak’s musi-
cal metaphor. Rumi’s or Emre’s mysticisms could be invoked as a countermove-
ment to a rigid fundamentalist Islam, which might explain their modern appeal, as
Yeşilada points out (Yeşilada 2012: 81). In Deutschsein, Şenocak combines the uni-
versal values of the Enlightenment with Anatolian and Islamic mysticism; and
through this association he also creates a dialog between “Deutschsein und Tü-
rkischsein” (Uysal Ünalan 2013: 84-85) Mysticism can be a component of the Romantic movement, and Şenocak
seems to be influenced by both. When he describes the atmospheric sounds of
being German (“die atmosphärischen Klänge des Deutschseins,” D 71), this
statement could be an evocation of Wagner’s music that he just discussed; and he
also refers to the mystical search of the outsider to become part of a new environ-
ment. When he invokes the poetic concept of a kinship of souls (“Seelen-
verwandtschaft,” D 48), he formally refers to Stefan George’s idea of a spiritual
inner unity in Geheimes Deutschland. But he may indirectly also imply an imagined
kinship of souls between Germans and Turks, possibly the kindred spirits of Ger-
man Romanticism and Turkish or Islamic mysticism. Şenocak searches for a rationality that is not spiritually dried up (“eine spirituell
nicht vertrocknete Rationalität,” D 177). His vision of a German identity is highly
aestheticized, poetic, and musical. In his language and his world view, mysticism
and modernity are compatible. Şenocak’s concept of the Enlightenment is a well-
tempered one, to continue a musical metaphor, one that understands its dialectic
and leaves room for spirituality and emotionality. In Deutschsein, Şenocak infuses 136 Vera Stegmann his poetic and musical call for a new Enlightenment with mysticism and certain
features of Romanticism. his poetic and musical call for a new Enlightenment with mysticism and certain
features of Romanticism. Works Cited Bannas, Günter (2014): “Kein Platz für Vornamen,” in: Frankfurter Allgemeine
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Philosophische Fragmente, Frankfurt. Kermani, Navid (2014): Wer ist Wir? Deutschland und seine Muslime, Munich. ermani, Navid (2014): Wer ist Wir? Deutschland und seine Muslime, Munich. --- (2012): “Triumph des Vulgärrationalismus,” in: Süddeutsche Zeitung, 2.08.2012,
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literaturkritik.de/public/ rezension.php?rez_id=15972 [last accessed:
04.09.2016]. Stranakova, Monika (2011): “Ein Land tritt auf der Stelle,” accessible at: http://
literaturkritik.de/public/ rezension.php?rez_id=15972 [last accessed:
04.09.2016]. Uysal Ünalan, Saniye (2013): Interkulturelle Begegnungsräume: Neue
Identitätskonstruktionen in der türkisch-deutschen Gegenwartsliteratur, Würzburg. Yeşilada, Karin E. (2012): Poesie der Dritten Sprache: Türkisch-deutsche Lyrik der zweiten
Generation, Tübingen. Zaimoglu, Feridun (2008): Liebesbrand, Köln. ermani, Navid (2014): Wer ist Wir? Deutschland und seine Muslime, Munich. Zaimoglu, Feridun (2008): Liebesbrand, Köln. Transkulturelles Lernen mit mehrsprachigen
Bilderbüchern Serap Atagül/Christian Müller Einleitung In den letzten Jahren ist eine steigende Anzahl an Veröffentlichungen mehrspra-
chiger Bilderbücher zu verzeichnen, die bisher noch nicht auf Aspekte transkultu-
rellen Lernens untersucht wurden. Sie laden Rezipienten dazu ein, im Identifizieren
von Transkulturalität Unterschiede und Schnittmengen deutscher und türkischer
Kultur zu entdecken, ihre Kompetenzen in Visual Literacy zu erweitern und sich an
sprachliche Varietäten von Deutsch und Türkisch anzunähern. p
In diesem Beitrag, der sich an der Textsorte ‚Review Essay‘ orientiert, wird for-
schungsmethodisch in deskriptiver Herangehensweise eine Auswahl an deutsch-
türkischen Bilderbüchern beleuchtet. Auf Grundlage dieses Vorgehens lassen sich
mögliche Arbeitsfelder für ein transkulturelles Lernen gewinnen: Als Untersu-
chungsschwerpunkte werden u. a. das Kennenlernen und die Akzeptanz unter-
schiedlicher Kulturen und Sprachen, lebensweltliche Gemeinsamkeiten und Zu-
sammenhänge sowie ein bewusster, kritischer Umgang mit Klischees und Vorurtei-
len angestrebt. Über das transkulturelle Lernen hinaus halten die Bilderbücher auch
sprach- und literaturdidaktische Implikationen bereit, auf die aus Platzgründen nur
vereinzelt eingegangen werden kann; das Gleiche gilt für die didaktisch-
methodischen Erwägungen zur Mehrsprachigkeit. Serap Atagül/Christian Müller 140 Als Untersuchungsgegenstände dienen die Bilderbücher Arkadaş Olalım Mı / Sollen
wir Freunde werden (2002) von Neşet Erol und Mustafa Delioğlu, Fatma und / ile
Martha (2005) von Zeynep Bassa, Sinan und Felix. Mein Freund Arkadaşım (2014)
von Aygen Sibel Çelik und Barbara Korthues und Leyla und Linda feiern Ramadan. Leyla ve Linda Ramazanı kutluyorlar (2011) von Arzu Gürz Abay und Sibel Demirtaş,
deren erstmalige wissenschaftliche Betrachtung und die aus dieser gewonnenen
transkulturellen Lernpotentiale einen Beitrag zur frühzeitigen Akzeptanz und
Wertschätzung der deutschen, türkischen und weiterer Kulturen leisten könnten. Als Untersuchungsgegenstände dienen die Bilderbücher Arkadaş Olalım Mı / Sollen
wir Freunde werden (2002) von Neşet Erol und Mustafa Delioğlu, Fatma und / ile
Martha (2005) von Zeynep Bassa, Sinan und Felix. Mein Freund Arkadaşım (2014)
von Aygen Sibel Çelik und Barbara Korthues und Leyla und Linda feiern Ramadan. Leyla ve Linda Ramazanı kutluyorlar (2011) von Arzu Gürz Abay und Sibel Demirtaş,
deren erstmalige wissenschaftliche Betrachtung und die aus dieser gewonnenen
transkulturellen Lernpotentiale einen Beitrag zur frühzeitigen Akzeptanz und
Wertschätzung der deutschen, türkischen und weiterer Kulturen leisten könnten. Transkulturelles Lernen Im Jahr 1993 konstatiert Bredella, es sei „eine Abstraktion, die fremde Kultur im
Gegensatz zur eigenen bestimmen zu wollen. Es gibt schon immer Überschnei-
dungen, wie es Unterschiede gibt“ (Bredella 1993: 34). Kulturelles Lernen ‚zwi-
schen‘ verschiedenen Kulturen wird bis heute zumeist unter interkulturellem Ler-
nen subsumiert; Welsch fragt mit dem Begriff ‚Transkulturalität‘ (vgl. Welsch 1994,
1995) nicht nach dem ‚Zwischen‘, nicht nach dem Nebeneinander und der Additi-
on von Kulturen im bikulturellen Sinne, sondern konturiert mit seiner Konzeption
die Überschreitung und Verbindung von verschiedenen Kulturen sowie einer hier-
durch möglichen Identitätsentfaltung (vgl. Welsch 1994, 1995). An Welsch an-
knüpfend formuliert Wintersteiner das Postulat einer transkulturellen literarischen
Bildung. Er bezeichnet hierbei „kulturelle Mischung, Multikulturalität und Mehr-
sprachigkeit nicht [als] den Ausnahmezustand, sondern [als] den Normalzustand“. In diesem Beitrag wird Wintersteiner im Besonderen entsprochen, da er Transkul-
turalität als „einen positiven Wert darstellt, der [...] Monokulturalität und Monolin-
guismus ablösen“ (Wintersteiner 2006: 15) wird oder – je nach subjektiver Betrach-
tungsweise – bereits abgelöst hat. Die Ermöglichung transkulturellen Lernens setzt
eine entsprechende Einstellung, Überzeugung und wenigstens eine Haltung voraus,
die im besten Falle von Authentizität geprägt sein sollte, sodass Transkulturalität
außerhalb und innerhalb der Schule gemeinsam ‚gelebt‘ werden kann. Transkultu-
relles Lernen geht unseres Erachtens darüber hinaus, „Schülerinnen und Schüler
für das Konzept der Transkulturalität zu sensibilisieren“ (Walden 2010: 30). Für
Kinder und Jugendliche stellt sich allzu oft die Frage nach einer Entscheidung für
eine einzige Kultur oder Sprache nicht mehr, oder sie haben sie sich nie gestellt, da
sie die Koexistenz von Kulturen und Sprachen nicht als solche wahrnehmen, son-
dern in einem selbstverständlichen Akt der Transkulturalität a priori verbunden
haben. Kinder und Jugendliche dürfen sich unterschiedlichen Kulturen zugehörig
fühlen, können und dürfen verschiedene Sprachen sprechen. Ihr transkulturelles
Handeln nehmen sie weder als Konzept noch in der Alltagswirklichkeit als etwas
Besonderes wahr, da eine transkulturelle Praxis für sie selbstverständlich ist. Das
bedeutet nicht, dass transkulturelles Handeln per se existiert und transkulturelles
Lernen obsolet wird – im Gegenteil: Es ist und bleibt eine Suche nach (auch
sprachlichen) Unterschieden und Gemeinsamkeiten transkultureller Art (vgl. Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 141 Rösch 2001: 111) mit dem Ziel der Akzeptanz aller Kulturen. Der Einsatz mehr-
sprachiger Bilderbücher als transkulturelle Lerngegenstände kann diese Suche be-
günstigen. Rösch 2001: 111) mit dem Ziel der Akzeptanz aller Kulturen. Der Einsatz mehr-
sprachiger Bilderbücher als transkulturelle Lerngegenstände kann diese Suche be-
günstigen. 1 Für drei unseres Erachtens sehr gelungene lernförderliche Unterrichtsmodelle, die hier nicht im
Einzelnen beleuchtet, jedoch genannt werden sollen‚ siehe Decker / Oomen-Welke (2008), Dirim
(2009) und Vach (2009). 2 Die Bilderbücher wurden ausgewählt nach den Kriterien der Mehrsprachigkeit – bestehend aus
Texten in deutscher und deren Übersetzungen in türkischer Sprache – und Transkulturalität. Die
Anzahl von vier Bilderbüchern ist dem einzuhaltenden Höchstumfang dieses Beitrags geschuldet; die
Einbindung weiterer mehrsprachiger Bilderbücher wäre folglich möglich gewesen. An dieser Stelle sei
auch auf andere zu untersuchende Bilderbuchformate hingewiesen, die sprachliches – über das Deut-
sche und Türkische hinaus –, literarisches und transkulturelles Lernen fördern könnten: Das digitale
Bilderbuch Glück der Autorin Elisabeth Simon mit Illustrationen von Lena Ellermann (online ver-
fügbar unter: http://www.amira-pisakids.de/#book=12&p=1 [letztes Zugriffsdatum: 25.10.2016])
auf dem Portal des kostenfreien Leseförderprogramms ‚Amira‘ kann auf verschiedenen Sprachen –
darunter auch auf Türkisch – rezipiert werden. Die Lektüre und das zur Verfügung stehende interak-
tive Quiz können transkulturelle Anschlussmöglichkeiten für den Deutschunterricht bereithalten. Transkulturelles Lernen g
g
Kindern und Jugendlichen kann transkulturelles Erkennen und Handeln nicht
immer intuitiv gelingen. Sie müssen in ihrer Identitätsbildung – ein weiteres Ziel
des transkulturellen Lernens – unterstützt und angeleitet werden. Transkulturalität
kann einen Transfer von Aspekten einer Kultur in eine andere anstreben, „etwa bei
Übersetzungen, die den Verstehens- und Kommunikationsprozess in der Sprache,
in die übersetzt wird, mit reflektieren und bewusst gestalten“ (Rösch 2007: 53). Die
Übersetzungen in den mehrsprachigen Bilderbüchern begünstigen einen solchen
Transfer, da die Texte auf Deutsch und Türkisch gelesen und beleuchtet werden
können. Obwohl sie nicht alle auf ihre Lernpotentiale bezüglich Mehrsprachigkeit
analysiert werden können, sollen mehrsprachige Bilderbücher in diesem Beitrag als
„kulturvermittelnde Texte [gelten], die einem anderskulturellen Lesepublikum eine
ihnen fremde Kultur nahe zu bringen versuchen“ (Rösch 2007: 53), denen be-
stimmte transkulturelle Implikationen inhärent sind, die für den Deutschunterricht
bisher noch nicht perspektiviert wurden. Halten Elemente des DaF- und DaZ-
Unterrichts im Lehramtsstudium immer mehr Einzug, finden sich noch selten
Unterrichtsmodelle, die beispielsweise die deutsche und türkische Sprache, die
verschiedenen und gemeinsamen kulturellen Aspekte für den Deutschunterricht
lernförderlich aufbereiten.1 Transkulturelles Lernen bedeutet nicht zwangsläufig, eine Übernahme anderer
Kulturen anzustreben (vgl. Rösch 2007: 53). Es soll jedoch auch keine Abgrenzung
von anderen Kulturen stattfinden. So kann zunächst eine „bikulturelle Doppelexis-
tenz“ (Rösch 2007: 53) ausgebildet werden, die unterschiedliche kulturelle Aspekte
in sich trägt und sich auf dem Wege eines transkulturellen Lernprozesses zu einer
transkulturellen Identität weiterentwickelt. Auch bei der Erreichung dieses Ziels
können Bilderbücher von Anfang an unterstützend wirken. Die möglichen Einblicke genuin bikultureller Art im Nebeneinander von zwei
Kulturen und auch die isoliert betrachtbaren interkulturellen Dimensionen zwi-
schen der deutschen und türkischen Kultur, die die vorliegenden mehrsprachigen
Bilderbücher bieten, werden unter die transkulturellen Lernpotentiale subsumiert,
sodass „das ‚verbindende Gemeinsame‘, das kulturübergreifende Rezeptionen
ermöglicht, nicht in der Zugehörigkeit zu einer bestimmten allgegenwärtigen Kul-
tur zu suchen [ist], sondern in den gemeinsamen Elementen, die durch die Kultu-
ren hindurch gehen“ (Iljassova-Morger 2009: 52). Serap Atagül/Christian Müller 142 Mehrsprachige Bilderbücher im Spiegel transkulturellen Lernens 3 Die Bildrechte waren bedauerlicherweise nicht zu erhalten. Mehrsprachige Bilderbücher im Spiegel transkulturellen Lernens Im Folgenden werden vier mehrsprachige Bilderbücher2 bezüglich ihrer transkultu-
rellen Lernpotentiale für eine heterogene Grundschulklasse vorgestellt. Dies be-
deutet, dass alle Schüler als Mitglieder der hiesigen Gesellschaft in die Überlegun-
gen einbezogen sind, unabhängig von ihrem kulturellen oder sprachlichen Hinter-
grund. Dementsprechend verweist Rösch zum Umgang mit Bilderbüchern im
Literaturunterricht explizit darauf, dass kein Schüler stigmatisiert werden solle, alle
die gleichen Aufgaben bekommen sollten, dass jeder frei in seiner Interpretation ist
sowie fähig, Deutungen der Mitschüler zu erkennen (vgl. Rösch 1997: 194). Nur in
einer auf einer transkulturellen Haltung gründenden Unterrichtsplanung und
-durchführung können Lehrpersonen aus sprachdidaktischer Sicht einer „mono-
lingualen Fokussierung des Deutschunterrichts“ (Engin / Olsen 2009: 8) entge-
genwirken und – aus literaturdidaktischer Perspektive – „transkulturelle Erfah-
rungsmomente“ (Engin / Olsen 2009: 12) an literarischen Gegenständen, wie den
ausgewählten mehrsprachigen Bilderbüchern, bereitstellen. Die Teilkapitel zu den
transkulturellen Implikationen enthalten teilweise sprach- und literaturdidaktische
Überlegungen, die für den Deutschunterricht von Interesse sein könnten. An die
jeweiligen Inhaltsangaben anschließend werden u. a. die Visualisierung, die Figu-
ren- und Handlungsgestaltung, die Mehrsprachigkeit (dargestellt am Beispiel Sinan
und Felix) und das Erkennen der Unterschiede und Gemeinsamkeiten der deut-
schen und türkischen Kultur thematisiert. Daraus lassen sich dann transkulturelle
Lernprozesse entwickeln. Mehrsprachige Bilderbücher werden als literarische Tex-
te präsentiert, denen als Lerngegenstand eine passende Eignung zugedacht wird, da
sie unterschiedliche Sprachen vereinen und anhand der Texte sowie deren Visuali-
sierungen verschiedene Kulturen schon für Rezipienten im Grundschulalter er-
fahrbar werden. Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 143 Arkadaş Olalım Mı? Sollen wir Freunde werden?
Neşet Erol / Mustafa Delioğlu (2002) Neşet Erol erzählt eine Geschichte, die – begleitet von stetem Zweifel und Vorur-
teilen – in eine Freundschaft mündet. Markus besucht eine neue Schule und wird
seinen Klassenkameraden vorgestellt. Yusuf, der alleine sitzt, bietet Markus einen
freien Platz neben sich an, den er gerne annimmt. In der Pause stellt sich Yusuf
Markus mit dem Namen „Josef“ vor. Markus betrachtet Yusuf verblüfft und merkt
bezüglich Yusufs Teint an, er sei doch so braun gebrannt, „fast schwarz“. Darauf-
hin macht Yusuf Markus auf dessen helle Haut und blonden Haare aufmerksam:
Sie lachen und beschließen Freunde zu werden. Am nächsten Schultag hört Mar-
kus, wie sein Freund Josef von Holger, einem anderen Klassenkameraden, mit
„Yusuf“ angesprochen wird. Markus spricht Holger darauf an, der laut auflacht, als
er hört, dass Markus Yusuf „Josef“ nennt. Markus wird daraufhin aufgeklärt, dass
„Josef“ der deutsche und „Yusuf“ der türkische Vorname sei. Dies erkenne man
an seinem äußeren Erscheinungsbild – schwarze Augen und schwarze Haare. Ver-
ärgert packt Markus seine Schultasche und setzt sich zu Rainer. Als Yusuf ihn hier-
zu befragt, entgegnet ihm Markus, dass er mit Türken nicht beisammensitzen
möchte und beendet seine Freundschaft mit ihm. Yusuf ist traurig darüber, aber
ihm ist ein solches Verhalten seiner Mitschüler bekannt. Die Ansichten von Mar-
kus’ Vater sind geprägt von Vorurteilen und Klischees und scheinen Markus zu
beeinflussen: Türken nähmen den Deutschen die Arbeitsplätze weg, türkische
Frauen verhüllten ihre Gesichter sowie Türken und Deutsche sollten jeweils lieber
unter sich Freundschaften schließen. Yusuf hingegen träumt von einer glücklichen
Freundschaft. Im weiteren Verlauf der Geschichte wird Markus von einem Hund
gebissen und Yusuf eilt zur Hilfe herbei. Beide müssen ins Krankenhaus eingelie-
fert werden. Dort bedanken sich Markus’ Eltern bei Yusuf und nähern sich Yusufs
Eltern an. Der Autor stellt des Weiteren dar, dass sich die Schüler der Klasse näher
kennenlernen, Freundschaften schließen und sich gegenseitig helfen. Letztlich
werden sie die „freundlichsten, fleißigsten und erfolgreichsten Kinder an der Schu-
le“ ( Erol / Delioğlu 2002: 37). Transkulturelle Implikationen p
Der Illustrator zeigt auf einer Doppelseite3 (Erol / Delioğlu 2002: 18-19) eine
Schulklasse, in der sich Kinder unterhalten, die möglicherweise einen unterschied-
lichen kulturellen und sprachlichen Hintergrund haben. Im Hinblick auf transkul-
turelles Lernen ist die Doppelseite geeignet, den Rezipienten zu verdeutlichen,
„dass die Gesellschaften aus disparaten Kulturen, aus verschiedenen sozialen
Schichten, Geschlechtern und ethnischen Gruppen bestehen und kulturelle Hybri-
dität zum wichtigsten Kennzeichen wird“ (Engin / Olsen 2009: 4). Dies kann im
Deutschunterricht durch einen Austausch der Eindrücke über Verhaltensweisen Serap Atagül/Christian Müller 144 der Figuren Yusuf und Markus geschehen. Die unterschiedlichen Bedürfnisse und
Wünsche von Yusuf können aus der Innensicht nachempfunden und somit als
Erzählanlass genutzt werden, in dessen Rahmen die Menschen als Individuum und
Persönlichkeit anerkannt werden: Jeder darf so sein, wie er ist. der Figuren Yusuf und Markus geschehen. Die unterschiedlichen Bedürfnisse und
Wünsche von Yusuf können aus der Innensicht nachempfunden und somit als
Erzählanlass genutzt werden, in dessen Rahmen die Menschen als Individuum und
Persönlichkeit anerkannt werden: Jeder darf so sein, wie er ist. J
Die beiden Hauptfiguren Yusuf und Markus stehen auf der Doppelseite visuell
nicht im Vordergrund, wie das zu vermuten wäre, sondern sind in der Klassenge-
meinschaft integriert abgebildet. Auf dem Bild scheint eine entspannte, freund-
schaftliche Atmosphäre zwischen den Kindern zu herrschen, die durch den Text
unterstützt wird. Die Bild-Text-Beziehung suggeriert – wird sie in transkultureller
Haltung gelesen –, dass jedem Individuum Bedeutung zukommt, unabhängig von
Herkunft, Sprache oder kulturellen Unterschieden. Dies unterstreichen auch die
offenen Fragen, die den Leser aus der Lektüre entlassen und als eine den Text
schließende Moral gedeutet werden könnten: Freundschaften zu schließen kann manchmal schwierig sein und braucht
oftmals Zeit. Ist es aber nicht viel wichtiger, dass Menschen einander ken-
nen lernen, ihre gegenseitigen Vorurteile abschaffen und den Willen ha-
ben, Freunde zu werden? Haben solche Bemühungen nicht Erfolg und
Glück? (Erol / Delioğlu 2002: 39) Die letzten Worte Erols können auf Grund der außergewöhnlichen Sprachver-
wendung „Haben solche Bemühungen nicht Erfolg und Glück?“ (Erol / Delioğlu
2002: 39) zu Irritationen führen. Möglicherweise wurde hier vom Türkischen ins
Deutsche übersetzt, da beispielsweise „her zaman“ (Erol / Delioğlu 2002: 38)
gebraucht wird und das nötige Wort „immer“ [eigene Übersetzung; S.A. / C.M.]
im deutschen Text keine Erwähnung findet. Transkulturelle Implikationen Dieser Satz könnte, bezugnehmend
zur vorherigen Seite, folgendermaßen verstanden werden: Die Bemühungen, ei-
nander kennenzulernen, Vorurteile abzubauen und Freundschaften zu schließen,
können zum Erfolg in der Schule beitragen, da sich die Schüler gegenseitig unter-
stützen (vgl. Erol / Delioğlu 2002: 36-39) – Erols Text weist hier verschiedene
Wege aus, die zu transkulturellem Lernen führen. g
Die von Erol im Text angesprochenen Vorurteile sind in diesem Bilderbuch
vor allem durch stereotypisierte Darstellungen gekennzeichnet: Yusuf, der Mit-
schülern vornehmlich durch seine schwarzen Haare, dunkle Augenfarbe und Haut
auffällt sowie Markus, der „hellblonde Haare, hellblaue Augen und eine weiße
Hautfarbe [hat], so als hätte er nie einen Sonnenstrahl abbekommen“ (Erol /
Delioğlu 2002: 3). Die Wahrnehmung Yusufs seitens Markus ist beeinflusst durch
die Vorurteile des Vaters, der darauf hinweist, dass man mit „Türken [...] keine
Freundschaften schließen [könne]. Die Deutschen sollen mit den Deutschen und
die Türken mit den Türken Freunde werden“ (Erol / Delioğlu 2002: 19). Auffal-
lend ist auch der Versuch Yusufs, durch eine seinen türkischen Namen verschlei-
ernde Änderung in den deutschen Namen „Josef“, eine Freundschaft mit Markus Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 145 aufzubauen. Markus’ empörte Reaktion auf die Klarstellung, dass „Josef“ eigentlich
„Yusuf“ heißt, lässt sich aus seinen Bemerkungen ablesen: aufzubauen. Markus’ empörte Reaktion auf die Klarstellung, dass „Josef“ eigentlich
„Yusuf“ heißt, lässt sich aus seinen Bemerkungen ablesen: Ich dachte, er wäre ein deutscher Junge. Ich hätte es eigentlich an seinen
schwarzen Haaren, den schwarzen Augen und der bräunlichen Haut er-
kennen müssen. [...] Er will sich wie ein deutscher Junge aufführen, nur
um mein Freund zu werden. Ich lasse mich nicht verarschen. (Erol /
Delioğlu 2002: 13) Yusufs Nutzung eines deutschen Namens suggeriert, er wolle sich der deutschen
Gesellschaft und seinem schulischen Umfeld zugehörig fühlen. Yusuf fühlt sich
zudem in der Schule nicht willkommen, aber „so etwas passierte ihm nicht zum
ersten Mal. Seine deutschen Mitschüler wollten nichts mit ihm zu tun haben, aber
sie sagten auch nichts Schlimmes“ (Erol / Delioğlu 2002: 15). Ein auf bikulturelle
Identitätsbildung gründendes transkulturelles Lernen könnte hier an die Überwin-
dung der kulturellen Grenzen anknüpfen, die Yusuf anzustreben versucht. Jedoch
ist „die Entwicklung einer bikulturellen Identität [...] abhängig von einem Sich-
Zurechtfinden in beiden Kulturkreisen und der Interaktion in beiden gesellschaftli-
chen Systemen“ (Kalkavan 2013: 257). 5 Diese und die folgenden kapitalisierten Wörter sind aus dem Originaltext zitiert. 4 Die Bildrechte waren leider nicht zu erhalten. 6 Kritisch zu betrachten sind einige im Folgenden genannten Elemente des Bilderbuchs: Die Buchsei-
ten sind zum Teil mit Schrift überfüllt und wirken unübersichtlich, was die Lesefreude einschränken
könnte. Grotesk auf Grund ihrer Bedeutungslosigkeit wirkt vor allem eine Kreatur, der Fatma auf
dem Schulweg begegnet und sagt: „BRING MICH ZU DEINEM ANFÜHRER!“ (Bassa 2005: 14).
Was sich die Autorin hierbei dachte, bleibt im Verborgenen. Für eine weitere große Irritation kann
das in Fatmas Unterricht von ihrer Lehrerin eingesetzte Bilderbuch sorgen. Das Cover ist identisch
mit dem in diesem Beitrag vorgestellten Bilderbuch. Auf unlogische Weise wird hier das intradiegeti-
sche (vgl. hierzu Genette 1989) Bilderbuch im Bilderbuch behandelt. Transkulturelle Implikationen Ein solches Sich-Zurechtfinden stellt für
Yusuf und – bei der Übertragung in die Realität – auch für Kinder eine immens
große Herausforderung dar, für deren Bewältigung sie im Deutschunterricht mit
entsprechenden transkulturellen Lernaufgaben bedacht werden können. Aus litera-
turdidaktischer Perspektive böten sich hier Aufgaben zur Perspektivenübernahme
Yusufs an. Auf diese Weise kann die Verleugnung der eigenen Identität beim Leser
Irritationen hervorrufen, die einen Anstoß zu transkulturellem Lernen in der Ana-
lyse der Identitätsbildung der literarischen Figur geben. Weiter führt die Perspekti-
venübernahme dieser Figur zu einer Reflexion der eigenen Identitätsbewusstheit. Letztlich, diese Entwicklung ist mit Schülern diskutierbar, werden Yusuf und Mar-
kus Freunde, trotz des Verleugnungsversuchs des Namens, trotz aller Unterschiede
und in der transkulturellen Überwindung der Vorurteile. Fatma und / ile Martha. Zeynep Bassa (2005) Zeynep Bassa erzählt in diesem comichaften Bilderbuch4 von zwei Mädchen, Fat-
ma und Martha, die sich in der Schule kennenlernen und sich trotz ihrer kulturell
unterschiedlich geprägten Lebensweisen befreunden. Eines Tages, als Fatma auf
dem Weg nach Hause ist, wird sie auf einige Jungen aufmerksam, die eine Haus-
wand mit rassistischen Bemerkungen wie „TÜRKEN RAUS5“ (Bassa 2005: 5) und Serap Atagül/Christian Müller 146 „DEUTSCHLAND DEN DEUTSCHEN“ (Bassa 2005: 5) bemalen. Unreflektiert
singt Fatma das Gelesene vor sich hin, bis sie entsetzt bemerkt, dass sie dies selbst
betrifft, da sie sich der türkischen Kultur zugehörig zu fühlen scheint. Daraufhin
überlegt sie sich, wie es wäre, Deutschland verlassen zu müssen, um in der Türkei
zu leben. Sie denkt zunächst über die positiven Seiten des Lebens in der Türkei
nach, bemerkt jedoch, dass sie in der Türkei ihr deutsches Lebensumfeld und vor
allem auch ihre Familie vermissen würde. Sie erinnert sich an einen Hinweis, was
zu tun sei, um in Deutschland bleiben zu können: Integration. Sie beschließt, eine
„ECHTE DEUTSCHE“ (Bassa 2005: 13) zu finden, um Antworten auf ihre Fra-
gen zu diesem Thema zu bekommen. Auf ihrer Suche begegnet sie auf dem
Schulweg einem polnischen und einem asiatischen Mädchen, die auf Grund von
Fatmas distanzlos vorgetragenen Fragen flüchten. Fatmas Lehrerin spricht sie auf
ihre traurige und nachdenkliche Stimmung an. Fatma öffnet sich ihr gegenüber:
„ICH MÖCHTE INTÄKRATZION [sic!] MACHEN. ABER WEISS NICHT,
WIE DAS GEHT“ (Bassa 2005: 16). Ihre Lehrerin klärt sie auf, dass Integration
ein gemeinsames Zusammenleben und Akzeptanz voraussetzt und macht sie auf
Martha, eine neue Mitschülerin, aufmerksam. Transkulturelle Implikationen Es beginnt eine herzliche Freund-
schaft zwischen Fatma und Martha, die auch von Vorurteilen in Alltagssituationen
und Einstellungsunterschieden von Familienmitgliedern begleitet wird, wie im
folgenden Abschnitt deutlich wird.6 Transkulturelle Implikationen p
Fatmas Lehrerin veranlasst das Kennenlernen zwischen Fatma und Martha. Dabei
unterhalten sie sich über ihre Wohnorte: „WIR WOHNEN IN DER BAHN-
HOFSTRASSE ZWISCHEN DEM DÖNERLADEN UND ALDI [sic!] UND
DU?“ sagt Fatma, worauf Martha antwortet, dass sie „IN DER KASTA-
NIENALLEE AM WALD“ (Bassa 2005: 17) wohne. Die erste Unterhaltung zeigt
die räumliche Separation und damit zusammenhängend eine Ungleichheit der Le-
bensverhältnisse auf. Fatma wird bereits auf den ersten Seiten des Buches mit einer
Aldi-Tüte abgebildet (Bassa 2005: 9), was nicht nur das Schüren von Vorurteilen,
sondern auch Unbehagen bei Lesern auslösen könnte. Diese und andere klischee-
beladenen Darstellungen in Bild und Text fordern Rezipienten dazu heraus, die
Bild-Text-Kombinationen mit Hilfe einer transkulturellen Haltung zu überwinden. Im weiteren Verlauf fragt Fatma Martha, ob sie heute zu ihr kommen dürfe; sie
erhält eine Absage mit folgender Begründung: 147 Transkulturelles Lernen mit mehrsprachigen Bilderbüchern HEUTE HABE ICH BALLET [sic!], MORGEN FLÖTE, ÜBERMOR-
GEN MUSS ICH MIT MAMA EINKAUFEN GEHEN UND NEUE
KLAMOTTEN KAUFEN. FREITAGS ÜBE ICH MATHE. NUR
SAMSTAG-NACHMITTAG [sic!] HABE ICH NICHTS VOR. (Bassa
2005: 17) Auch hier könnte eine Klischeehaftigkeit in der Zuschreibung eines pedantisch
geplanten Tagesablaufs der deutschen Familie zu erkennen sein, die keine spontane
Verabredung zulässt. Der bedrückte und verärgerte Gesichtsausdruck Marthas
könnte dem Betrachter ihre Unzufriedenheit über diese Planung verraten. Das
Gespräch und die Bildsequenz auf Seite 17 werden mit der Bestätigung der Verab-
redung für den kommenden Samstag beendet. Die Bilder regen dazu an, Klischees
und Vorurteile bewusst kritisch wahrzunehmen und als Sprechanlass im Deutsch-
unterricht in sprachdidaktischer Hinsicht lernförderlich aufzubereiten. Die Schüler
treten bei der Betrachtung der Identitäten der literarischen Figuren Fatma und
Martha in einen kommunikativen Austausch. Dies kann „als Impuls für das Erar-
beiten kulturspezifischer Eindrücke“ (Spiegel 2009: 173) dienen und soll von den
Schülern selbst bewertet und diskutiert werden, da es wichtig ist, sich „von beste-
henden Klischeevorstellungen [...] zu lösen“ (Tekinay 2001: 89). g
[ ]
(
y
)
Die Erzählung wird „AM SAMSTAG...“ (Bassa 2005: 18) fortgesetzt und
Martha stellt ihre „NEUE TÜRKISCHE FREUNDIN FATMA“ (Bassa 2005: 18)
ihrer Mutter vor, die ihr vorwurfsvoll antwortet: „MARTHA, DU HAST MIR
ABER GAR NICHT GESAGT [sic!] DASS DEINE NEUE FREUNDIN TÜR-
KIN IST. NA JA, WAS SOLL’S ... HALLO!“ und fährt fort, dass Fatma „ABER
SCHÖNES HAAR [habe], SO FEST UND RABENSCHWARZ“ (Bassa 2005:
19). Auch hier wird wiederholt mit stereotypisierten Bildern gespielt und auf die
dunkle Haarfarbe Fatmas fokussiert, wohingegen Martha und ihre Mutter in blon-
der Haarfarbe dargestellt werden. Auffällig ist auch, dass die nationale Identität
Fatmas – und später auch Marthas – explizit betont wird. Im Hinblick auf trans-
kulturelles Lernen ist die Unterteilung in ‚Eigenes‘ und ‚Fremdes‘ bezüglich natio-
naler Identitäten, wie es hier geschieht, kontraproduktiv, „da diese Zuschreibungen
in Einwanderungsgesellschaften als überholt angesehen werden müssen und nicht
den Bedürfnissen der Schüler entgegenkommen“ (Engin / Olsen 2009: 14). Schülern kann in der Diskussion solcher Bilder und Texte deutlich gemacht
werden, dass es schon immer nationales Identitätsdenken gab und wohl immer
geben wird, eine transkulturelle Haltung jedoch dazu beitragen kann, dass sich
solche Denkstrukturen nicht negativ auf die Gesellschaft auswirken. Die negativen Einstellungen gegenüber nationalen Identitäten prägen auch den
weiteren Handlungsverlauf. Die abwertende Aussage von Marthas Mutter gegen-
über Türken „LEIDER STEHT HYGIENE BEI DEN TÜRKEN NICHT IM-
MER AN ERSTER STELLE“ (Bassa 2005: 19), wird durch kindliche Naivität
sowie Wohlwollen überdeckt und wird von Martha nicht hinterfragt. HEUTE HABE ICH BALLET [sic!], MORGEN FLÖTE, ÜBERMOR-
GEN MUSS ICH MIT MAMA EINKAUFEN GEHEN UND NEUE
KLAMOTTEN KAUFEN. FREITAGS ÜBE ICH MATHE. NUR
SAMSTAG-NACHMITTAG [sic!] HABE ICH NICHTS VOR. (Bassa
2005: 17) Auf das Beja- Serap Atagül/Christian Müller 148 hen der Frage von Marthas Tante Christa, ob Fatma und ihre Mutter Deutsch
sprechen können, führt sie fort: „ABER DEINE OMA BESTIMMT NICHT! GIB IHR DIESE ADRESSE... ...DA KANN SIE ENDLICH MAL DEUTSCH
LERNEN!“ (Bassa 2005: 21). Das Nichtbeherrschen der deutschen Sprache wird
zur persönlichen Demütigung genutzt und findet sich auch im Gespräch zwischen
Fatma, Martha und Fatmas Opa sowie später auch mit der Tante wieder (Bassa
2005: 23, 25). Auf dem Weg zum „TÜRKISCHEN LADEN“ begegnen die Mäd-
chen Fatmas Opa, der stereotyp mit langem Bart, unterschiedlich gemusterter
Kleidung, einer Gebetshaube und Gebetskette am Handgelenk abgebildet ist. Mit
erhobenem Zeigefinger belehrt er die Mädchen, dass sie „SCHÖN SPIELEN“
und auf Schweinefleisch, Schnaps und Bier, was Deutsche wohl „VIEL“ konsu-
mieren, verzichten sollen (Bassa 2005: 23). Auf die Frage, was Fatmas Opa damit
meine, erklärt ihr Fatma: „NA, JA, WIR DÜRFEN KEIN SCHWEINEFLEISCH
ESSEN UND KEIN BIER UND SO’N ZEUG TRINKEN“ und begründet ihre
Aussage mit dem Koran, „EIN BUCH, IN DEM ALLES STEHT [sic!] WAS DU
TUN DARFST UND WAS NICHT“ (Bassa 2005: 24). Es wird anschließend eine
Analogie zur Bibel hergestellt, denn „ES IST AUCH EIN BUCH, IN DEM
STEHT [sic!] WAS DU DARFST UND WAS NICHT“ (Bassa 2005: 24). Diese
Textstellen eröffnen Anknüpfungspunkte zum transkulturellen Lernen in den teils
verletzenden, klischeehaften Aussagen sowie – hier erstmals angesprochenen –
möglichen religiösen Gemeinsamkeiten. g
g
„IM TÜRKISCHEN LADEN“ stellt Fatma ihrer Tante Hatice ihre „DEUT-
SCHE FREUNDIN MARTHA“ vor (Bassa 2005: 25). Verärgert erklärt sie Fatma,
sie solle aufpassen, denn „DEUTSCHE MÄDCHEN [...] SPIELEN MIT JUNGS
[...]“ und türkische Mädchen dürfen nicht mit Jungen spielen, da das „NICHT
RICHTIG“ (Bassa 2005: 25) sei. Die Mädchen verwerfen auf Grund der belehren-
den Bemerkungen bikultureller Art ihren ursprünglichen Plan, gemeinsam ein
„BISSCHEN BAKLAVA UND EIN BISSCHEN LOKUM [...]“ zu essen mit den
Worten „MEIN HUNGER IST VERGANGEN!“ und „MEINER AUCH!“
(Bassa 2005: 26). Den ständigen Separationsversuchen begegnen Fatma und
Martha, indem sie Hand in Hand den Laden verlassen – ein deutliches Zeichen
transkultureller Haltung. Fatma will sich ihrer Freundschaft noch einmal gewiss
werden und fragt: „SIND WIR DENN NOCH FREUNDE?“ (Bassa 2005: 27). „NA KLAR!“, antwortet Martha und sie schwören, dass sie „FÜR IMMER
FREUNDE“ bleiben wollen (Bassa 2005: 27). HEUTE HABE ICH BALLET [sic!], MORGEN FLÖTE, ÜBERMOR-
GEN MUSS ICH MIT MAMA EINKAUFEN GEHEN UND NEUE
KLAMOTTEN KAUFEN. FREITAGS ÜBE ICH MATHE. NUR
SAMSTAG-NACHMITTAG [sic!] HABE ICH NICHTS VOR. (Bassa
2005: 17) Das Bilderbuch endet mit einer
ähnlichen Bildsequenz wie zu Anfang: Auf der letzten Seite des Buches ist die zu
Beginn gezeigte Wand noch einmal abgebildet, mit der von Fatma ergänzten Auf-
schrift, die Nationalitäten und Kulturen überschreitend und verbindend wirkt:
„TÜRKEN NIX [sic!] RAUS. FATMA UND MARTA [sic!] SINT [sic!] GUTE
FREUNDE“ (Bassa 2005: 30). Für transkulturelles Lernen besonders in den Blick zu nehmen sind die aus-
schließlich ablehnenden Haltungen der Erwachsenen gegenüber der jeweiligen
Kultur. Diesen steht die transkulturelle Haltung der Kinder entgegen, die das Posi- Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 149 tive und Gemeinsame beider Kulturen mit ihrer eng gelebten Freundschaft heraus-
stellen. Im Literaturunterricht kann die Auseinandersetzung mit den transkulturel-
len Haltungen der Figuren Fatma und Martha die negativen bikulturellen Haltun-
gen der Erwachsenen obsolet werden lassen. Auf diese Weise können Schüler
Identifikationsmöglichkeiten angeboten bekommen und lernen, unterschiedliche
Kulturen zu akzeptieren. Leyla und Linda feiern Ramadan. Leyla ve Linda Ramazanı kutluyorlar. Arzu Gürz Abay / Sibel Demirtaş (2011) Das Bilderbuch erzählt die Geschichte einer Freundschaft zwischen Leyla und
Linda, die in Köln leben und sich im Urlaub während der Fastenzeit in einer türki-
schen Hafenstadt namens Ayvalık treffen. In den Sommerferien besucht Leyla ihre
Großeltern in der Türkei und Linda reist mit ihrer Mutter auf eine griechische
Insel, die sich unweit von Ayvalık befindet. Für Leyla ist dieser Urlaub besonders
aufregend, weil sie sich dort in der Zeit des Ramadans befindet und dessen Tradi-
tionen kennenlernt. Sie freut sich zudem auf die Ankunft ihrer Freundin Linda. Auch Linda lernt muslimische Bräuche und
türkische Speisen kennen und darf das „Zu-
ckerfest“ (Gürz Abay / Demirtaş 2011: 28),
das auch als Ramadan-Fest zu bezeichnen ist,
miterleben. Abb. 1: Buchcover: Gürz Abay, Arzu:
Leyla und Linda feiern Ramadan. Leyla
ve Linda Ramazanı kutluyorlar. Abb. 1: Buchcover: Gürz Abay, Arzu:
Leyla und Linda feiern Ramadan. Leyla
ve Linda Ramazanı kutluyorlar. Serap Atagül/Christian Müller 150 Transkulturelle Implikationen Transkulturelle Implikationen Transkulturelle Implikationen Abb. 2: Gürz Abay, Arzu: Leyla und Linda feiern Ramadan. Leyla ve Linda Ramazanı kutluyorlar,
S. 12-13. Abb. 2: Gürz Abay, Arzu: Leyla und Linda feiern Ramadan. Leyla ve Linda Ramazanı kutluyorlar,
S. 12-13. Die vorliegende Doppelseite des Bilderbuchs (s. Abb. 2) zeigt eine Szenerie in der
Küche der türkischen Familie in Ayvalık: Auf der linke Abbildungshälfte sind Ley-
las Opa Halim, ihre Oma und ihre Eltern zu sehen, die gemeinsam den Tisch de-
cken. Die rechte Seite der Abbildung gibt den Blick durch das offene Fenster auf
die Straße frei. Leyla schaut „dem Trommler aus dem Fenster zu“ (Gürz Abay /
Demirtaş 2011: 12). Als Gegenstände türkischer Kultur sind die Teekanne und die
Teegläser mit Untertassen, die auf dem Tisch stehen, zu erkennen. Der Krug mit
den dazugehörigen Bechern und die Obstschale auf der Kommode neben dem
Fenster sind oftmals Bestandteile eines traditionellen Haushalts in der Türkei. Der
Trommler hingegen, der einen roten Fes – eine Kopfbedeckung aus rotem Filz, die
vor allem zu Zeiten des Osmanischen Reichs getragen wurde –, eine rote Weste
und spitze Schuhe trägt, bedarf einer Erklärung: Es ist eine alte Tradition, während der Ramadanzeit [...] [vor der Morgen-
dämmerung; S.A. / C.M.] von einem Trommler geweckt zu werden. In
den alten Zeiten gab es schließlich keinen Wecker. Mit dem lauten Tam-
tam der Schlegel und zwischen den fröhlichen Melodien singt der Tromm-
ler Reime, die er von seinen Vorfahren gelernt hat [...]. (Gürz Abay / De-
mirtaş 2011: 10) Der Leser lernt – wie Linda im Bilderbuch – auch im Weiteren über die kulturellen
Traditionen, auch im transkulturellen Sinn, wenn Leyla diese auf das Leben in
Deutschland transferiert: Deutschland transferiert: Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 151 Jede Nacht während des Ramadans geht ein Trommler in den Straßen
umher, um uns aufzuwecken. Er singt dazu witzige Reime und spielt ganz
laut auf seiner Trommel. Stell dir vor, da spielt in Deutschland jemand
Trommel auf der Straße; mitten in der Nacht! (Gürz Abay / Demirtaş
2011: 26) Jede Nacht während des Ramadans geht ein Trommler in den Straßen
umher, um uns aufzuwecken. Er singt dazu witzige Reime und spielt ganz
laut auf seiner Trommel. Stell dir vor, da spielt in Deutschland jemand
Trommel auf der Straße; mitten in der Nacht! (Gürz Abay / Demirtaş
2011: 26) Jede Nacht während des Ramadans geht ein Trommler in den Straßen
umher, um uns aufzuwecken. Transkulturelle Implikationen Er singt dazu witzige Reime und spielt ganz
laut auf seiner Trommel. Stell dir vor, da spielt in Deutschland jemand
Trommel auf der Straße; mitten in der Nacht! (Gürz Abay / Demirtaş
2011: 26) Die Unvoreingenommenheit, die Neugier und die Kulturoffenheit Lindas schaffen
transkulturelle Lernmöglichkeiten, die von Schülern adaptierbar sind und sich u. a. in Abbildung 3 widerspiegeln: Abb. 3: Gürz Abay, Arzu: Leyla und Linda feiern Ramadan. Leyla ve Linda Ramazanı kutluyorlar,
S. 20-21. Abb. 3: Gürz Abay, Arzu: Leyla und Linda feiern Ramadan. Leyla ve Linda Ramazanı kutluyorlar,
S. 20-21. Halim erklärt Linda den Gebetsruf (im Türkischen ‚Ezan‘ genannt): Halim erklärt Linda den Gebetsruf (im Türkischen ‚Ezan‘ genannt):
Es ist der Gebetsrufer, oder Müezzin genannt. Er ruft uns täglich fünf Mal
vom Minarett aus zum Gebet. Nun wissen wir, dass es Zeit ist, unseren
Gebetsteppich auszurollen und zu beten. (Gürz Abay / Demirtaş 2011:
20) 20) Der Text wird in einer Gedankenblase illustriert, in der der Gebetsrufer eine Ge-
betshaube als religiöses Symbol trägt. Die bildlichen Elemente sind hier meist ana-
log zum Text dargestellt, was dem Leser das Verstehen erleichtern kann. Auf den Lichterketten, die zwischen zwei Minaretten befestigt sind, ist auf tür-
kischer Sprache „Hoşgeldin Ramazan“ (Gürz Abay / Demirtaş 2011: 21) zu lesen,
was sich mit ‚Herzlich Willkommen Ramadan‘ [eigene Übersetzung; S.A / C.M.]
übersetzen lässt. Leylas Opa erklärt Linda auch, warum gebetet wird und dass beim
Gebet der Körper „nach Mekka“ (Gürz Abay / Demirtaş 2011: 22) ausgerichtet
ist, eine Stadt in Saudi Arabien. Es ist der Ort, wo die heilige Kaaba ist, ein Haus 152 Serap Atagül/Christian Müller Gottes. Alle Muslime auf der ganzen Welt beten in Richtung Kaaba. So sind sie
mehrmals am Tag in ihrem Gebet vereint (Gürz Abay / Demirtaş 2011: 22). Das Kennenlernen der türkischen Kultur vollzieht sich nicht ausschließlich
anhand religiöser Aspekte, sondern auch im gemeinsamen Essen nach dem Ge-
betsruf: Die Familie und die Gäste setzten sich gerade an den reich gedeckten
Tisch, als Linda erschrocken einen Schrei ausstößt und mit einem Sprung
fast vom Stuhl fällt: ein lauter Kanonenschuss! Statt eines knurrenden Ma-
gens haben jetzt alle ein lächelndes Gesicht, denn der Kanonenschuss ver-
kündet das Ende der Fastenzeit (Gürz Abay / Demirtaş 2011: 24). 7 Ein weiteres Bilderbuch mit transkulturellen Lernpotentialen derselben Autorin befand sich wäh-
rend der Erstellung dieses Beitrags im Druck und trägt den Titel ‚Sinan, Felix und die wilden Wörter‘
(Pulheim 2016). Transkulturelle Implikationen Auf dem Tisch sind türkische Speisen wie „Börek mit Spinat und Joghurt” (Gürz
Abay / Demirtaş 2011: 28), gefüllte Weinblätter, Fladenbrot, schwarzer Tee und
auch in Ayvalık angebaute Oliven zu sehen, die Linda schon „oft bei Leyla in Köln
gegessen [hat], daher kennt sie bereits viele Gerichte der türkischen Küche“ (Gürz
Abay / Demirtaş 2011: 28). Mimik und Gestik der Figuren wirken glücklich und
verdeutlichen dem Rezipienten ein selbstverständliches Miteinander der Kulturen. Transkulturelle Gemeinsamkeiten zeigen sich auch im Alltagsleben der Mädchen,
denn Leyla und Linda gehen in Deutschland in den gleichen Kindergarten und
besuchen auch den gleichen Tanzkurs. Sie spielen oft zusammen und die Mütter
verstehen sich gut. (Gürz Abay / Demirtaş 2011: 14) Dem Leser wird Linda somit als eine Figur präsentiert, die die türkische Kultur
nicht als fremde ansieht, diese akzeptiert und in ihr Leben integriert. Auch für
Leyla scheint es keinerlei ‚kulturelle Hürden‘ zu geben. Transkulturelles Lernen mit mehrsprachigen Bilderbüchern Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 153 Sinan und Felix. Mein Freund Arkadaşım
Aygen-Sibel Çelik / Barbara Korthues (2014) Sinan und Felix. Mein Freund Arkadaşım
Aygen-Sibel Çelik / Barbara Korthues (2014) Abb. 4: Buchcover:
Çelik, Aygen-Sibel: Sinan
und Felix. Mein Freund
Arkadaşım. Aygen-Sibel Çelik erzählt von verschiedenen Alltagssituationen der Freundschaft
zwischen Sinan, einem Deutsch und Türkisch sprechenden Jungen, und dem
Deutsch sprechenden Felix.7 An einem Sommertag
überlegen sich Sinan und Felix, was sie unternehmen
wollen. Während dieses Gesprächs schießt Murat, eine
weitere Figur, seinen Ball an Sinans Kopf. Daraufhin
unterhalten sich Sinan und Murat auf Türkisch. Felix
versteht kein Wort und fühlt sich ausgeschlossen. Sinan
überredet Felix zu einem gemeinsamen Fußballspiel mit
Murat. Hülya, die mit ihrem neuen Fahrrad und ihrem
Hund in die Handlung Einzug hält, spricht ebenso Tür-
kisch mit Sinan und Murat, was Felix erneut sauer wer-
den lässt. Felix schießt Murats Ball versehentlich in den
See und fällt beim Versuch, den Ball herauszuholen,
hinein. Sinan und Hülya helfen Felix aus dem Wasser,
wohingegen Murat nur auf seinen Ball fokussiert ist
und den beiden nicht hilft. Völlig durchnässt stellt sich
Felix vor Murat und ruft: „Banane!“, was auf Türkisch
bedeutet: „Mir doch egal!“ (Çelik / Korthues 2014: o. S.). Der Begriff „Banane“ des Textes wird auf dem
Buchcover bildlich illustriert: Sinan sitzt am linken Rand und Felix rechts auf einer
überdimensionalen Banane. Der Untertitel des Bilderbuches „MEIN FREUND“
bzw. „ARKADAŞIM“ steht unterhalb der sitzenden Kinder, wobei der deutsche
Begriff unter Sinan und der türkische unter Felix zu finden ist. Die enge Freund-
schaft wird mit den letzten Worten der Erzählung noch einmal verdeutlicht:
„‚Komm!‘, sagte Sinan. ‚Wir gehen!‘ [sic!] Felix war froh, dass er und Sinan sich so
gut verstanden. Sinan war eben sein bester Freund.“ (Çelik / Korthues 2014: o. S.) Abb. 4: Buchcover:
Çelik, Aygen-Sibel: Sinan
und Felix. Mein Freund
Arkadaşım. Abb. 4: Buchcover:
Çelik, Aygen-Sibel: Sinan
und Felix. Mein Freund
Arkadaşım. Serap Atagül/Christian Müller 154 Transkulturelle Implikationen
Abb. 5: Çelik, Aygen-Sibel: Sinan und Felix. Mein Freund Arkadaşım, o. S. Transkulturelle Implikationen Transkulturelle Implikationen Transkulturelle Implikationen Abb. 5: Çelik, Aygen-Sibel: Sinan und Felix. Mein Freund Arkadaşım, o. S. Auf der präsentierten Doppelseite des Bilderbuches auf Abbildung 5 vollzieht sich
die erste Begegnung der Jungen mit Hülya bilingual: Murat begrüßt sie mit den
Worten: „Selam, Hülya!“ (Çelik / Korthues 2014: o. S.). Felix kommt „sich richtig
doof vor“ (Çelik / Korthues 2014: o. S.), da er ihn nicht verstehen kann. Auch in
anderen Situationen wird der Leser auf die Enttäuschung von Felix aufmerksam:
„Felix verstand kein einziges Wort. Das machte ihn wütend. 8 Hervorhebungen im Text. Sinan und Felix. Mein Freund Arkadaşım
Aygen-Sibel Çelik / Barbara Korthues (2014) Sinan war doch sein
bester Freund! Worüber hatte er da mit Murat gesprochen?“ (Çelik / Korthues
2014: o. S.). Wenn sich Murat und Sinan auf Türkisch unterhalten, fühlt sich Felix
ausgeschlossen und denkt, dass Murat „bestimmt absichtlich“ so handelt (Çelik /
Korthues 2014: o. S.). Murat versucht ihn zu beruhigen, er solle „mal cool“ (Çelik
/ Korthues 2014: o. S.) bleiben, denn „Türkisch ist gar nicht so schwer!“ (Çelik /
Korthues 2014: o. S.). Als transkulturelle Implikation kann hier die Aufforderung
zum Lernen einer fremden Sprache als Mittel zur Verständigung aufgegriffen wer-
den. Seinen ersten Versuch in türkischer Sprache macht Felix, nachdem auch Sinan
Hülya mit „Selam!“ begrüßt und hinzufügt „Ich heiße Sinan.“ (Çelik / Korthues
2014: o. S.). „Ach so! Jetzt hatte Felix verstanden. ‚Selam, ich bin der Felix.‘“ (Çelik
/ Korthues 2014: o. S.). Çelik greift hier eine alltägliche Situation im Leben der
Kinder und Erwachsenen auf, da das Bilderbuch „neben der Mehrsprachigkeit der Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 155 Kinder und Jugendlichen mit Migrationshintergrund auch die lebensweltliche
Mehrsprachigkeit von einheimischen Kindern und Jugendlichen“ (Engin
/ Olsen 2009: 7) berücksichtigt und somit ein transkulturelles Lernen anhand des
Erkennens von vergleichbaren sprachlichen Situationen wie einer Begrüßung er-
möglicht: Türkisch und Deutsch werden hier in einen Begrüßungssatz selbstver-
ständlich eingebunden. Diese gemischte Sprachenverwendung wird nicht hinter-
fragt oder abgelehnt. Zur näheren Untersuchung des Sprachgebrauchs lassen sich
deutsche und türkische Interjektionen und deren unterschiedliche Intonation –
diese wird den Rezipienten durch die visualisierte Buchstabengröße und den
-umfang erleichtert – auf ihre Transkulturalität hin überprüfen: ‚Ei, ei, ei!8‘, rief Sinan auf Türkisch. ‚Hilfe, er will mich fressen!‘
‚Ei, ei, ei!‘, sagte Felix auf Deutsch. ‚Ich wusste gar nicht, dass du Angst vor Hunden hast!‘
‚Schnuffi, aus!‘, rief Hülya. ‚Er will doch nur mit dir spielen.‘
‚Jaaaaa!‘, sagte Sinan auf Türkisch. ‚Von wegen!‘
‚Jaaa!‘, sagte Felix auf Deutsch. ‚Das stimmt!‘
(Çelik / Korthues 2014: o. S.) In einer Vokabelliste sind deutsche und türkische Begriffe aus dem Text gegen-
übergestellt und mit einer Aussprachehilfe versehen, was Rezipienten zur Übung
der Sprachen und ihrer Besonderheiten, zur Überwindung sprachlicher Unter-
schiede und zum Erkennen von Gemeinsamkeiten motivieren könnte. Zudem
unterstützen lyrische Texte das sprachliche transkulturelle Lernen auf einigen Sei-
ten des Bilderbuchs: Trifft man einen, den man mag,
sagt man ‚Hallo!‘ und ‚Guten Tag!‘
Begrüßung steht auf dem Programm. Auf Türkisch sagt man sich ‚Selam!‘ (Çelik / Korthues 2014: o. Sinan und Felix. Mein Freund Arkadaşım
Aygen-Sibel Çelik / Barbara Korthues (2014) S.) Trifft man einen, den man mag,
sagt man ‚Hallo!‘ und ‚Guten Tag!‘
Begrüßung steht auf dem Programm. Auf Türkisch sagt man sich ‚Selam!‘ (Çelik / Korthues 2014: o. S. sagt man ‚Hallo!‘ und ‚Guten Tag!‘ Auf Türkisch sagt man sich ‚Selam!‘ (Çelik / Korthues 2014: o. S.) Zur Vertiefung dieses Lernens könnten Schüler im Deutschunterricht dazu aufge-
fordert werden, sich selbst an einer kreativen Schreibaufgabe zu versuchen, indem
sie lyrische Texte mit den Wörtern der Vokabelliste entwerfen zur Hybridisierung und Mischung von Sprachen im schulischen wie im All-
tagskontext. Damit wird die Grenze zwischen dem ‚Eigenen‘ und dem
F
d
‘
f
h b
d
h ißt d
Ab
i
Sp Hybridisierung und Mischung von Sprachen im schulischen wie im All-
tagskontext. Damit wird die Grenze zwischen dem ‚Eigenen‘ und dem
‚Fremden‘ aufgehoben, das heißt, dass Abgrenzungen von ‚eigener Spra- ‚Fremden‘ aufgehoben, das heißt, dass Abgrenzungen von ‚eigener Spr Serap Atagül/Christian Müller 156 che‘ und ‚fremde(n) Sprache(n)‘ als überholt erkannt werden müssen [...]. (Engin / Olsen 2009: 7) che‘ und ‚fremde(n) Sprache(n)‘ als überholt erkannt werden müssen [...]. (Engin / Olsen 2009: 7) che‘ und ‚fremde(n) Sprache(n)‘ als überholt erkannt werden müssen [...]. (Engin / Olsen 2009: 7) Das den Text und die Bilder ergänzende Angebot an Sprachspielen, Worträtseln
und Steckbriefen über Sinan und Felix kann zusätzliches Interesse an sprachlichem
transkulturellem Lernen wecken. Rezipienten werden des Weiteren Visualisierungen dargeboten, die transkultu-
relles Lernen über die Sprache hinaus ermöglichen können: Am linken Seitenrand
der Abb. 5 ist Till Eulenspiegel zu sehen, der lächelnd auf den am rechten Seiten-
rand sichtbaren Nasreddin Hoca blickt. Till Eulenspiegel und Nasreddin Hoca sind
zwei Schelme aus deutschen und türkischen Volksgeschichten. Ihre transkulturel-
len Gemeinsamkeiten, die von Rezipienten intertextuell bzw. intermedial erarbeitet
und verglichen werden können, bestehen in ihren jeweiligen scharfsinnigen Scher-
zen und Streichen, aber auch ihren Lebensweisheiten. Schlussbetrachtung Auf Grund
dessen kann der Einsatz von Bildergeschichten – und in der Folge auch von mehr-
sprachigen Bilderbüchern – unabhängig von der Erstsprache einer Förderung
mündlichen Nacherzählens dienlich sein. Zur weiteren Sprachförderung könnte die
Aussprache und das Hören von Versen, Reimen und klangähnlichen Wörtern bei-
tragen, die beispielsweise in ‚Sinan und Felix‘ zu finden sind. Als Gegenstände
diverser Sprechanlässe ermöglichen mehrsprachige Bilderbücher eine Wortschatz-
erweiterung, da sprachlich und bildlich kulturelle Gemeinsamkeiten und Unter-
schiede aufgeführt werden, die Schüler zu einer Diskussion anregen. Des Weiteren
könnte die Betrachtung der unterschiedlichen Typographien von Interesse für eine
Sprachsensibilisierung sein. Beziehungen dargelegt werden konnte, ist die Ausbildung einer Bildlesekompetenz
bei der Bilderbuchrezeption ebenso von Bedeutung wie die zum Textverstehen
nötigen Lesekompetenzen. Bildlesen fördert die Imaginationsbildung – hier vor
allem auch zur Perspektivenübernahme – sowie auch sprachliche Kompetenzen
(vgl. Dehn 2008: 227; vgl. Dehn 2011: 48). Für den Deutschunterricht ist eine
integrative didaktische Perspektivierung von mündlichen Sprachkompetenzen im
Literaturunterricht interessant – auch in zwei unterschiedlichen Sprachen. In einer
Untersuchung mit einem Kind mit Türkisch als Erst- und Deutsch als Zweitspra-
che zu einer mündlichen Nacherzählung einer deutsch-türkischsprachigen Bilder-
geschichte stellt Kalkavan fest, dass sich die Nacherzählungen in beiden Sprachen
hinsichtlich der verbalen Wiedergabestrategien inhaltlich und bezüglich ihrer narra-
tiven Struktur nicht signifikant unterscheiden (Kalkavan 2013: 115). Auf Grund
dessen kann der Einsatz von Bildergeschichten – und in der Folge auch von mehr-
sprachigen Bilderbüchern – unabhängig von der Erstsprache einer Förderung
mündlichen Nacherzählens dienlich sein. Zur weiteren Sprachförderung könnte die
Aussprache und das Hören von Versen, Reimen und klangähnlichen Wörtern bei-
tragen, die beispielsweise in ‚Sinan und Felix‘ zu finden sind. Als Gegenstände
diverser Sprechanlässe ermöglichen mehrsprachige Bilderbücher eine Wortschatz-
erweiterung, da sprachlich und bildlich kulturelle Gemeinsamkeiten und Unter-
schiede aufgeführt werden, die Schüler zu einer Diskussion anregen. Des Weiteren
könnte die Betrachtung der unterschiedlichen Typographien von Interesse für eine
Sprachsensibilisierung sein. Neben den didaktischen Überlegungen zu literarischem und sprachlichem Ler-
nen wurde in diesem Beitrag die grundsätzliche Bedeutung transkulturellen Ler-
nens dargestellt. Jeder Mensch – auch schon im Kindesalter – darf selbst entschei-
den, ob ihm etwas eigen und fremd ist, ob er von außen, z. B. anhand der Bilder-
buchfiguren, an ihn herangetragene Trennungsmöglichkeiten von Kulturen und
Sprachen wahrnimmt und überwindet. Schlussbetrachtung Diese Freiheit der Einnahme und Aus-
übung einer transkulturellen Haltung ist gerade in aktuellen Zeiten der durch Mig-
rationsbewegungen zunehmenden Heterogenität unserer Gesellschaften von gro-
ßer Bedeutung, die im Deutschunterricht von Anfang an thematisiert werden soll-
te. Ein didaktisch begründeter Einsatz mehrsprachiger Bilderbücher kann hierzu
einen Beitrag leisten. Schlussbetrachtung Transkulturelles Lernen gewinnt im Zusammenleben verschiedener Kulturen eine
immer größere Bedeutung. Mehrsprachige Bilderbücher, die sprachliche und kultu-
relle Unterschiede und Gemeinsamkeiten anhand von Text und Bild aufzeigen,
sind transkulturelle Lerngegenstände par excellence. Schon an den abgebildeten
(Abb. 1 und 4) und nicht abgebildeten Cover sowie bei den zum Großteil bilingua-
len Titeln wird das Potential für transkulturelles Lernen offenbar: Alle Figuren
werden durch ihre Namen, ihre Haarfarben und teilweise auch durch ihre Teints
nach deutscher und türkischer Herkunft stereotyp präsentiert und auf diese Weise
separiert – besonders beachtlich ist dies, da die vier Bilderbücher von verschiede-
nen Autoren, Illustratoren und Verlagen produziert wurden. Auffallend verbin-
dend ist das zentrale Motiv, das allen hier vorgestellten Bilderbüchern gemein ist:
das beidseitige (Er-)Leben der mit kulturellen Hürden verbundenen und letztlich
engen deutsch-türkischen Freundschaften, bei denen sich transkulturelles Lernen
u. a. in den kindlichen Figurenhandlungen beobachten lässt. g
g
Die Figuren und deren Handlungen eröffnen Rezipienten bei der multiper-
spektivischen Suche nach Transkulturalität literar-ästhetische Erfahrungen anhand
von Text und Bild. Die Übernahme der Perspektiven literarischer Figuren (vgl. Spinner 2006: 6-16; vgl. Freitag 2007: 197-226) als ein Ziel des Literaturunterrichts
lässt sowohl einen kulturellen Perspektivenwechsel als auch eine Reflexion beider
Kulturen zu. Ein solcher Literaturunterricht kann ein Nährboden für eine Identifi-
zierung kultureller Unterschiede und Gemeinsamkeiten sein sowie eine Akzeptanz
der möglichen Vermischung von Kulturen anbahnen. Rein bildbasiertes transkul-
turelles Lernen erfolgt mit mehrsprachigen Bilderbüchern über eine anzustrebende
Kompetenzerweiterung in Visual Literacy, indem Rezipienten über das Bildlesen zu
einem Perspektivenwechsel und damit zu einem Verstehen von kulturellen Aspek-
ten angeleitet werden. Wie an den Verweisen auf die Visualisierung und Text-Bild- 157 Transkulturelles Lernen mit mehrsprachigen Bilderbüchern Beziehungen dargelegt werden konnte, ist die Ausbildung einer Bildlesekompetenz
bei der Bilderbuchrezeption ebenso von Bedeutung wie die zum Textverstehen
nötigen Lesekompetenzen. Bildlesen fördert die Imaginationsbildung – hier vor
allem auch zur Perspektivenübernahme – sowie auch sprachliche Kompetenzen
(vgl. Dehn 2008: 227; vgl. Dehn 2011: 48). Für den Deutschunterricht ist eine
integrative didaktische Perspektivierung von mündlichen Sprachkompetenzen im
Literaturunterricht interessant – auch in zwei unterschiedlichen Sprachen. In einer
Untersuchung mit einem Kind mit Türkisch als Erst- und Deutsch als Zweitspra-
che zu einer mündlichen Nacherzählung einer deutsch-türkischsprachigen Bilder-
geschichte stellt Kalkavan fest, dass sich die Nacherzählungen in beiden Sprachen
hinsichtlich der verbalen Wiedergabestrategien inhaltlich und bezüglich ihrer narra-
tiven Struktur nicht signifikant unterscheiden (Kalkavan 2013: 115). Çelik, Aygen-Sibel / Korthues, Barbara (2014): Sinan und Felix. Mein Freund
Arkadaşım, Pulheim. Primärliteratur Bassa, Zeynep (Autorin und Illustratorin) (2005): Fatma und / ile Martha,
Hückelhoven. Bassa, Zeynep (Autorin und Illustratorin) (2005): Fatma und / ile Martha,
Hückelhoven. Çelik, Aygen-Sibel / Korthues, Barbara (2014): Sinan und Felix. Mein Freund
Arkadaşım, Pulheim. Çelik, Aygen-Sibel / Korthues, Barbara (2014): Sinan und Felix. Mein Freund
Arkadaşım, Pulheim. 158 Serap Atagül/Christian Müller Erol, Neşet / Delioğlu, Mustafa (2002): Arkadaş Olalım Mı? Sollen wir Freunde
werden?, Hückelhoven. Gürz Abay, Arzu / Demirtaş, Sibel (2011): Leyla und Linda feiern Ramadan. Leyla ve
Linda Ramazanı kutluyorlar, Langenhagen. Rösch, Heidi (1997): Bilderbücher zum interkulturellen Lernen, Baltmannsweiler. Sekundärliteratur Bredella, Lothar (1993): „Ist das Verstehen fremder Kulturen wünschenswert?“, in:
Bredella, Lothar / Christ, Herbert (Hg.): Zugänge zum Fremden, Gießen,11-36. Decker, Yvonne / Oomen-Welke, Ingrid (2008): „Ich bin ich und du bist du – ich
heiße Jussuf und wie heißt du?“, in: Grundschule Deutsch 18, 32-35. Dehn, Mechthild (2008): „Unsichtbare Bilder. Visual Literacy als Aufgabe des
Deutschunterrichts?“, in: Plath, Monika / Mannhaupt, Gerd (Hg.): Kinder –
Lesen – Literatur. Analysen – Modelle – Konzepte, Baltmannsweiler, 1-32. Dehn, Mechthild (2011): „Elementare Schriftkultur und Bildungssprache“, in:
Fürstenau, Sara / Gomolla, Mechtild (Hg.): Migration und schulischer Wandel:
Mehrsprachigkeit, Wiesbaden, 129-151. Dirim, Inci (2009): „‚Der Wal‘ heißt auf Türkisch ‚balina‘“, in: Grundschule Deutsch
23, 11-13. Engin, Havva / Olsen, Ralph (2009): „Transkulturelles Lernen im
Deutschunterricht“, in: Engin, Havva / Olsen, Ralph (Hg.): Interkulturalität und
Mehrsprachigkeit, Baltmannsweiler, 1-17. Freitag, Britta (2007): „Literarische und transkulturelle Kompetenzen bei der
Figurenkonstruktion und Perspektivenübernahme: Ein Rollenspiel im
aufgabenorientierten Literaturunterricht“, in: Bredella, Lothar / Hallet,
Wolfgang (Hg.): Literaturunterricht, Kompetenzen und Bildung, Trier, 197-226. Genette, Gérard (1989): Paratexte. Das Buch vom Beiwerk des Buches. Aus dem
Französischen von Dieter Hornig, Frankfurt a. M. / New York. Iljassova-Morger, Olga (2009): „Transkulturalität als Herausforderung für die
Literaturwissenschaft und Literaturdidaktik“, in: DAAD (Hg.): Das Wort. Germanistisches Jahrbuch Russland, Zwickau, 37-57. Kalkavan, Zeynep (2013): „Verbale Strategien in erst- und zweitsprachlichen
Nacherzählungen von Kindern mit Türkisch als Erstsprache: Ein Fallbeispiel“,
in: Becker, Tabea / Wieler, Petra (Hg.): Erzählforschung und Erzähldidaktik heute. Entwicklungslinien, Konzepte, Perspektiven, Tübingen, 99-119. ösch, Heidi (1997): Bilderbücher zum interkulturellen Lernen, Baltmannsweiler. Transkulturelles Lernen mit mehrsprachigen Bilderbüchern 159 Rösch, Heidi (2001): „Das interkulturelle Paradigma in Deutschdidaktik und
Pädagogik“, in: Rosebrock, Cornelia / Fix, Martin (Hg.): Tumulte. Deutschdidaktik zwischen den Stühlen, Baltmannsweiler, 106-124. Rösch, Heidi (2001): „Das interkulturelle Paradigma in Deutschdidaktik und
Pädagogik“, in: Rosebrock, Cornelia / Fix, Martin (Hg.): Tumulte. Deutschdidaktik zwischen den Stühlen, Baltmannsweiler, 106-124. Rösch, Heidi (2007): „Interkulturelle Literatur lesen – Literatur interkulturell
lesen“, in: Fäcke, Christiane / Wangerin, Wolfgang (Hg.): Neue Wege zu und mit
literarischen Texten, Baltmannsweiler, 51-62. Simon, Elisabeth / Ellermann, Lena: Glück, verfügbar unter: http://www.amira-
pisakids.de/#book=12&p=1 [letztes Zugriffsdatum: 25.10.2016]. Spiegel, Carmen (2009): „‚Iih, Schwein!‘ – Kulturspezifische Konzepte im sozialen
Raum ‚Schule‘, in: Engin, Havva / Olsen, Ralph (Hg.): Interkulturalität und
Mehrsprachigkeit, Baltmannsweiler, 165-176. Spinner, Kaspar H. (2006): „Literarisches Lernen“, in: Praxis Deutsch 33, 6-16. Sekundärliteratur Tekinay, Alev (2001): „Die ‚Tee-Grenze‘ oder Darstellung der
Fremdwahrnehmung in der deutschen Gegenwartsliteratur – Bedeutung für
den interkulturellen Deutschunterricht“, in: Hummelsberger, Siegfried (Hg.):
Didaktik des Deutschen als Zweitsprache und Interkulturelle Erziehung. Theorie,
Schulpraxis und Lehrerbildung, Baltmannsweiler, 82-122. Vach, Karin (2009): „Tilki Çalışmayı Nasıl Öğrendi oder Wie der Fuchs zu arbeiten
lernte“, in: Grundschule Deutsch 24, 14-17. Walden, Daniela (2010): „Transkulturelles Lernen im Deutschunterricht“, in:
Rupp, Gerhard / Boelmann, Jan / Frickel, Daniela (Hg.): Aspekte literarischen
Lernens. Junge Forschung in der Deutschdidaktik, Berlin, 29-42. Welsch, Wolfgang (1994): „Transkulturalität. Lebensformen nach der Auflösung
der Moderne“, in: Luger, Kurt / Renger, Rudi (Hg.): Dialog der Kulturen. Die
multikulturelle Gesellschaft und die Medien, Wien, 147-169. Welsch, Wolfgang (1995): „Transkulturalität. Zur veränderten Verfaßtheit heutiger
Kulturen“, in: Zeitschrift für Kulturaustausch 45(1), Stuttgart, 39-44. Wintersteiner, Werner (2006): Transkulturelle literarische Bildung. Die „Poetik der
Verschiedenheit“ in der literaturdidaktischen Praxis, Innsbruck / Wien / Bozen. 1 Der Roman verkaufte sich nach Angaben von Selim Özdoğan bislang rund 40.000 mal.
2 Verfügbar unter http://worte2014.tumblr.com [letztes Zugriffsdatum: 26.10.2016]. Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Daniel Schreiner Der Kölner Schriftsteller Selim Özdoğan machte bereits 1995 mit seinem Erst-
lingswerk Es ist so einsam im Sattel, seit das Pferd tot ist in Deutschland auf sich auf-
merksam. Der Roman gilt bis heute als ein Kultbuch, das sich weiterhin stetig ver-
kauft.1 Mit den nachfolgenden Romanen Nirgendwo & Hormone, Mehr, Ein gutes
Leben ist die beste Rache und dem Buch zu Fatih Akıns Film Im Juli gelang es ihm,
sich als vielseitiger Schriftsteller der deutschen Literaturszene zu positionieren. Selim Özdoğan hat zudem für einige Jahre eine Kolumne in der Wochenzeitschrift
Die Zeit verfasst, in der er sich zu politischen und kulturellen Themen in der BRD
äußerte. Auf der Internetpräsenz tumblr erreicht Özdoğan jenseits seiner Romane
mit seinen Audioblogs ein größeres Publikum.2 Obwohl seine späteren Romane
Die Tochter des Schmieds und Heimstraße 52, die sich mit dem Schicksal einer türki-
schen Familie in der Türkei und in Deutschland beschäftigen, von einzelnen Re-
zensionen in Zeitungen und Internetforen gelobt wurden, ist über ihn im deut-
schen universitären Bereich im Vergleich zu Emine Sevgi Özdamar, Zafer Şenocak
oder Feridun Zaimoğlu nur wenig geschrieben und geforscht worden. Im Jahr
2010 wurde Selim Özdoğan für den deutschen Science Fiction Preis für seinen
Roman Zwischen zwei Träumen nominiert. Nach einem längeren Aufenthalt in der
Türkei erschien 2016 sein neuester Roman Wieso Heimat, ich wohne zur Miete. Im Juli
2016 nahm Selim Özdoğan zudem das erste Mal am Ingeborg-Bachmann- Der Kölner Schriftsteller Selim Özdoğan machte bereits 1995 mit seinem Erst-
lingswerk Es ist so einsam im Sattel, seit das Pferd tot ist in Deutschland auf sich auf-
merksam. Der Roman gilt bis heute als ein Kultbuch, das sich weiterhin stetig ver-
kauft.1 Mit den nachfolgenden Romanen Nirgendwo & Hormone, Mehr, Ein gutes
Leben ist die beste Rache und dem Buch zu Fatih Akıns Film Im Juli gelang es ihm,
sich als vielseitiger Schriftsteller der deutschen Literaturszene zu positionieren. Daniel Schreiner 162 Literaturwettbewerb in Klagenfurt teil. Derzeit arbeitet er an einem Roman mit
dem Arbeitstitel Wo noch Licht brennt, welches den letzten Teil der Trilologie mar-
kiert, die er mit Die Tochter des Schmieds und Heimstraße 52 begonnen hatte. Im
Herbst 2016 war der Autor für ein Semester als Writer in Residence an der University
of Michigan in Ann Arbor tätig und in den USA auf Lesereise. Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Im folgenden Interview – das ich im Rahmen meiner Dissertation über Strate-
gien kultureller und politischer Partizipation in türkisch-deutscher und mexika-
nisch-amerikanischer Literatur mit ihm geführt habe – gibt Selim Özdoğan Aus-
kunft über seine vielseitige Prosa sowie seine Erfahrungen mit dem deutschen
Literaturbetrieb und diskutiert politische Fragen der deutsch-türkischen Erfah-
rungswelt. — Anfang Juli 2016 warst du beim Ingeborg-Bachmann-Literaturwettbewerb „Tage der
deutschsprachigen Literatur“ eingeladen. Wie bist du dorthin hingekommen und welche Erfah-
rungen hast du dort gemacht? Ich glaube, ich werde als ein Autor wahrgenommen, der an so etwas eher nicht
teilnehmen würde. Was ja Verweigerung ist und somit eine Grenze. Die will ich ja
in meinem Leben nicht mehr … Alles tendiert bei mir dahin, die Grenzen aufzulö-
sen. So gesehen schien der Wettbewerb eine gute Möglichkeit, um eine Fahrkarte
durch den gesamten Literaturbetrieb zu bekommen, und ich habe mich daher dort
mit einem Text beworben. Hat halt leider nicht geklappt. Ich glaube, man muss,
wenn man dorthin geht, für sich selber auch eine klare Vorstellung haben, was dort
geschieht. Für mich ist der Ingeborg-Bachmann-Literaturwettbewerb eine Veran-
staltung der Literaturkritik, bei der man sich der Deutungshoheit von sieben Ju-
roren ausliefert. Mit dieser Vorstellung ist es mir nicht schwer gefallen, teilzuneh-
men. Man akzeptiert, dass das eine Veranstaltung ist, die Literaturkritik, in kanoni-
sche Bildung investierte Zeit und Elitarismus legitimiert. Das ist der Rahmen, der
vorgegeben ist. In dem bewege ich mich. Ich begebe mich also freiwillig in einen
gesetzten Rahmen. Ich würde es auch noch mal machen, aber dann mit einem
Text, dessen Schwäche ich nicht schon im Vorfeld sehen kann. Ich wusste, man
kann meinem Text vorwerfen, dass er zu viel will bzw. nicht genau weiß, was er
will. Ich habe gehofft, dass das nicht passiert, aber es ist dennoch eingetreten. — In einem deiner Audioblogs hörte ich den Satz „Ich möchte mit dir reden! Austausch!“ Die-
sen Wunsch nach Kommunikation bzw. die Beschäftigung mit dem Thema des menschlichen
Austauschs bzw. des gestörten Austauschs kommt in vielen deiner Romane vor. Was bedeutet
Austausch für dich und woher kommt die große Relevanz dieses Themas für deine Prosa? Weil Menschen soziale Wesen sind. Mehr Relevanz braucht das Thema nicht. Wir
alle sind soziale Wesen. Aber wir bleiben total oft – und das ist das Interessantere
für mich – an einer komischen Oberfläche hängen. Es gibt bestimmte Sachen, die
wie Kontakt aussehen, aber nichts mit Kontakt zu tun haben. Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Ein billiges, aber
zeitgenössisches Beispiel dafür ist Facebook. Du bist in Kontakt mit Leuten, aber
das ist nicht das, was ich unter Kontakt verstehe. Es geht ja nicht um einen reinen 163 Interview mit Selim Özdoğan Informationsaustausch, es geht um etwas, was darunter liegt. Die Verbindung, die
stattfindet, ist idealerweise eine andere. Ich fand beispielsweise Gespräche über das
Wetter früher scheiße, da wir alle sehen, wie das Wetter heute ist. Und wie du das
empfindest, ist doch scheiß egal … Aber wenn das deine Möglichkeit ist, in Kon-
takt mit dem Gegenüber zu treten, ist das doch super. Es geht nicht um das Wet-
ter, sondern um die Wärme, die Freundlichkeit, die Nähe, das Interesse, das man in
die Worte legen kann. Es geht auch um dein Wesen. Darum geht es ja immer. Du
willst dich mit Leuten verbinden oder verbunden fühlen. Ich glaube, das ist ein
universelles Bedürfnis für die allermeisten. Es gibt Ausnahmen, es gibt immer
Ausnahmen. — Gül und Fuat aus deinem Roman „Heimstraße 52“ reden nicht miteinander und leben
aneinander vorbei. Wieso ist das so? — Gül und Fuat aus deinem Roman „Heimstraße 52“ reden nicht miteinander und leben
aneinander vorbei. Wieso ist das so? Es gibt keinen Raum dafür, es gibt allein kulturell schon keinen Raum dafür. Das
ist etwas relativ Modernes, eine Partnerschaft, die auf tatsächlicher Kommunikati-
on aufbaut und nicht darauf, dass man irgendwann die Eigenheiten des Partners
verstanden hat und sich dementsprechend verhält. Man kann ja ein beliebiges
deutsches Beispiel von vor 50, 60 Jahren nehmen und hat die gleiche Form der
Nichtkommunikation. Wo hätten sie es denn auch lernen sollen? Es gab keinen
Raum! Die andere Frage ist jedoch, muss es den geben? Ist das, was damals pas-
siert ist, unbedingt falsch? Nein! Das Bedürfnis nach Kommunikation hast du
vielleicht woanders erfüllt bekommen. Ich finde das nicht unbedingt schlimm. Es
ist nicht meine Vorstellung von einer Partnerschaft, aber das Konzept von Part-
nerschaft hat sich auch gewandelt. Und man glaubt ja immer, dass das, was man
heute glaubt, richtig ist, und das, was man früher geglaubt hat, falsch ist. — Du bist in Köln-Mühlheim geboren worden. Warst du im Sommer 2014 auf der Kundge-
bung „Birlikte“, bei der an den Nagelbombenanschlag der NSU in der Keupstraße vor 10 Jah-
ren erinnert wurde? Wie fandest du die Veranstaltung und wie hast du den damaligen Anschlag
erlebt? Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Die Frage ist häufig nicht „Wie können wir mit
ihnen reden?“, sondern „Wie können wir für sie etwas Gutes tun“, was sie schon
wieder in so eine doofe Opferrolle hineindrängt. p
g
Punkt 2: Dieser ganze NSU-Komplex, zu dem es viel zu sagen gäbe … viel-
leicht so ... ich hab trotz NSU nie Angst vor Rechts gehabt in diesem Land. Das
kann damit zu tun haben, dass ich von meiner Erscheinung her nicht eindeutig als
jemand identifizierbar bin, von dem man sagen könnte: „Boah, der gehört hier
aber nicht hin“. Der NSU ist eine überschaubare Größe, die man einordnen kann. Wovor ich eigentlich viel mehr Angst habe, ist diese ganze Mitte, die sich halt nicht
für rechts hält. Das ist für mich ein Riesenproblem. Davor habe ich Angst. Das
sind die Leute, die einem Sarrazin Raum geben. Das sind ja keine Rechten, die wen
einladen, sondern das sind immer Leute … Wenn der Satz schon so anfängt: „Ver-
stehen sie mich nicht falsch, ich hab ja selbst türkische Freunde.“ Alles, was da-
nach kommt, ist Rassismus. Aber dieser Mensch hält sich nicht für rechts. Tut der
wirklich nicht, der meint das auch nicht böse, das ist mir klar. Aber das kann ich
nicht einordnen. Ich weiß nicht, wie viele es davon gibt. Ich habe absolut Angst
davor. Ein weiteres Beispiel dafür ist, wie Begriffe das Bewusstsein und die Gesin-
nung dahinter nicht verbessern, sondern verbergen. Das hab ich ja quasi live miter-
lebt. Am Anfang waren wir Gastarbeiter, dann sind wir Ausländer geworden, dann
sind wir Migranten geworden, dann sind wir zu Menschen mit Migrationshinter-
grund geworden und dann sind wir – unterwegs nach 2001 – auch noch Muslime
geworden. Aber vergessen wir das. Von ‚Menschen mit Migrationshintergrund‘ bis
‚Dönermord‘ war der Weg so kurz, dass ich denke, was ist denn passiert? Es ändert
sich nichts in den Köpfen dieser Menschen. ‚Dönermorde‘ war für ein paar Tage
okay und dann hat man es stillschweigend unter den Tisch fallen lassen. Punkt 2: Dieser ganze NSU-Komplex, zu dem es viel zu sagen gäbe … viel-
leicht so ... ich hab trotz NSU nie Angst vor Rechts gehabt in diesem Land. Das
kann damit zu tun haben, dass ich von meiner Erscheinung her nicht eindeutig als
jemand identifizierbar bin, von dem man sagen könnte: „Boah, der gehört hier
aber nicht hin“. Der NSU ist eine überschaubare Größe, die man einordnen kann. 3 Selim Özdoğan hat den Begriff als Überschrift für eine Glosse über den Terminus ‚Migrationshin-
tergrund‘ verwendet. Der Text erschien in der Wochenzeitung Die Zeit („Unser Vibrationshinter-
grund“, 13.05.2009) und in dem von Deniz Utlu herausgegeben Kulturmagazin Freitext (Freitext Nr.
18/2011). Die Kategorisierung ‚Menschen mit Migrationshintergrund‘ muss demnach als diskursiver Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Ich habe damals schon nicht mehr auf der Rheinseite gewohnt und ich habe das so
erlebt, wie ich die meisten Sachen erlebe … das läuft immer so ein bisschen an mir
vorbei. Ich bin kein Nachrichtenkonsument. Das ist ein komplexes Thema, da
kommen mehrere Fässer zusammen, die man da aufmachen kann. Punkt 1 muss man vielleicht warnend voraus schicken. Du musst dir überlegen
… ich bin gar nicht in Mühlheim geboren, das steht nur in Wikipedia, aber ich bin
da aufgewachsen. Ich bin da zur Schule gegangen. Birlikte war ein Riesenkulturpro-
gramm, da sind jede Menge Menschen aufgetreten. Ich stand dann irgendwann da
und dachte, warum hat mich eigentlich keiner gefragt. Warum fragen die nieman-
den, der von hier kommt? Irgendjemand von denen – nicht alle müssen mich ken-
nen – aber irgendjemand hätte darauf kommen können, dass man auch … Alles,
was ich jetzt sage, muss mit Vorsicht genossen werden. Vielleicht bin ich ja nur Daniel Schreiner 164 beleidigt. Jedenfalls habe ich angefangen zu überlegen, ob das nicht auch ein Indiz
dafür ist, dass man gerne über die Leute, aber nicht mit ihnen redet. beleidigt. Jedenfalls habe ich angefangen zu überlegen, ob das nicht auch ein Indiz
dafür ist, dass man gerne über die Leute, aber nicht mit ihnen redet. Das heißt nicht, alle müssen türkische Freunde haben und bei denen zu Besuch
gewesen sein und mit ihnen Tee getrunken haben. Nein! Aber es ist auch ein Zei-
chen von fehlendem Kontakt auf eine Art Ob das jetzt auf Birlikte zutrifft oder g J
g
g
g
dafür ist, dass man gerne über die Leute, aber nicht mit ihnen redet. Das heißt nicht, alle müssen türkische Freunde haben und bei denen zu Besuch
gewesen sein und mit ihnen Tee getrunken haben. Nein! Aber es ist auch ein Zei-
chen von fehlendem Kontakt auf eine Art. Ob das jetzt auf Birlikte zutrifft oder
nicht ist ja letztendlich zweitrangig. Die Frage ist häufig nicht „Wie können wir mit
ihnen reden?“, sondern „Wie können wir für sie etwas Gutes tun“, was sie schon
wieder in so eine doofe Opferrolle hineindrängt. Das heißt nicht, alle müssen türkische Freunde haben und bei denen zu Besuch
gewesen sein und mit ihnen Tee getrunken haben. Nein! Aber es ist auch ein Zei-
chen von fehlendem Kontakt auf eine Art. Ob das jetzt auf Birlikte zutrifft oder
nicht ist ja letztendlich zweitrangig. Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Aber man muss dieser Mitte den rassistischen Spiegel vorhalten. Und ich bin
nicht so, dass ich sage, die sind alle böse. Nein, die wollen mir nichts Böses. Das ist
mir bewusst, die haben aber auch kein Bewusstsein für eine Augenhöhe, auf der
man sich begegnen könnte. Menschen sagen: „Aber wenn ich in Italien bin, bin ich
auch mit Zuschreibungen von außen konfrontiert, das ist normal, das ist mensch-
lich.“ Ja, aber der Unterschied ist, dass ich hier nicht fremd bin, ich gehöre dazu,
ich bin hier nicht als Tourist. — Es tut mir leid, aber ich muss mit dir über „Vibrationshintergründe“3 reden. Deine Prosa
verweigert sich kraftvoll der Vereinnahmung und Einsortierung in irgendwelche Konzepte von 165 Interview mit Selim Özdoğan ‚Migrationsliteratur‘ und Bindestrich-Identitäten. Wie gelingt dir das? Wie lange wird es dauern,
dass wir alle so frei und grenzenlos sehen können und übereinander sprechen können, ohne je-
manden ‚identitär‘ einzusortieren? Serdar Somuncu hat gestern auf dem Birlikte-Festival auch
sehr optimistisch [Selim lacht: Der spricht optimistisch? So kann ich mir den gar nicht
vorstellen!] von einer neuen deutschen Identität der Vielen gesprochen. Wo geht es hin? g p
g
Es hat lange gedauert bis ich verstanden habe, dass Erwartungen, die man auf-
grund meines Namens hat, zu verweigern, auch Unfreiheit bedeutet. Auf einmal
macht man Dinge – die man gerne machen würde – aus dieser Verweigerungshal-
tung nicht. Dass ich anders wahrgenommen werde, wird mir mit diesem Namen
erhalten bleiben. Da führt kein Weg dran vorbei. Und so lange wir Identität als
etwas begreifen, das sich klar abgrenzen lässt, werden wir auch mit diesem Identi-
tätsbegriff nie weiter kommen. Die Kunst war es, einfach irgendwann zu sagen:
Ich verweigere nicht und ich erfülle nicht. Ich bin frei davon. Meine literarische Bildung ist ja autodidaktisch. Ich habe gelesen, was mich in-
teressiert und habe dann lange Jahre geglaubt, ich wäre auf eine Art und Weise viel
weniger gebildet als viele meiner Kollegen, bis ich festgestellt habe: „Also okay, ich
kenne vieles nicht, was die gelesen haben, aber die kennen auch nicht vieles, was
ich gelesen habe. Die haben den Namen noch nie gehört!“ Ich kann die Budden-
brooks einordnen, aber ich fand es langweilig zu lesen und habe es weggelegt. Die
haben keine Ahnung, wenn ich sage, ich habe Angst und Schrecken in Las Vegas von
Hunter S. Thompson fünfmal gelesen. Das haben die nie gehört. Ist aber auch
egal. Diskriminierungsvorgang verstanden werden. Siehe dazu auch die treffende Analyse der Schriftstelle-
rin Lena Gorelik: „Ein wenig später sprach man von Menschen mit Migrationshintergrund, um einen
Namen für Menschen wie mich zu haben. Man gestand den Ausländern also mittlerweile zu, auch –
oder in erster Linie – Mensch zu sein. Ein echter Fortschritt. Das befanden dann auch Wissenschaft-
ler, Politiker, Öffentlichkeitsmacher für politisch korrekt, und der Begriff blieb, bis er wie eine anste-
ckende Krankheit um sich griff, sich verbreitete wie ein Virus. Jedermann erleichtert, sich endlich
unproblematisch zu dieser problematischen Masse von Menschen äußern zu können, nicht für eine
Sekunde in Erwägung ziehend, dass diese Menschen, für die man einen so schönen politisch korrek-
ten Namen gefunden hatte, so heterogen sind, wie es Gruppen nur sein können.“ (Gorelik, Lena
(2012): Sie können aber gut Deutsch, München, S. 33) Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Ich bin ja letzten Endes ein sehr amerikanisch geprägter Autor und kein
deutsch oder türkisch geprägter Autor. Ich verstehe Literatur auch als etwas Inter-
nationales. Und ja, Literatur ist auch immer etwas Nationales, weil wir sie A nun
mal in einer Sprache schreiben müssen und weil sie B auch etwas über ein Land
aussagt, dessen Sprache sie benutzt. Aber ganz am Ende ist Literatur international
und wir könnten uns auch gesellschaftlich dahinentwickeln, dass Vielfalt gelebt
wird und nicht nationalstaatliche Trennungen gemacht werden. Aber ich fürchte,
die Literatur wird dabei hinterherhinken. Mein Lieblingsbeispiel ist Hiphop. Hip-
hop wird als internationale Kultur verstanden, aber wir haben heute außer ein paar
Feuilletonisten niemanden, der mit dem Finger auf die Hiphoper mit Migrations-
hintergrund zeigt und sagt „Die haben eine andere kulturelle Herkunft.“ Man muss
die Musik nicht mal gut finden, aber das sind in der Innensicht alles deutsche Hip-
hoper. Die bringen deutschen Hiphop nach vorne, unabhängig von ihrer Herkunft. Daniel Schreiner 166 Das liegt A daran, dass die das auch als internationale Kultur begreifen und dass
die alle auch amerikanischen Hiphop hören, und B daran, dass Vielfalt einfach
schon gelebt wird. Das ist ja so. Das können wir nicht mehr wegdiskutieren. Das
wird ja tatsächlich gelebt. In der Literatur, das sind ja gerade die Leute, die gerne in
der Regel auf sowas Stumpfes wie Hiphop herabschauen und das für dumm halten
und unmusikalisch und was weiß ich. Der Wille zur Differenzierung führt halt
auch zu Schubladendenken. Das liegt in der Natur der Sache begründet, dass die
Leute, die wir als Literaten für dumm halten, auf eine Art schon zwei Schritte wei-
ter sind. Sehr genaue rationale Unterscheidungen führen zu Ausgrenzung und zur
Betonung der Andersartigkeit. — Du hast in deinen Audioblogs verstärkt deine Meinung zu Maxim Billers These der „lang-
weiligen deutschen neuen Literatur“4 wiedergegeben und die Position bezogen, dass man Literatur
nicht ihre Herkunft vorwerfen kann, dass Literatur Grenzen und Schranken aufhebt. Was
treibt dann stattdessen jemanden wie Akif Pirinçci5 um? Denkst du über sowas nach oder lässt
dich das kalt und nervt es dich gar, deswegen gefragt zu werden, nur weil du auch einen türki-
schen Nachnamen hast? Das interessiert mich ja, das ist nicht schlimm. Ich kann mich nur nicht richtig
darüber aufregen. Man kann das so und so sehen. 5 Akif Pirinçci war der erste türkisch-deutsche Bestsellerautor. Sein Katzen-Krimi Felidae aus dem
Jahr 1989 wurde sogar ins Englische übersetzt und verfilmt. Seit einigen Jahren ist Pirinçci nun als
Verfasser verschwörungstheoretischer ‚Sachbücher‘ einmal mehr erfolgreich. Sein Buch Deutschland
von Sinnen. Der irre Kult um Frauen, Homosexuelle und Zuwanderer (2014) und die Folgeveröffentlichungen
sind allesamt frauenfeindlich, rassistisch und homophob. 4 Biller, Maxim (2014): „Letzte Ausfahrt Uckermark“, in: Die Zeit, 20.02.2014. — Ein Buch will vermarket werden. Gibt es moralische Grenzen für dich, die du nicht über-
schreiten willst? Da ist aber eine sehr schwer zu beantwortende Frage. Moral ist ja in der Regel ein
willkürlich gesetzter Wert. Es hat keinen Wert, dass ich eine andere Vorstellung
von Literatur habe und mir denke: „Aber das Literaturverständnis dieser anderen
Leute ist halt falsch.“ Ich komme nirgendwo hin damit. Ich bin nicht derjenige, der
die Spielregel bestimmt. Also wenn du bei einem Spiel mitspielen willst, musst du
dich auf eine Art auch an die Spielregeln halten. Dass man korrumpierbar ist, kann
schon sein. Aber der Gradmesser für: „Würdest du das machen oder halt nicht“,
ist nicht moralisch, sondern eine Frage nach: „Bleibt das Schreiben eine schöne
Beschäftigung für mich? Hilft das Schreiben Grenzen aufzulösen?“ Wenn die
Antwort ja ist, ist alles gut, wenn es um etwas anderes geht, kann ich es nicht ma-
chen. — Im Roman „DZ“ kritisiert Ziggys Tochter die Verlogenheit der europäischen Gesellschaft
und träumt vom wilden Leben in der DZ. Was ist es, was sie kritisiert und was davon siehst du
schon heute in Europa angelegt? p
g
g
Das ist ein ganz grundsätzliches Problem, glaube ich, von Gesellschaften, die zu
groß werden. Irgendwann gehen halt Kontakte verloren. Und Dinge, die keine
Kontakte sind, werden dir als Kontakt verkauft. Die gesamte Kulturbranche, wir
können das auch Unterhaltungsindustrie nennen, ist ja letzten Endes ein Beispiel
dafür, dass in dem System, in dem wir leben, eine gewisse – Verlogenheit ist nicht
das Wort dafür – eine gewisse Mehrdeutigkeit enthalten ist. Nehmen wir das Buch
als Kulturgut. Es ist nur ein Produkt der Unterhaltungsindustrie, das muss man
vermarkten, das muss sich so und so oft verkaufen. Der Autor will davon leben. Der Verlag will davon leben. Das unterscheidet sich im Grunde nicht von einer
Seife, die du verkaufst. Aber dann haben wir hier die Möglichkeit … du kannst mit
dem Text in Kontakt treten. Der löst etwas bei dir aus und das ist es, was der Au-
tor, der am Ausgangspunkt der Kette steht, sich wünscht. Am anderen Ende der
Kette ist der Leser, der sich vielleicht das selbe wünscht und dazwischen ist es
einfach nur ein zu vermarktendes Produkt. Punkt! Und solange das ein zu ver-
marktendes Produkt ist, interessiert nicht der Kontakt, sondern der Markt interes-
siert. Dadurch kommt sowas zustande, was man Verlogenheit nennt. Ich will Kon-
takt, aber ich will die Bücher auch verkaufen. Interview mit Selim Özdoğan
Erfüllen und Verweigern von Erwartungshaltungen Ich sehe das als Verlogenheit:
Wir geben den Leuten die Möglichkeit, sich über Pirinçcis Position zu empören,
indem wir darüber berichten. Das hat auch einen verstärkenden Effekt für Leute,
die anderer Meinung sind. Aber ich glaube, dass diesen Leuten viel zu viel Auf-
merksamkeit geschenkt wird. Das ist die Medienwelt, in der wir leben. Warum
kriegt denn dieser Mann so viel Platz? Und drei Wochen bei Amazon auf Platz 1
gewesen zu sein ist überhaupt kein hinreichender Grund. Es gibt andere Bücher,
die bei Amazon auch auf Platz 1 gewesen sind. Das hat aber niemanden interes-
siert. Woher kommt das? Wieso kriegen die so viel Raum? Warum durfte Thilo
Sarrazin überall hingehen? Ich verstehe nie genau, wie diese Sachen zusammen-
hängen … So ist aber Feuilletondenken in Deutschland. Natürlich habe ich auch
Theorien dazu. Aber der Weg in meinem Kopf von einem Comedian wie Bülent
Ceylan zu Akif Pirinçci ist nicht so weit. Die dürfen das. Die haben den richtigen
Namen und die dürfen dann Sachen sagen, die eigentlich tabu sind und als rassis-
tisch gewertet werden. Wir benutzen diese Leute, um bestimmte Positionen zu
vertreten, um bestimmte Haltungen zu behaupten. Wenn Akif Pirinçci in einer
Rede einen strafrechtlich nicht relevanten KZ-Vergleich bringt und sein Verlag
daraufhin seine Bücher aus dem Programm nimmt, ist das marketingtechnisch eine 167 Interview mit Selim Özdoğan kluge Entscheidung des Verlages, aber es steht niemand auf und fragt nach der
Freiheit der Kunst. Danach fragen wir, wenn wir einen linken Platzhalter haben. So
existieren diese Figuren, um öffentlich Haltung zu behaupten. 1964 bekommt Sid-
ney Poitier den Oscar, dann kann man behaupten, Schwarze sind ja gar nicht be-
nachteiligt. Dann müssen sie jahrelang den Clown machen wie Eddie Murphy. Dann kommt Denzel Washington Jahrzehnte später und wird eigentlich im Grun-
de wieder als Platzhalter missbraucht. — Ein Buch will vermarket werden. Gibt es moralische Grenzen für dich, die du nicht über-
schreiten willst? — Ein Buch will vermarket werden. Gibt es moralische Grenzen für dich, die du nicht über-
schreiten willst? Das geht nicht genau in die Richtung
deiner Frage … Aber ich glaube, so entstehen diese Sachen. Es kommen dann Daniel Schreiner 168 einem Dinge verlogen vor, weil Interessen erst mal behauptet werden, aber wenn
man nachfragt, geht es doch um etwas Anderes. Das Interesse der Drogenpolitik
ist ja nicht: „Wir wollen aufgeklärte mündige Bürger haben, die Dinge selber für
sich entscheiden können“, sondern das Interesse der Drogenpolitik ist irgendetwas
anderes, aber wir tun so, als würde es um die Gesundheit der Bürger gehen. — Der Ungrund, ein Wort, das ja auf den Philosophen Jakob Böhme zurückgeht, bezeichnet
den Ursprung aller Dinge und bei dir heißt es „Am Anfang war Klang“! — Der Ungrund, ein Wort, das ja auf den Philosophen Jakob Böhme zurückgeht, bezeichnet
den Ursprung aller Dinge und bei dir heißt es „Am Anfang war Klang“! Die Idee scheint auch in „Zwischen zwei Träumen“ durch. Die Romane „DZ“ und „Zwischen
Zwei Träumen“ wirken auf mich wie Zwillingsschwestern, die zusammengehören. Ist das so und
was sind das für Gesellschaften, in denen die beiden Romane spielen? Die Idee scheint auch in „Zwischen zwei Träumen“ durch. Die Romane „DZ“ und „Zwischen
Zwei Träumen“ wirken auf mich wie Zwillingsschwestern, die zusammengehören. Ist das so und
was sind das für Gesellschaften, in denen die beiden Romane spielen? f
f
,
p
Dass die so ein bisschen ähnlich sind, weiß ich, aber was passiert ist … ich habe
zwei Antworten dazu in meinem Kopf. Die Idee, dass du Träume aufzeichnen
kannst, ist ja nicht meine. Ganz eindeutig. Da kommen die meisten Menschen von
ganz alleine drauf. Bei Zwei Träumen ging es mir darum, was kann man daraus bas-
teln? Dass das dann eine spirituelle Komponente bekommt, liegt in meinem Cha-
rakter begründet. Das Schöne war, auf einmal eine freie Fläche zu haben. Norma-
lerweise, die anderen Sachen, die ich geschrieben habe, hatten immer einen festen
Bezugsrahmen, den wir Realität nennen, an die sich die Handlung halten musste. In der Heimstraße kann nicht plötzlich einer anfangen zu fliegen. Das ist nicht
schlimm. Da ist immer noch viel Platz, du musst dich halt nur an gewisse Grenzen
halten. Bei Zwischen zwei Träumen habe ich festgestellt, „Boah ist das geil, jetzt
kannst du machen, was du willst!“ Es war sehr schön zu schreiben und ich wollte
das noch einmal haben. — Ein Buch will vermarket werden. Gibt es moralische Grenzen für dich, die du nicht über-
schreiten willst? Ich würde das ein weiteres Mal so machen − dass diese
beiden Romane alternative Realitäten behandeln und drogenlastig sind, ist dabei
eher ein Zufall. Mein jüngster Roman Wieso Heimat, ich wohne hier doch nur zur Miete,
geht in eine ähnliche Richtung. In ihm wird aber deutlich weniger mit veränderten
Bewusstseinszuständen gearbeitet, sondern mit kulturellen Realitäten, die ja auch
jeder hat. Das ist dein Wahrnehmungszustand. Bei Drogen ist das den Leuten
einfacher zu erklären, aber es gibt auch Wahrnehmungszustände eines Veganers
oder eines regelmäßigen Burger-Essers und ihren Blick auf Deutschland. All das
sind ja auch verschiedene Perspektiven. — Nochmal zum Ungrund zurück …. Klang … das ist so ein Klischee-Standardspruch, aber du wirst viele Autoren fin-
den, die doch lieber Musiker geworden wären, wenn sie gekonnt hätten, doch dann
fehlte das Zutrauen oder Talent. Aber Sprache ist letzten Endes immer auch
Klang. Das wird in Deutschland nicht unbedingt wahrgenommen. Da wird auch
gerne behauptet, die deutsche Sprache sei nicht melodisch. Das ist für mich immer
ein Hinweis darauf, die eigene Unfähigkeit der Sprache anzulasten. Sprache ist für
mich immer Klang und man kann das auch auf verschiedene Art und Weise erklä-
ren. Wenn du im Nebenraum Leute hast, die sich unterhalten, aber du kannst die
Wörter nicht verstehen, kriegst du trotzdem eine Ahnung, was dort für eine Stim- Interview mit Selim Özdoğan 169 mung herrscht. Für mich ist Klang total wichtig. Schreiben ist immer auch: Ich
muss die Sätze zu Ende schreiben, die müssen passen, die müssen stimmen. Du
kannst sehr viel über Klang wettmachen, was dir an – oh ein Eigentor – sprachli-
cher Qualität fehlt. mung herrscht. Für mich ist Klang total wichtig. Schreiben ist immer auch: Ich
muss die Sätze zu Ende schreiben, die müssen passen, die müssen stimmen. Du
kannst sehr viel über Klang wettmachen, was dir an – oh ein Eigentor – sprachli-
cher Qualität fehlt. — Die Übersetzung der Tontafel in DZ war sprachlich sehr erfindungsreich … — Die Übersetzung der Tontafel in DZ war sprachlich sehr erfindungsreich … Ja, aber es hat irgendwann auch keinen Spaß mehr gemacht. Ich musste eine Spra-
che finden, die sich deutlich von der Alltags- und Schriftsprache unterscheidet und
immer noch lesbar ist. — Hat dir das Türkische dabei geholfen? Dass
Portugal, Deutschland, Spanien, Italien, Griechenland nicht so eine astreine demo- Daniel Schreiner 170 kratische Geschichte in den letzten 100 Jahren hatten, ist auch egal. Wir haben das
erfunden, wir kennen uns damit aus. Und die anderen haben das gefälligst auch
einzuführen.“ Dabei geht es in erster Linie um wirtschaftliche Interessen. Ich weiß
ja auch keinen Weg daraus oder eine bessere Gegenwelt, aber so sind das letzten
Endes nur Details, die anders sind, sowohl Europa als auch die DZ sind beides im
Grunde repressive Systeme. — Hast du einen gewissen Anspruch, mit deiner Arbeit etwas verändern zu wollen? a du einen gewi en
n pruch, mi deiner
rbei e wa verändern zu wollen? Nein, ich möchte nicht die Leute verändern, aber ich möchte in den Kopf von
Leuten. Es gibt kein Ziel dahinter außer Kontakt. Die Bücher sind nur ein Aus-
druck von meinem täglichen Leben. Und da wünsche ich mir schon ein anderes
Bildungssystem, das dem einzelnen mehr Autorität und mehr Autonomie beibringt
und zugesteht. Essen ist ein gutes Beispiel. Verbraucherschutzverbände klagen
darüber, dass die Verpackung Falsches verspricht. Die Klage ist berechtigt. Aber
wir können alle lesen und schreiben. Wir können alle dieses Ding im Supermarkt
umdrehen und sehen, was denn die Zutaten sind. So viel ‚Autoritäten in Frage
stellen‘ würde ich mir schon wünschen. Meine Arbeit als Autor könnte dazu füh-
ren, den Leuten einen Geschmack von Freiheit zu geben. Aber der Anspruch ist
jetzt nicht: „Ich will die Leute ändern“. Ich will, dass wir in einer besseren Welt
leben. Ich weiß nicht, wie eine bessere Welt hier aussehen sollte, aber ich weiß, es
wird einfach sehr viel Scheiße als Lebensmittel verkauft. Dann hast du auf der
anderen Seite dogmatische Veganer, ich denke dann: „Ey Alter, du bist Großstadt-
kind, sonst wärst du nicht so.“ Leute, die Fleisch essen, sind nicht per se schlecht. Und die, die sich vegan ernähren, sind nicht alle gut. Von diesem komischen
Schwarzweißdenken einfach mal wegzukommen und von dieser Überzeugung
wegzukommen, dass man selber auf jeden Fall alles richtig macht. Nur deshalb
kommen wir auf so Ideen wie Demokratieexport. — Hat dir das Türkische dabei geholfen? Nein, da helfen … Referenzpunkte könnte ich dir insgesamt so viele aufzählen, das
ist aber egal. Die Idee, dass du eine Sprache hast, die alles über Verben ausdrückt,
finde ich naheliegend. Da bin ich aber nicht alleine darauf gekommen, die habe ich
von Borges. Dann habe ich ein Buch gelesen von einem ehemaligen Missionar:
Dan Everett, Das glückliche Volk, so heißt das auf Deutsch, oder Das glücklichste
Volk. Der versucht die Sprache des Stammes in Brasilien, bei dem er gelebt hat, die
eine ganze besondere Struktur hat, zu beschreiben. In wie viele Richtungen kann
Sprache denn gehen. Brauchen wir Wörter für Farben …? — Welche Werte sind in diesen zukünftigen Welten der Romane „DZ“ und Zwischen zwei
Träumen wichtig? Wie definieren sich die Menschen dort? Die Protagonisten reisen viel und sind
an exotischen Orten, nationale Identitäten scheinen weniger eine Rolle zu spielen? Nationale Identitäten sind ja etwas sehr Künstliches, die zur Abgrenzung dienen. Das hat mich nie interessiert. Eine kulturelle Identität ist da etwas anderes, ist aber
beide Male nicht Thema. In Zwischen zwei Träumen wollte ich eine Welt beschreiben,
die sich von der heutigen unterscheidet, auch in den unerheblichen Details. In DZ,
das ist ja egal, ob wir dieses Europa oder die DZ haben, es ist hier wie dort ein
kapitalistisches System, das regiert. Wir verkaufen Träume, wir verkaufen Drogen,
das sieht nach Freiheit aus. Weil etwas da drüben illegal ist, bei uns legal ist. Aber
letztlich entscheidet der Marktwert und der ist wichtiger als die Freiheit. — Ist diese Zukunft so viel anders als unsere Gegenwart? Nein, ich finde nicht. Was einfach damit zu tun hat, dass wir immer noch ein kapi-
talistisches System haben, und das diktiert. Das ist kein Unterschied und das ist ja
die Welt, in der wir leben. Es ist immer total schön mit so Worthülsen wie Demo-
kratie und Meinungsfreiheit um sich zu schmeißen, aber man muss sich nichts
vormachen. Das zählt alles nichts. Das sind nicht die vorrangigen Interessen. Auch
wenn so getan wird, als würden wir weltweit Demokratie befürworten. Erst wollte
man das Christentum überall in der Welt verbreiten. Und dann sagte man: „Das
war ein Fehler … machen wir jetzt nicht mehr, fanden wir im Nachhinein auch
doof, aber Demokratie als Wert finden wir okay, das wollen wir einführen. — Hat dir das Türkische dabei geholfen? — Vielen Dank für das Gespräch! Ich danke! — Hat dir das Türkische dabei geholfen? — Krishna Mustafa, der Protagonist in deinem jüngsten Roman „Wieso Heimat, ich wohne zur
Miete“ bricht ebenfalls mit diesem Schwarzweißdenken und stellt mit seiner Naivität und Neu-
gierde Erwartungshaltungen und stereotype Sichtweisen in der Türkei und der BRD generell in
Frage … Wieso Heimat, ich wohne zur Miete lebt davon, einseitige Rollenzuschreibungen sicht-
bar zu machen und nicht nur die eine Seite sichtbar zu machen, sondern gleichzei-
tig zu zeigen, dass die eigene Sicht auf die Rolle, die man spielt, auch nicht richtig
sein muss. Und es somit eigentlich immer bei jedem ‚Entweder-oder‘ noch eine
dritte Möglichkeit gibt. Wir haben uns aber angewöhnt zu glauben, wer nicht auf
der einen Seite steht, der steht auf der anderen. Dichotomien zu denken hat Tradi-
tion, aber ich glaube, dass der Reflex zu urteilen sich mit der Beschleunigung der
Medien verstärkt hat. Und dass eine Normierung stattfindet, die wenig Zwischen-
töne zulässt. Identitäten werden medial fabriziert. 60% der Türken in Deutschland
haben Erdoğan gewählt. Das ist die medial verbreitete Wahrheit, die ein Bild vom
Türken entwirft. Die statistische Wahrheit ist, dass ca. 3 Millionen türkisch- Interview mit Selim Özdoğan 171 stämmige Menschen in Deutschland wohnen, von denen 1,4 Millionen aufgrund
von Alter und Pass wahlberechtigt in der Türkei sind. Von diesen 1,4 Millionen
haben 34% gewählt. Von diesen 34% haben 59,7% Erdoğan gewählt. Das sind ca. 220.000 Menschen. Was ja nicht ganz 60% von 3 Millionen sind. So wird ein ein-
seitiges Bild generiert. stämmige Menschen in Deutschland wohnen, von denen 1,4 Millionen aufgrund
von Alter und Pass wahlberechtigt in der Türkei sind. Von diesen 1,4 Millionen
haben 34% gewählt. Von diesen 34% haben 59,7% Erdoğan gewählt. Das sind ca. 220.000 Menschen. Was ja nicht ganz 60% von 3 Millionen sind. So wird ein ein-
seitiges Bild generiert. Das Beispiel illustriert dies nicht zu 100 Prozent, aber es zeigt, wie die Medien
grundsätzlich arbeiten und du dann nur eine Seite wahrnimmst. Niemand sagt, die
Mehrheit hat nicht gewählt. Krishna Mustafa aus Wieso Heimat ist jemand, der das
vorgefertigte Bild aus Neugierde und aus seiner eigenen Logik heraus hinterfragt. Dabei bemerkt er, wie wenig Substanz diese Bilder haben. Er kann erkennen, dass
die Fragen, die an ihn herangetragen werden, oft ins Leere greifen, weil sie die
Wahlmöglichkeit von vornherein beschränken und polare Lösungen suggerieren. Die Welt ist rund, es gibt nicht die eine oder die andere Seite, Grenzen sind immer
nur willkürlich gesetzt. Werkverzeichnis Selim Özdoğan Es ist so einsam im Sattel, seit das Pferd tot ist, Berlin, 1995. Es ist so einsam im Sattel, seit das Pferd tot ist, Berlin, 1995. Nirgendwo & Hormone, Berlin, 1996. Es ist so einsam im Sattel, seit das Pferd tot ist, Berlin, 1995. Nirgendwo & Hormone, Berlin, 1996. Nirgendwo & Hormone, Berlin, 1996. Passen die Schuhe, vergisst man die Füße, Hamburg, 2012. Kopfstand im Karma-Taxi, Bekenntnisse eines Pranajunkies, Winterthur, 2012.
DZ, Innsbruck, 2013. Kopfstand im Karma-Taxi, Bekenntnisse eines Pranajunkies, Winterthur, 2012. 1 Diese Unterscheidungen in der Bildanalyse stammen aus der kunsthistorischen Theorie von Erwin
Panofsky. Aufgrund des Einflusses dokumentarischer Methoden werden sie häufiger in
sozialwissenschaftlichen Analysen verwendet. Für eine gute Darstellung dieser Methode: Burkard,
Michel
(2013)
„Fotografien
und
ihre
Lesarten.
Dokumentarische
Interpretation
von
Bildrezeptionsprozessen“, in: Bohnsack et al. (Hg.): Die dokumentarische Methode und ihre
Forschungspraxis. Grundlagen qualitativer Sozialforschung, S. 99-131. Ein gutes Leben ist die beste Rache, Berlin, 1998. Ein gutes Leben ist die beste Rache, Berlin, 1998. Ein gutes Leben ist die beste Rache, Berlin, 1998. Mehr, Berlin, 1999. Mehr, Berlin, 1999. Im Juli, Hamburg, 2000. Ein Spiel, das die Götter sich leisten, Berlin, 2002. Trinkgeld vom Schicksal, Berlin, 2003. Die Tochter des Schmieds, Berlin, 2005. Tourtagebuch, Regensburg, 2006. Zwischen zwei Träumen, Bergisch Gladbach, 2009. Ein Glas Blut, Hamburg, 2010. Heimstraße 52, Berlin, 2011. Der Klang der Blicke, Innsbruck, 2012. 172 Daniel Schreiner Kopfstand im Karma-Taxi, Bekenntnisse eines Pranajunkies, Winterthur, 2012. DZ, Innsbruck, 2013. Kopfstand im Karma-Taxi, Bekenntnisse eines Pranajunkies, Winterthur, 2012. Was wir hörten, als wir nach der Wahrheit suchten, Köln, 2013. Wieso Heimat, ich wohne zur Miete, Innsbruck, 2016. Almut Küppers/Barbara Pusch/Pınar Uyan
Semerci (Hg.) (2016): Bildung in transnationalen
Räumen Education in Transnational Spaces,
Wiesbaden (297 S.) Emre Arslan In einer Anzeigenkampagne der ‚Deutschlandstiftung Integration‘ zwischen 2009
und 2011 strecken öffentlich bekannte Migranten aus Deutschland ihre Zungen
heraus. Auf dem Plakat steht das folgende Motto: „Raus mit der Sprache. Rein ins
Leben“. Nur so viel (oder besser: so wenig) Information reicht für viele Menschen
in Deutschland aus, um zu verstehen, worum es in dieser Kampagne geht: Sprache
ist der Schlüssel, der die Tür zur Integration in Deutschland aufschließt. Die Stif-
tung erstrebte anscheinend eine möglichst sichere Vermittlung der Botschaft mit
folgenden Zeichen: Die Zungen unserer ‚Vorzeigemigranten‘ sind rot-schwarz-
gold bemalt. Die Übersetzung dieses vor-ikonographischen Wissens in das Ikono-
graphische1 sollte sogar für ungebildete Migranten eindeutig sein. Auf den Zungen
der glücklichen und stolzen Migranten ist nämlich eine deutsche Flagge sichtbar. Mit der Sprache ist somit die deutsche Sprache und nicht eine beliebige, wie etwa 174 Emre Arslan die Herkunftssprache der Migranten, gemeint. Mit dieser zusätzlichen Hilfe ist der
Botschaft nicht zu entgehen: Migranten sollen Deutsch lernen, dann sind sie gut
integriert und erfolgreich. die Herkunftssprache der Migranten, gemeint. Mit dieser zusätzlichen Hilfe ist der
Botschaft nicht zu entgehen: Migranten sollen Deutsch lernen, dann sind sie gut
integriert und erfolgreich. g
g
Die wissenschaftliche Analyse kann natürlich nicht bei der bloßen Wiedergabe
des ikonographischen Wissens bleiben, da es sich dabei vorwiegend um die Ikono-
logie eines Bildes handelt. Somit ist die wissenschaftliche Frage wie folgt zu stellen:
Was verrät diese Anzeigenkampagne unabhängig von ihrer beabsichtigten Bot-
schaft über unseren Zeitgeist? Der vorliegende Sammelband von Küppers, Pusch
und Semerci bietet eine solide Grundlage, um die ikonologische Ebene solcher
Bilder oder Diskurse zu durchdringen.2 Hegemoniale Diskurse und Strukturen
sowohl in Deutschland als auch in der Türkei – trotz ihrer wichtigen Unterschiede –
sind sehr stark von einer monolingualen und mononationalen Ideologie geprägt. Als
Folge hinterlassen diese dominanten Ideologien und Praxen eine große Lücke, weil
sie die reale transnationale Wirklichkeit von Menschen nicht erkennen und aner-
kennen. Dieser Sammelband kann auch als ein Versuch für die Schließung dieser
Lücke betrachtet werden. Mehrere Beiträge im Buch belichten unsichtbar bleiben-
de Erlebnisse, Erfahrungen, Probleme und Potenziale der realen Menschen insbe-
sondere im Bereich der Bildung im transnationalen Raum. 2 Passenderweise wird gerade diese Anzeigekampagne in einem der Beiträge im Buch behandelt. Julia
Split verdeutlicht in ihrem Beitrag die Differenzen zwischen der o. g. Suggestion der Kampagne und
der Realität der in Callcentern arbeitenden türkischen Rückkehrer in Istanbul (S. 65-66). Almut Küppers/Barbara Pusch/Pınar Uyan
Semerci (Hg.) (2016): Bildung in transnationalen
Räumen Education in Transnational Spaces,
Wiesbaden (297 S.) Im Beitrag von Susanne Becker über die Sprachvermischung (oder wie es in ih-
rem Beitrag heißt: Translanguaging) im deutsch-türkischen transnationalen Raum
werden nicht nur die Abwertungsmechanismen durch eine monolinguale Ideologie
geschildert, sondern auch Beispiele der unsichtbar bleibenden, aber nützlichen
Funktionen vom „Translanguaging“ geliefert: „Neben dem Arbeitsmarkt kann
Translanguaging in lokalen transnationalen Räumen wie türkischen Supermärkten
und Friseursalons in Deutschland als Ware gehandelt werden. Hier wird Translan-
guaging jenseits der tatsächlichen Kommunikationsmöglichkeiten als Marker von
Ethnizität zur Ware. So antizipieren Kunden mit einem „Süpermarket“ eventuell
Produkte, die sie in einem „Supermarkt“ nicht bekommen würden“ (S. 48). Auch
Julia Split setzt sich mit der widersprüchlichen Position der Migrantensprachen
zwischen der alltäglichen Bedeutung der persönlichen Ebene und einer ideologi-
schen Funktion auf der nationalen Ebene auseinander. Während Rück- und Ab-
wanderer in deutschen Firmen in der Türkei ihre Deutschkenntnisse als Werte auf
dem Markt einbringen können, müssen sie beispielsweise in der Kommunikation
mit Kunden ihre türkischen Namen verheimlichen und einen deutschen Namen
annehmen (S. 65). Auf europäischen und nationalen Ebenen wird durchaus ein starker Mehrspra-
chigkeitsdiskurs gepflegt. Kutlay Yağmur legt in einer quantitativen Analyse jedoch
dar, dass die Migrantensprachen in fast allen Ländern Europas im Verhältnis zu
den ‚Prestigesprachen‘ sehr wenig anerkannt und unterstützt werden (S. 105). Am
Beispiel des Türkischunterrichts bestätigen Christoph Schroeder und Almut Küp- Bildung in transnationalen Räumen 175 pers im deutschen Kontext diese Tendenz. Die Autoren berichten über den Status
und die Herausforderungen der türkischen Sprache im deutschen Bildungssystem
und folgern, dass das Bildungspotenzial dieser Sprache hier nicht erkannt wird. Die
Beiträge von Seyitahmed Tokmak über Schulen in freier Trägerschaft und von
Beatrix Albrecht über die interkulturelle Öffnung in der hannoverschen Albert-
Schweitzer-Schule geben seltene Beispiele des Nutzens dieses Potenzials wieder. Almut Küppers wertet in ihrem Beitrag die Prozesse und Wirkungen des deutsch-
türkischen bilingualen Unterrichtskonzepts der Albert-Schweitzer-Schule unter der
Leitung von Beatrix Albrecht in Hannover aus. Besonders wertvoll macht ihre
Analyse die starke Einbeziehung der Kinderperspektive, die im nationalistischen
Diskurs häufig ausgeblendet wird: „Anders als bei Politikern, Bildungsplanern,
Schulleitern, Eltern oder Lehrern sind die Handlungen der Kinder kaum beein-
flusst von Ideologien, Weltanschauungen oder Überzeugengen. … In ähnlicher
Weise begegnen Kinder Sprachen. Sie kümmern sich nicht um deren Status in der
Gesellschaft oder um deren Wert als kulturelles Kapital, sondern sind grundsätz-
lich neugierig darauf, die Sprachen zu lernen und zu benutzen“ (S. 183). Almut Küppers/Barbara Pusch/Pınar Uyan
Semerci (Hg.) (2016): Bildung in transnationalen
Räumen Education in Transnational Spaces,
Wiesbaden (297 S.) Die über-
zeugende Analyse von Küppers zeigt die sinnvollen sozialen, emotionalen und
intellektuellen Effekte des Konzepts auf die Kinder. Küppers berichtet gleichzeitig
von einem frustrierenden Weg der Schulentwicklung, insbesondere wegen der
hohen ideologischen Einwände und Hürden. Ähnliche Ergebnisse findet man auch
im Beitrag von Sarah Fürstenau: Die Erfahrungen mit dem von ihr initiierten Pro-
jekt MIKS (Mehrsprachigkeit als Handlungsfeld Interkultureller Schulentwicklung. Eine Interventionsstudie in Grundschulen) belegen erneut die verhaltene und
skeptische Haltung von Schulleitung, Lehrkräften und Eltern (auch mit türkischen
Hintergrund) gegenüber einer Einbeziehung der Migrantensprachen in den Schul-
alltag. Trotz der zunehmenden Realität der Transnationalisierung zwischen Deutsch-
land und der Türkei bleibt die mononationale und monolinguale Ideologie auch in
der Türkei sehr verbreitet. Während Sarah Rottmann die negativen Effekte dieser
Ideologie auf Kinder von Rückkehrern aus Deutschland feststellt, beobachtet Mü-
ge Ayan Ceyhan solche Auswirkungen auf kurdische Kinder in türkischen Schulen. Beide Beiträge zeigen dabei, dass die vorherrschende nationalistische Ideologie in
türkischen Schulen zu einer starken Unterdrückung der realen Potenziale, Emotio-
nen und Erlebnisse der Kinder führt. Weitere zwei Beiträge im Buch setzen sich
gerade mit dieser engstirnigen nationalistischen Ideologie in der Türkei auseinan-
der. Kenan Çayır analysiert Schulbücher in der Türkei und stellt fest, dass diese ein
stark sicherheitsorientiertes Weltbild propagieren und einen Argwohn gegenüber
Transnationalisierung und Globalisierung fördern. Batuhan Aydagül berichtet über
einen ‚„Education Think-and-do-Tank“, der sich an türkischen Schulen mit dem
Ziel der Förderung des kritischen Denkens engagiert. Sein Beitrag verdeutlicht
nicht nur die Bedeutung solcher Aktivitäten für die Schule, sondern auch die star-
ken Widerstände gegen eine kritische Auseinandersetzung mit dem herrschenden
Schulethos seitens der Schulleitung und der Lehrkräfte. Die engstirnige nationalis- Emre Arslan 176 tische Ideologie in der Türkei trägt der immer intensiver gewordenen deutsch-
türkischen Transnationalisierung der Bildung nicht Rechnung. Die Beiträge von
Karl-H. Ressmeyer und Nilüfer Tapan / Sevinç Hatipoğlu behandeln die Bedeu-
tung der deutschen Schulen in der Türkei für Interkulturalität und Transnationali-
sierung. Im Hochschulbereich ist diese Tendenz sogar noch prägnanter: Şeyda Ozil
berichtet über die seit langem bestehenden türkisch-deutschen Beziehungen im
Hochschulwesen am Beispiel der Germanistikabteilung der Istanbul Universität
und stellt fest: „Insgesamt lässt sich aufgrund der vielfältigen Entwicklungen und
Kooperationen zwischen der Türkei und Deutschland mittlerweile von institutio-
nalisierten transnationalen Beziehungen sprechen“ (S. 254). Almut Küppers/Barbara Pusch/Pınar Uyan
Semerci (Hg.) (2016): Bildung in transnationalen
Räumen Education in Transnational Spaces,
Wiesbaden (297 S.) İzzet Furgaç berichtet
über die Türkisch-Deutsche Universität in Istanbul als eine vertiefte Institutionali-
sierung des transnationalen Raums zwischen beiden Ländern und Wiebke Bach-
mann behandelt hauptsächlich die Bemühungen des Deutschen Akademischen
Austauschdienstes (DAAD) in der Türkei. Der Beitrag von Süheyla Schroeder und
Karin Schmidt über Bahçeşehir University Campus in Berlin offenbart die Gegen-
seitigkeit dieser Entwicklung. g
g
Wie die Beiträge im Sammelband zeigen, ist eine enorme und vielschichtige
deutsch-türkische Transnationalisierung nicht nur auf der Alltags-, sondern auch
auf institutioneller Ebene, vor allem im Bereich von Bildungseinrichtungen festzu-
stellen. Sie verdeutlichen jedoch auch gleichzeitig, dass dies nicht automatisch zu
einem kritischen Hinterfragen der veralteten Ideologien oder Denkweisen führt. Anscheinend fehlt vielen Menschen u. a. Wissen, um ihre altbekannten und
dadurch bequemen Denkmuster zu ändern, obwohl diese nicht mehr den erlebten
Realitäten entsprechen. Kulturwissenschaftliche und bildungstheoretische Konzep-
te sind unverzichtbar, um Wissen über die realen Prozesse zu erlangen. Wie Adel-
heid Hu diagnostiziert, ist das Wissenschaftsfeld dieser Aufgabe bisher kaum ge-
recht geworden: „Insgesamt gesehen ist festzustellen, dass der Diskurs über Inter-
nationalisierung überraschend wenig unter Einbezug kulturwissenschaftlicher oder
auch bildungstheoretischer Konzepte geführt wird, und dies, obwohl Migration,
Globalisierung, Multikulturalismus, Interkulturalität und Identität Kernthemen der
Kulturwissenschaften wie auch der Bildungswissenschaften sind. Wenn überhaupt
kulturelle Aspekte ins Spiel kommen, scheinen essentialisierende und reinfizierende
Konzepte von Kulturen und kulturellen Zugehörigkeiten an der Tagesordnung zu
sein“ (S. 260). Der Sammelband von Küppers, Pusch und Semerci ist ein wichtiger
und gelungener kultur- und bildungswissenschaftlicher Beitrag zum Diskurs der
Transnationalisierung. Um die realen Geschichten und Prozesse menschlicher
Verhältnisse hinter dem nationalistischen Nebel zu enthüllen, benötigen wir jedoch
deutlich mehr solcher Beiträge. Beiträgerinnen und Beiträger Arslan, Emre: Dr., Universität Siegen; Arbeitsschwerpunkte: Soziale Ungleichheit,
Migration, Bildung und Nationalismus Arslan, Emre: Dr., Universität Siegen; Arbeitsschwerpunkte: Soziale Ungleichheit,
Migration, Bildung und Nationalismus Atagül, Serap: Pädagogische Hochschule Ludwigsburg;
Arbeitsschwerpunkte: Inter- und transkulturelle Literaturdidaktik Dayıoğlu-Yücel, Yasemin: Dr., DAAD-Lektorin, İstanbul Üniversitesi;
Arbeitsschwerpunkte: Interkulturelle Literaturwissenschaft, Environmental
Humanities, Literatur und Digitale Medien Dickinson, Kristin: Assistant Prof., University of Michigan; Research Areas:
German Turkish Literary and Cultural Relations, Translation Studies, Mono-
and Multilingualism Gramling, David: Assistant Prof., University of Arizona; Research Areas:
Multilingualism Studies, Comparative Literature, Translation Studies Hepkaner, İlker: Ph.D. Candidate in Middle Eastern and Islamic Studies, New
York University; Research Areas: Heritage Studies, Visual Studies, Nationalism
in Turkey and Palestine/Israel Hofmann, Michael: Prof. Dr., Universität Paderborn; Arbeitsschwerpunkte:
Literatur der Aufklärung und der Weimarer Klassik, Interkulturelle
Literaturwissenschaft, deutsch-türkische Literatur Laut, Jens Peter: Prof. Dr., Universität Göttingen; Arbeitsschwerpunkte: Vor- und
frühislamisches türkisches Zentralasien, Kemalismus, moderne türkische
Literatur Beiträgerinnen und Beiträger 178 Müller, Christian: Pädagogische Hochschule Ludwigsburg;
Arbeitsschwerpunkte: Literatur- und Mediendidaktik, Kinder- und
Jugendliteraturforschung, Theaterdidaktik Ozil, Şeyda: Prof. Dr., İstanbul Üniversitesi; Arbeitsschwerpunkte: Deutsch-
türkische Studien, angewandte Sprachwissenschaft, Grammatik und Syntax Ozil, Şeyda: Prof. Dr., İstanbul Üniversitesi; Arbeitsschwerpunkte: Deutsch-
türkische Studien, angewandte Sprachwissenschaft, Grammatik und Syntax
Özdoğan Selim: Autor Ozil, Şeyda: Prof. Dr., İstanbul Üniversitesi; Arbeitsschwerpunkte: Deutsch
türkische Studien, angewandte Sprachwissenschaft, Grammatik und Syntax
Özdoğan, Selim: Autor türkische Studien, angewandte Sprachwissenschaft, Grammatik und Synta
Özdoğan, Selim: Autor Schreiner, Daniel: M.A., Universität Bonn; Arbeitsschwerpunkte: Mexikanisch-
amerikanische Literatur, türkisch-deutsche Literatur, rituelles und
therapeutisches Erzählen Schwalm, Martina: Ph.D. Candidate, University of Arizona; Research Areas:
Turkish-German Studies, Intercultural Literature, World Literature Seviner, Zeynep: Assistant Prof., Bilkent Üniversitesi; Research Areas: 19th and
20th Century Ottoman and Turkish Literatures, Translation Theory, Literature
and Visual Culture Sönmez, Sevengül: Bilgi Üniversitesi, Editor, Scholar, Archivist; Research Areas:
Literary History, Biography, Sabahattin Ali Stegmann, Vera: Associate Prof., Lehigh University; Research Areas: 20th and 21st
Century German Literature, Drama and Film, Bertolt Brecht, Transnational
Literature in German Uca, Didem: Ph.D. Candidate, University of Pennsylvania; Research Areas:
Intercultural Literature and Film, Intersectional Feminist Studies, Narratology Zierau, Cornelia: Dr., Oberstudienrätin, Universität Paderborn;
Arbeitsschwerpunkte: Interkulturelle Literaturwissenschaft und -didaktik,
zeitgenössische Kinder- und Jugendliteratur, sprachsensibler
Literaturunterricht Wissenschaftlicher Beirat Jannis Androutsopoulos (Spach- und Medienwissenschaft, Universität Hamburg)
Christian Dawidowski (Literaturdidaktik, Universität Osnabrück)
Dilek Dizdar (Interkulturelle Germanistik und Übersetzungswissenschaft,
Universität Mainz)
Deniz Göktürk (German and Film Studies, University of California, Berkeley)
Ortrud Gutjahr (Neuere deutsche Literatur und Interkulturelle
Literaturwissenschaft, Universität Hamburg)
Mahmut Karakuş (Neuere deutsche Literaturwissenschaft, Istanbul Universität)
Ersel Kayaoğlu (Neuere deutsche Literaturwissenschaft, Istanbul Universität)
Kader Konuk (Institut für Turkistik, Universität Duisburg-Essen)
Nilüfer Kuruyazıcı (Neuere deutsche Literaturwissenschaft, Istanbul Universität)
Claus Leggewie (Kulturwissenschaftliches Institut Essen)
Norbert Mecklenburg (Neuere deutsche Literaturwissenschaft,
Universität zu Köln)
Christoph Schroeder (Sprachwissenschaft, Universität Potsdam)
Canan Şenöz-Ayata (Sprachwissenschaft, Istanbul Universität)
Katrin Sieg (German and European Studies, Georgetown University)
Azade Seyhan (German and Comparative Literature, Bryn Mawr College)
Levent Tezcan (Culture Studies, Tilburg University) Jannis Androutsopoulos (Spach- und Medienwissenschaft, Universität Hamburg)
Christian Dawidowski (Literaturdidaktik, Universität Osnabrück) Dilek Dizdar (Interkulturelle Germanistik und Übersetzungswissenschaft,
Universität Mainz) Deniz Göktürk (German and Film Studies, University of California, Berkeley) Ortrud Gutjahr (Neuere deutsche Literatur und Interkulturelle
Literaturwissenschaft, Universität Hamburg) Ersel Kayaoğlu (Neuere deutsche Literaturwissenschaft, Istanbul Universität) Nilüfer Kuruyazıcı (Neuere deutsche Literaturwissenschaft, Istanbul Universität) Norbert Mecklenburg (Neuere deutsche Literaturwissenschaft,
Universität zu Köln) I
m Mittelpunkt dieses Bandes steht das Werk des türkischen Autors und
Übersetzers aus dem Deutschen Sabahattin Ali, der mit seinem Roman
Kürk Mantolu Madonna (Die Madonna im Pelzmantel) zu posthumem
Ruhm gelangte. Der Roman, der zum Großteil in Deutschland spielt, und
andere seiner Werke werden unter Aspekten der Weltliteratur, (kultureller)
Übersetzung und Intertextualität diskutiert. Damit reicht der Fokus weit
über die bislang im Vordergrund stehende interkulturelle Liebesgeschichte
in der Madonna hinaus. Weitere Beiträge beschäftigen sich mit Zafer Şenocaks Essaysammlung
Deutschsein und dem transkulturellen Lernen mit Bilderbüchern. Ein Interview mit Selim Özdoğan rundet diese Ausgabe ab. ISBN: 978-3-86395-297-6
ISSN: 2198-5286
|
https://openalex.org/W2403825653
|
http://revistas.unisimon.edu.co/index.php/desarrollogerencial/article/download/481/469
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es
|
LAS ESTRATEGIAS DE MARKETING ECOLÓGICO EN CADENAS MINORISTAS, EN UNA PERSPECTIVA INTERNACIONAL
|
Desarrollo gerencial
| 2,014
|
cc-by
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119
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
LAS ESTRATEGIAS DE MARKETING ECOLÓGICO EN CADENAS MINORISTAS, EN UNA
PERSPECTIVA INTERNACIONAL
GREEN MARKETING STRATEGIES IN RETAIL CHAINS: FROM AN INTERNATIONAL PERSPECTIVE
James Pulido Novoa
Ernesto Fidel Cantillo Guerrero
Universidad minute de Dios, Sede Costa Norte
RECIBIDO: Febrero 2 de 2014
ACEPTADO: abril 12 de 2014
RESUMEN
El objetivo del presente artículo de investigación consistió en identificar, desde una perspectiva internacional, las
distintas estrategias de mercadeo ecológico, aplicadas por un grupo de cadenas comerciales minoristas. La técnica de
investigación fue la revisión documental y se utilizó el método analítico. El trabajo se desarrolló en tres etapas: en la
primera, se seleccionaron 50 fuentes de información; posteriormente, se construyó un mapeo con los términos claves
pertinentes al objeto de estudio; por último, se redactó este artículo. Se estudiaron las estrategias de comunicación
dirigidas a los consumidores y clientes, con relación a la oferta de productos y servicios amigables para el medio
ambiente. Como resultado de investigación, se determinó que las cadenas minoristas internacionales con sede en
Colombia realizan una labor de mercadeo ecológico mediante una estrategia clara de comunicación con los
consumidores. Se concluyó que las cadenas comerciales minoristas asumen las tendencias del mercadeo afines a los
intereses del Estado colombiano (Plan estratégico nacional de mercados verdes de la república de Colombia), y que
dichas cadenas juegan un papel fundamental como intermediarios comerciales de productos y servicios amigables para
el medio ambiente.
Palabras clave: Marketing ecológico, producto eco amigable, cadenas minoristas, responsabilidad medioambiental.
Referenciar este articulo (APA)
Pulido Novoa, James & E. Cantillo-Guerrero. Las estrategias de marketing ecológico en cadenas minoristas, en una
perspectiva internacional, En desarrollo Gerencial, revista de la facultad de ciencias económicas,
administrativas y contables de la universidad Simón Bolívar. 6 (1) P.118-143.
Director regional de la costa Norte, Corporación Universitaria Minuto de Dios, jpulido@uniminuto.edu
Magister en mercadeo, Coordinador de Investigación, Corporación Minuto de Dios , Barranquilla,
Ernerto.cantillo@uniminuto.edu
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
.http://portal.unisimonbolivar.edu.co:82/rdigital/desarrollogerencial/index.php/desarrollogerencial/issue/archive
120
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
ABSTRACT
The aim of this research article was to identify different green marketing strategies implemented by a group of
retail chains, from an international perspective. A documentary review was the technique used, along with the analytical
method. This article was developed in three stages; in the first stage 50 sources of information were selected; thereafter,
a mapping was built with the key terms relevant to the subject matter; and finally, the draft article. The strategies
focused on the communication to consumers and clients regarding the offer of products and/or amicable services to the
environment, were studied. As a result of this research, it was determined that retail chains with headquarters in
Colombia carry out green marketing work through a clear communication strategy with consumers. It was concluded
that market trends that agree with the interests of the Colombian government (plan estratégico nacional de mercados
verdes de la república de Colombia) converge with the commercial retail chains, which play an essential role as
commercial intermediaries of products and/or amicable services to the environment.
Key words: Green marketing, Eco-amicable product, Commercial Retail Chains, Environmental responsibility.
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
.http://portal.unisimonbolivar.edu.co:82/rdigital/desarrollogerencial/index.php/desarrollogerencial/issue/archive
121
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
de la empresa en las
actividades
INTRODUCCIÓN
primarias (Porter, 2002).
En su obra, Los diez pecados
capitales del marketing, Kotler (2004)
enuncia los grandes errores en los que
incurren las empresas en cuanto a la
dirección
comercial.
Dos
de
esos
pecados se enfocan en el consumidor y
en el mercado: el primero de ellos, nos
habla sobre las fallas comunes de una
empresa que no está lo suficientemente
orientada hacia el mercado y dirigida a
los consumidores; y el segundo se
refiere a las empresas que no logran
entender
del
potenciales.
todo
a
sus
clientes
Esta falta de orientación
hacia el mercado y el consumidor, de
las
cuales
puede
adolecer
un
determinado número de empresarios
De
modo
que
un
factor
determinante para generar valor y
competir es el análisis de las tendencias
de consumo, respecto a lo cual, las
empresas deben tomar en cuenta a los
consumidores que se preocupan por el
medio
ambiente,
pues,
justamente
representan una importante opción que
crece en distintos países del globo.
Así, en Alemania y Suecia, el consumo
de productos ecológicos crece a una tasa
anual de 14% y 20%, respectivamente.
En su
mayoría, los
consumidores
adquieren tales productos tanto en los
supermercados tradicionales como en
supermercados exclusivos de productos
ecológicos (Yáñez, 2009).
que olvidan medir las tendencias del
mercado objetivo y de realizar la
planeación y auditoría de mercadeo
acorde
con
el
escenario
del
consumidor, afecta a la cadena de valor
Investigaciones
hechas
en
Hong Kong informan también sobre el
creciente valor
que
adquieren
los
productos verdes en los consumidores
jóvenes (Lee, 2008); a lo que se debe
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
.http://portal.unisimonbolivar.edu.co:82/rdigital/desarrollogerencial/index.php/desarrollogerencial/issue/archive
122
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
agregar el mercado de la energía solar
consideradas son suficientes para el
fotovoltaica, que creció un 20% en el
planteamiento
2009 con respecto al 2008 (Solarbuzz,
específicas
2009).
desarrollo de este artículo: ¿Cuáles son
Cifras como las anteriores sugieren a las
empresas,
considerar
medioambiental
en
el
sus
factor
decisiones
estratégicas de carácter corporativo y de
marketing, dado que el consumidor,
preocupado por los recursos naturales y
la biodiversidad, desea, y en algunos
casos exige, que los productos sean
amigables con el medio ambiente
de
para
tres
preguntas
resolver
en
el
los supermercados o hipermercados
mundialmente conocidos, que se puedan
tomar como muestra de casos exitosos
en
mercadeo
ecológico?,
¿qué
compromisos de responsabilidad tienen
con el medio ambiente los principales
almacenes de cadena ubicados en
Colombia?,
¿ofrecen
servicios
en
que
su
productos
o
esencia
son
ecológicos y fabricados a través de
(Bravo, 2005).
procesos con materiales limpios?
Ahora bien, uno de los sectores
estratégicos de la economía que más
MÉTODO
contacto tiene con gran parte de los
consumidores colombianos es el de los
almacenes
de
empezando
La metodología utilizada en este trabajo
porque
diario,
caso
de
consistió en la revisión de fuentes
hipermercados, reciben la visita de
secundarias (libros, revistas científicas,
92.766 empleados quienes, a su vez, son
revistas en Internet, datos estadísticos
consumidores (DANE, 2010).
Este
de agencias) y en análisis de casos, a
motivo y las cuestiones previamente
través de las direcciones electrónicas de
a
cadena,
Diseño
en
el
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
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123
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
un determinado grupo de empresas
marketing ecológico.
dedicadas al negocio del “retail” o
identificación
cadenas comerciales minoristas.
establecidas, se reconocieron los medios
por
Participantes
los
de
A partir de la
las
cuales
estrategias
este
tipo
de
organizaciones realiza su labor de
Las cadenas minoristas nacionales e
internacionales que funcionan en la
ciudad de Barranquilla
responsabilidad social empresarial y, a
la
vez,
resulta
atractiva
para
el
comprador, al entender éste que las
organizaciones donde
Instrumentos
compran sus
productos son eco-amigables y brindan
La técnica de investigación utilizada fue
productos ecológicos de calidad.
la revisión documental
En
específico,
se
detectaron
Procedimiento
estrategias ecológicas aplicadas a los
El estudio se realizó en tres etapas: la
siguientes factores: Productos, procesos,
primera comprendió la selección de la
recursos físicos, optimización de la
fuentes secundarias; en la segunda etapa
cadena de suministro (logística inversa),
se realizó el análisis de los hallazgos y,
ubicación y eficiencia energética. Entre
en la última, la redacción del artículo.
los casos exitosos internacionales de
estrategias de marketing ecológico en
RESULTADOS
cadenas comerciales minoristas están La
Los hallazgos obtenidos en esta
corporación IKEA es una organización
investigación se centraron en el análisis
multinacional sueca, que se dedica a la
de
los
casos
internacionales
y
estrategias
de
venta minorista de muebles y objetos
colombianos
de
para el hogar.
Esta ha implementado
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
.http://portal.unisimonbolivar.edu.co:82/rdigital/desarrollogerencial/index.php/desarrollogerencial/issue/archive
124
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
estrategias de gestión ambiental, que
de simplemente desecharlos; de modo
hace públicas a través de acciones de
que, al final, las piezas se utilizan de
mercadeo ecológico como, por ejemplo,
recambio y se venden a precios
el diseño de paquetes planos (fabricados
reducidos
con materiales biodegradables), que
especialmente designadas.
facilitan
una
optimización
en
la
Esto
garantiza el transporte de un mayor
número de unidades por viaje, a la vez
que contribuye a la reducción de
emisiones anti-ambientales por el menor
consumo de combustible.
A lo
anterior se suma el manejo del código
de conducta con los proveedores, a
quienes se les exige sistemas de
transportes con combustibles limpios
(Políticas de reducción de residuos
sólidos IKEA Corporation, 2010).De
igual forma, IKEA realiza campañas de
punto
de
recogidas
de
residuos
eléctricos para los clientes y promueve
el reciclaje de los mismos, intentando
con esto reparar los productos en lugar
zonas
de
la
tienda
WALT-MART
distribución de espacios en el almacén y
en el vehículo transportador.
en
Esta
organización
trabaja
el
concepto del mercadeo ecológico desde
diferentes puntos de vista.
De
manera concreta, en su campaña de
comunicación
de las acciones de
mercadeo ecológico, figuran estrategias
como: El ahorro de millones de dólares,
cambiando bombillas de iluminación
incandescente
por
bombillas
CFL
(Romero, 2010); la renovación de sus
equipos de refrigeración de baja y
media temperatura, que eran iluminados
con
lámparas
fluorescentes
tradicionales, por refrigeradores con
iluminación proporcionada por LEDs,
que se activan por movimiento, los
cuales, estima la compañía, ahorrarían
cerca de US$2.6 millones en costos de
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
.http://portal.unisimonbolivar.edu.co:82/rdigital/desarrollogerencial/index.php/desarrollogerencial/issue/archive
125
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
energía, y contribuiría a la reducción de
como ocurre con la instalación de
emisiones de CO² en un costo cercanas
paneles solares fotovoltaicos en las
a 35 millones de libras anuales.
azoteas de los depósitos, comenzado por
Por
demás, Walt-Mark se ha propuesto
sus tiendas de California y Hawái.
objetivos como lograr que sus nuevas
este último se encuentra el mayor panel
tiendas usen 30% menos de energía que
solar.
las actuales, y ha convidado a cerca de
En
Casos colombianos de marketing
60.000 proveedores a producir bienes
ecológico
que no sean dañinos para el medio
minoritas
en
cadenas
comerciales
ambiente, exigiendo a estos mismos que
reduzcan el total del material de
Almacenes
Éxito
divulga
acciones ambientales, tales como:
empaque en un 5% para el año 2013
www.miplanetaexito.com. Esta
(Romero Castro, 2010).
Página Web es la encargada de
COSTCO
divulgar las acciones
Esta empresa utiliza estrategias
de mercadeo que incluyen un programa
para reducir las emisiones de gases de
efecto invernadero, a través de medidas
de
eficiencia
energética.
Costco
encontró que la electricidad comprada
alcanza el 84 % de su huella de carbono
en el 2007.
Su programa de energía
pone especial énfasis en diseños de
edificios
energéticamente
eficientes,
ambientales
realizadas por la empresa, lo cual es
quizás la mejor acción de mercadeo
ecológico hecha por un Retail en
Colombia.
Los colores que
identifican la página guardan una
relación
con
los
colores
institucionales del Grupo Éxito, y
en ella se encuentra información
actualizada de las actividades que
desarrolla
la
organización
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
.http://portal.unisimonbolivar.edu.co:82/rdigital/desarrollogerencial/index.php/desarrollogerencial/issue/archive
para
126
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
proteger
como
Es la primera cadena de
el medio ambiente, así
recomendaciones
consumidores
sobre
a
los
comercio de cubrimiento nacional que
acciones
empaca las compras de sus clientes en
positivas hacia el mismo.
bolsas plásticas biodegradables y bolsas
Publicación de las distintas
reutilizables.
Su uso representa
actividades de ahorro de recursos, a
puntos en la tarjeta de fidelización de
través del informe de sostenibilidad
sus clientes.
(Almacenes Exito S.A., 2010)
Actualmente las cadenas minoristas,
Los ingresos obtenidos por
valor de $4.950 millones en la venta de
productos
orgánicos
y
ecológicos,
representados en 105 referencias de
productos que se comercializan después
de comprobar que estos cumplen con
los
correspondientes
certificados
expedidos por la autoridad competente.
de Almacenes Éxitos, están haciendo
esfuerzos, para tener mayor cobertura,
han optado por realizar
alianzas
estratégicas, con las tiendas de barrios
del
sector
sur
proporcionando
logística,
para
a
de
Barranquilla,
estas
toda
promocionar
la
sus
productos de marca propia un día a la
semana, desde este escenario también,
La
consecución
de
la
14001/04
del
se proyecta la cultura del marketing
certificación
ISO
ecológicos e igualmente sucede con las
ICONTEC
en
Éxito
Bello
tiendas de barrios Express, las cuales
(departamento de Antioquia) por su
han venido fortaleciendo el sector de
acertada
gestión
ambiental,
tiendas de barrios.
convirtiéndose así en el primer almacén
de cadena de Colombia en lograrlo.
De
acuerdo
a
lo
expuesto
anteriormente Peralta (2011):
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
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127
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
“Tendero
experto:
bolsas de tela reutilizables en
Denominamos así a quien tiene
todos sus hipermercados, con el
una trayectoria en el negocio, es
propósito de que el comprador
el empresario que trata de sacar
lleve su mercado en ellas y
ventaja
racionalice
competitiva
a
su
su
consumo
de
negocio, con propósitos incluso
bolsas plásticas. Esta iniciativa
de expandir su mercado y
es la primera avalada por el
aplicando
de
Ministerio
de
Ambiente,
Marketing uno a uno y de
Vivienda
y
Desarrollo
desarrollo de mercado” P. 174.
Territorial.
Además,
Carrefour (actual Jumbo)
reutilización de una bolsa evita
En
estrategias
su
portal
web,
el uso de al menos 15 bolsas de
www.carrefour.com.co, sección
de
responsabilidad,
plástico por cada compra.
hace
referencia a su campaña “Bolsas
reutilizables Carrefour”, con la
que comunican la razón de la
actividad e incentivan a los
la
Por otro lado, en línea con los
principios del Pacto Mundial de las
Naciones Unidas en cuanto al respeto
del medio ambiente, las estrategias
ambientales de Carrefour comprenden:
clientes al uso de las mismas,
mostrándoles que las bolsas
en sus tiendas.
reutilizables son una solución
de largo plazo.
Por otra
Reducción del impacto ambiental
El desarrollo de productos que
parte, en Colombia, Carrefour
respeten el medio ambiente y la
ha sido pionero en ofrecer
biodiversidad.
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
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128
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
La reducción del 30% de
amplia
gama
de
productos
consumo de energía del grupo
comprometidos con el ahorro de
Carrefour al año 2020.
recursos (grifería, bombillos de baja
La sensibilidad y educación de
los
consumidores
y
de
sus
colaboradores sobre el consumo
responsable
y
el
desarrollo
potencia, canecas para clasificación
de
basuras,
pinturas
lámparas
ecológicas,
solares,
sellos
para
aislamiento térmico, entre otros).
Otro detalle para resaltar en la Web
sostenible.
de esta empresa es la campaña de
Home Center
En
su
acciones
página
Web,
esta
para
el
hogar.
organización
Mediante esta, la empresa no solo
presenta un pop-up dinámico, en el
promueve
la
compra
de
los
cual enseña la bolsa reutilizable con
productos que comercializa, sino
frases ecológicas y emotivas que
que también ofrece información
incentivan su uso. Adicionalmente,
valiosa
a
los
consumidores
y
Home Center maneja una línea
clientes sobre el ahorro de recursos
amplia de productos amigables para
relacionado con cada uno de los
el medio ambiente, así como de
bienes de sus hogares, como son:
productos
fabricados
por
electrodomésticos, muebles de sala,
empresarios
comprometidos
con
comedor,
planes de gestión ambiental.
regaderas
y
otros.
Todo
Finalmente, en la divulgación de su
esto se comprueba cuando uno
programa de responsabilidad social
ingresa al portal Web de la empresa
empresarial, Home Center incluye al
(www.homecenter.com.co), sección
medio ambiente, y precisa esto
hogar verde, donde se encuentra una
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
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129
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
como una responsabilidad de toda la
minorista con respecto al mes de
compañía,
septiembre del año anterior (DANE,
que
contribuye
a
preservarlo desde una óptica que
2010).
involucra las operaciones totales de
incluyen a los hipermercados y demás
la misma.
almacenes de cadena, que para este
Almacenes Olímpica
Tales estadísticas
articulo denominamos supermercados y
tiendas especializadas.
La única acción ambiental que
esta organización divulga es la adopción
del elefante de la Fundación Zoológico
de Barranquilla.
A pesar de que en sus
puntos de ventas manejan líneas de
productos orgánicos y ecológicos, esta
cadena de almacenes no saca provecho
de esta actividad para realizar más
acciones de mercadeo ecológico.
Marketing
supermercados
especializadas
ecológico
en
y
tiendas
las
direcciones
Al observar
electrónicas
establecimientos
como
SURTIFRUVER
de
de
FEDCO,
la
Sabana,
Almacenes YEP y La 14, se encontró
que ninguno de ellos realiza acciones de
mercadeo ecológico, debido a la falta de
divulgación
de
los
programas
ambientales llevadas a cabo en sus
organizaciones.
Estas
organizaciones
tampoco promueven
explícitamente productos o servicios
ecológicos, aunque de forma indirecta
podría considerarse como tal el enfoque
Cifras de la muestra mensual de
de SURTIFRUVER de la Sabana,
comercio al por menor, elaborada por el
tienda especializada en la venta de
DANE para el mes de septiembre del
frutas y verduras, que, en su página
2010, muestran un crecimiento en
18,6% de las ventas del comercio
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130
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
web, promueve el consumo de frutas y
condición de ser amigable para el medio
verduras por salud y bienestar.
ambiente.
Las empresas en cuestión
asumen
compromisos
de
Teniendo en cuenta variables
como
volumen
de
ventas
(BPR
responsabilidad medio ambiental para el
Benchmark, 2010) y presencia en el
cumplimiento
territorio
de
las
estrategias
nacional,
las
cadenas
anteriormente expuestas, y formulan
comerciales minoristas ubicadas en
políticas con base en la responsabilidad
Colombia
social y en un enfoque ecológico.
categorías: 1) hipermercados, y 2)
En general, las políticas de estas
supermercados
organizaciones consisten en mantener
especializadas.
una cadena de valor sostenible en el
importante aclarar que el presente
sistema de actividades del negocio.
estudio se limita a supermercados y
Para esto, se organizan desde el inicio
tiendas especializadas, que manejan
hasta el final de todo el ciclo de vida del
consumo masivo relacionados con la
producto, generando un valor agregado
canasta familiar, el cuidado y la belleza,
para todos los funcionarios y clientes de
descartando otra serie de productos que
la organización, quienes aseguran así un
se comercializan al por menor a través
éxito perdurable contribuyendo de esta
del formato de tiendas especializadas.
manera al bienestar de la humanidad y
del medio ambiente.
se
clasifican
y
en
dos
tiendas
En este sentido, es
En las estrategias de marketing
ecológico de este modelo de negocios,
Se logró identificar que las
se detectó claramente el compromiso de
organizaciones objeto de estudio poseen
los propietarios de las cadenas y los
en su oferta productos que reúnen la
consumidores con el medio ambiente.
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131
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
A continuación, se detallan también
Almacenes Éxito, estrategias de
aspectos relevantes sobre el particular
comunicaciones
en las organizaciones:
atractivas como la de Home Center,
novedosas
y
y la fuerte promoción de las bolsas
Las cadenas
minoristas,
al
reutilizables de Carrefour.
igual
que
empresariales,
otros
han
sectores
tomado
la
A los supermercados y otras
iniciativa de aplicar estrategias de
tiendas especializadas aún les falta
mercadeo ecológico, entendiendo
camino por recorrer en cuanto a
que esto no solo constituye un
acciones de marketing ecológico,
motivo extra de generación de
que les permitan ganar un espacio
ventas, sino que también muestra a
en la mente de los consumidores
la comunidad el compromiso social
ecológicos.
y ambiental que tienen con el
no estén a tiempo de incluir este
entorno, base indispensable para la
nicho de consumidores.
perdurabilidad de la empresa en un
Esto no significa que
Es importante destacar la
medio cada vez más competitivo.
labor realizada por el Grupo Éxito,
Como ejemplos, podemos citar a las
principal almacén de cadena en
cadenas Wal-Mart, IKEA y Costco.
Colombia.
La estrategia de
hipermercados
marketing ecológico realizada por
colombianos (de capital nacional y
esta organización constituye un
extranjero) no están muy distantes
modelo
de estas iniciativas en estrategias de
empresas, al reflejar en sus acciones
marketing ecológico, y cuentan con
un
campañas exitosas como la de
manifiesta en la publicación de
Los
a
fuerte
seguir
para
compromiso
otras
que
Desarrollo Gerencial, Vol. 6. No. 1- 119-144- Enero-Junio 2014 Universidad Simón Bolívar–Barranquilla – Colombia, ISSN: 2145-5147
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se
132
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
resultados
de
su
gestión,
recalentamiento global, la tecnología, el
información vigilada por la firma de
uso de bolsas plásticas, etc. Todo lo
auditoría Deloitte.
cual obliga a las empresas a ofertar
productos
DISCUSIÓN
y
servicios
que
no
incrementen ese riesgo y, en lo posible,
Según la Asociación Americana
contribuyan a erradicarlo.
Así las
de Marketing (AMA, por sus siglas en
cosas, cuando una organización decide
inglés), el marketing ecológico es el
ofrecer productos o servicios amigables
esfuerzo que hace una empresa para
para el medio ambiente, suele utilizar
producir,
promover,
empacar
y
estrategias de comunicación dirigidas a
recuperar los productos con un enfoque
los consumidores que evidencien sus
de
responsabilidad
y
sensibilidad
ecológica (Samper, 2009).
Por su
prácticas de mercadeo que se hallan
comprometidas con el cuidado del
parte, Polonsky (1997) lo define como
mismo.
De esta manera, las distintas
un conjunto de actividades diseñadas
variables generadoras de valor en las
para
generar
y
facilitar
cualquier
empresas se transforman, gracias a una
intercambio de bienes y servicios que
gestión ambiental
sostenible en la
permitan satisfacer las necesidades del
cadena, que nunca pierde la visión de
ser humano, a través del cuidado y
apuntar a un consumidor ecológico. El
protección del medio ambiente.
El
diseño de productos, el empaque, el
marketing ecológico implica, entonces,
embalaje y la publicidad de las líneas de
el
desarrollo
de
actividades
de
producción,
así
como
el
canales
de
preservación de un entorno ambiental,
aprovisionamiento,
los
que cada día corre más peligro de
distribución y las demás áreas de la
autodestrucción, por problemas como el
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133
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
organización han de involucrarse en la
actividades
estrategia de mercadeo ecológico.
impacto ambiental.
Desde principio de los años 80, el
marketing ecológico se convirtió en una
Razones para utilizar el
marketing ecológico
Como se viene exponiendo, al
tendencia fuerte de aplicación comercial
tratar de satisfacer las necesidades de
en el mundo, llegándose a considerar la
los
década del 90 como la revolución verde.
ecológica, recientemente las empresas
Sin embargo, durante esas décadas, se
han decidido desarrollar procesos de
vieron muchas campañas y productos
producción
relacionados que no obtuvieron los
desarrollo sustentable de la economía y
resultados
que
consumidores
que
implican
con
un
alto
conciencia
contribuyan
al
esperados
(Vazifehdust,
la sociedad (Salgado y Beltrán, 2009).
Javad & Gharib, 2011).
Aun así, es
En este marco, cobra valor el interés por
notable que, en las normas y en el
preservar
comportamiento de las personas, se ha
limitados, el uso eficiente de los
producido un cambio en sus actitudes
mismos, la reducción o reutilización de
hacia un estilo de vida verde, y es claro
los residuos, y demás acciones que se
que la gente intenta activamente reducir
enfocan a satisfacer una demanda que
el impacto sobre el medio ambiente
hoy exige hechos concretos, pero sin
(Cherian & Jacob, 2012). Estos cambios
que la empresa pierda su interés básico
conllevan a que las empresas realicen
para la perdurabilidad, la optimización
distintas acciones a su interior para
de sus recursos y el logro de objetivos
implementar las estrategias de mercadeo
económicos.
ecológico, más aún si el sector de la
satisfacción-optimización y consecución
organización
de objetivos, el mercadeo ecológico
se
relaciona
con
los
recursos
naturales
En este ciclo de
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134
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
pasa a ser una filosofía comercial
habilidad especial que logra desarrollar
relevante, por medio de la cual la
la empresa y que la ubica en una
empresa aprovecha la gestión ambiental
posición de preferencia a los ojos del
y el uso eficiente de recursos, realizada
mercado (Mapcal, 1997).
al interior de ella para divulgarla, bajo
los métodos del mercadeo, al entorno y
a los consumidores en general.
Fernández (2009) se refiere a otro
aspecto de gran relevancia en este tema:
la responsabilidad social corporativa,
Ya se ha advertido que, en
dado que las empresas deben reinvertir
algunos sectores, el mercadeo ecológico
voluntariamente
precisa un estricto cumplimiento (por
mejoramiento del ambiente interno y
ejemplo, en la minería); pero, en otros,
social, interactuando con los grupos de
es una oportunidad para proponer
interés como clientes, proveedores,
nuevos productos y explorar nuevos
empleados,
mercados
(ejemplo,
agricultura
comunidad general.
ecológica);
mientras
en
responsabilidad
social
empresas constituye la única forma de
cumple
la
ofertar sus productos (ejemplo, energías
lineamientos, que deben reflejarse en
limpias). Entonces, se puede decir que
los planes y políticas organizacionales,
las
los cuales deben incluir la dimensión
organizaciones
que
tienen
otras
variadas
razones para implementar el mercadeo
ambiental
ecológico,
pero
contribuir
consumidores,
para
y
al
la
Actualmente, la
empresarial
sociedad
(Aparicio,
unos
2009).
son
en
sí
Además,
permiten
a
las
establecer normas y políticas que
mismas obtener ventajas competitivas,
fomentan y exigen a las organizaciones
concepto entendido como la destreza o
cumplir con los requisitos en materia
oportunidades
todas
para
que
los
Estados
empiezan
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a
135
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
medioambiental.
Para esto, se puede
establecer si la empresa se encuentra
citar, en Colombia, la Ley 1333 del
preparada
2009, que establece el procedimiento
disponibilidad) para asumir el reto.
sancionatorio ambiental.
Si el diagnóstico brinda la tranquilidad
Por último,
(cultura-recursos-
se debe reconocer la relación costo-
suficiente
beneficio a favor de la empresa en esta
estrategia de mercadeo ecológico, la
materia.
Al respecto, el estudio
cual debe ir sincronizada con las
de Aberdeen Group (2008) resalta que
distintas estrategias establecidas en el
un 9% de los consumidores está
plan de marketing, a su vez, el plan de
dispuesto a pagar un precio superior por
acción de la estrategia de mercadeo
los productos ecológicos. Y es que los
ecológico debe contar con una serie de
consumidores de productos ecológicos
acciones dirigidas a la obtención de
van entendiendo que estos productos
mayores ingresos para afrontar el
poseen valor agregado, y para el
cambio cultural y de hábitos de
empresario estos productos constituyen,
consumo que han surgido en los
hoy por hoy, una oportunidad valiosa,
consumidores
mediante la cual
pueden obtener una
resumiéramos todo esto en un formato
rentabilidad por unidad más alta que
de ecuación, el modelo a proponer sería:
con los productos tradicionales.
para
del
implementar
siglo
XXI.
Si
Figura 1. Ecuación de incrementos de ventas en
mercados ecológicos
En el proceso de iniciar una
estrategia de marketing ecológico en
una cadena comercial minorista, el
primer paso sugerido es realizar el
una
Fuente: Elaboración de autores.
diagnóstico ambiental de la empresa, y
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136
Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
Acciones ecológicas en
el marketing MIX
favor del eco-marketing, tales como: la
El marketing MIX es el eco-producto
en sí y la pregunta que debe plantearse
al
respecto
es:
diferentes actividades desarrolladas a
¿Cómo
podemos
convertir una cadena minorista, que es
un canal de distribución, en un eco-
reducción de consumos de recursos
(energéticos, agua, gas), la organización
de brigadas de reciclaje en el barrio o
zona urbana en que se encuentra
ubicado, y el uso de las redes sociales.
producto? El análisis de casos permite
Por último, se recomienda a las
sugerir la hipótesis de que más que un
organizaciones utilizar los eco-canales:
eco-producto, la organización debe
La tienda, el supermercado, son canales
construir un eco-marca, es decir, una
de distribución, que como tales pueden
marca que se logre posicionar como
establecer acciones ecológicas en el
ambientalmente responsable en la mente
punto de venta (canecas para reciclaje),
de un consumidor, tal como lo hacen los
manejo efectivo de envases retornables,
hipermercados aquí reseñados.
adecuación eco-eficiente en las bodegas
Otra opción para estas organizaciones
de
es el eco-precio, ya que el retail, sector
Además, pueden exigir al mayorista
empresarial que compite ampliamente
demostraciones
con la variable precio, exige realizar
ambiental y, así, fomentarlo en los
acciones que optimicen el costo en los
demás actores de la cadena.
procesos, y que a su vez no incrementen
la estructura de precio al público.
A través de la eco-publicidad,
la organización ha de promulgar las
almacenamiento,
de
entre
otras.
responsabilidad
Al consistir la estrategia en
migrar de una planeación de mercadeo
tradicional
a
otra
de
mercadeo
ecológico, se requiere que cada uno de
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Las estrategias de marketing ecológico en cadenas minoristas, en una perspectiva internacional
los
miembros
que
conforman
o/archivos/guia_de_agricultura_
la
ecologica.pdf
empresa esté 100% comprometido con
ella, esto es, con la intención de
divulgar a los clientes y consumidores
en general las acciones que la empresa
lleva a cabo en pro del medio ambiente
Allen,
T
(2007).
could green marketing be a
sustainable
South
mercadeo ecológica.
maestría).
palabras, que la organización en su
competitive
advantage for retailers within
y actuar acorde con la planeación de
Con otras
Green-marketing,
Africa?
(Tesis
de
University
of
Pretoria, Pretoria.
totalidad asuma el gran reto de ser y
Almacenes
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https://openalex.org/W4288088364
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https://hal.science/hal-02326926/document
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English
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Large-Scale Characterization and Segmentation of Internet Path Delays with Infinite HMMs
|
arXiv (Cornell University)
| 2,019
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cc-by
| 13,029
|
To cite this version: Maxime Mouchet, Sandrine Vaton, Thierry Chonavel, Emile Aben, Jasper den Hertog. Large-Scale
Characterization and Segmentation of Internet Path Delays with Infinite HMMs. IEEE Access, 2020,
8, pp.16771-16784. 10.1109/ACCESS.2020.2968380. hal-02326926v2 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02326926
https://hal.science/hal-02326926v2
Submitted on 27 Jan 2020 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 Received December 4, 2019, accepted December 31, 2019, date of publication January 21, 2020, date of current version January 28, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2968380 MAXIME MOUCHET
1, SANDRINE VATON
1, THIERRY CHONAVEL
1,
EMILE ABEN
2, AND JASPER DEN HERTOG
2 MAXIME MOUCHET
1, SANDRINE VATON
1, THIERRY CHONAVEL
1,
EMILE ABEN
2, AND JASPER DEN HERTOG
2
1Lab-STICC, IMT Atlantique, 29280 Plouzané, France
2RIPE NCC, 1012 Amsterdam, The Netherlands
Corresponding author: Maxime Mouchet (maxime.mouchet@imt-atlantique.fr) RIPE NCC, 1012 Amsterdam, The Netherlands
Corresponding author: Maxime Mouchet (maxime.mouchet@imt-atlantique.fr) The work of Maxime Mouchet was supported by the Futur and Ruptures Program of Institut Mines Télécom for his Ph.D. ABSTRACT Round-Trip Times are one of the most commonly collected performance metrics in computer
networks. Measurement platforms such as RIPE Atlas provide researchers and network operators with an
unprecedented amount of historical Internet delay measurements. It would be very useful to process these
measurements automatically (statistical characterization of path performance, change detection, recognition
of recurring patterns, etc.). Humans are quite good at finding patterns in network measurements, but it can
be difficult to automate this and enable many time series to be processed at the same time. In this article
we introduce a new model, the HDP-HMM or infinite hidden Markov model, whose performance in trace
segmentation is very close to human cognition. We demonstrate, on a labeled dataset and on RIPE Atlas
and CAIDA MANIC data, that this model represents measured RTT time series much more accurately than
classical mixture or hidden Markov models. This method is implemented in RIPE Atlas and we introduce
the publicly accessible Web API. An interactive notebook for exploring the API is available on GitHub. INDEX TERMS Round-trip times, RIPE Atlas, hidden Markov models, nonparametric Bayesian models,
anomaly detection, time series clustering. FIGURE 1. RTT between two RIPE Atlas anchors from May 1st to May 5th,
2018. his work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ VOLUME 8, 2020
This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons. A. SCOPE OF THE PAPER Network management has traditionally been entrusted to
humans. But this is expensive, error-prone, and slow to adapt
to changes. The task of human experts is very complex
because of the large number and heterogeneity of equipments,
as well as the wide variety of applications. FIGURE 1. RTT between two RIPE Atlas anchors from May 1st to May 5th,
2018. We believe that the future of network management is
in automation, or driverless (autonomous) networks. Ref-
erences [1]–[4]. For self-driving networks to become real-
ity, recent machine learning techniques must be used to
extract information from network measurements and auto-
mate decision-making. Different needs should be addressed:
statistical characterization, prediction, detection of changes
or anomalies, classification, etc. The results must be reliable
and accurate in order to automate decision-making related to
network management or to security and resilience, and the
analysis should be scalable and fully automated (no human
intervention). Delay is an important performance metric. In particular,
it is easy to measure Round Trip Time (RTT) and data is read-
ily available from measurement infrastructures at the Internet
scale like RIPE Atlas [5]. Humans are quite good at finding
patterns in this latency data, as you can see for yourself
in Figure 1, but this is difficult to automate. An automated
solution would allow many time series to be processed at the
same time. In this article we propose to use a Hierarchical Dirichlet
Process Hidden Markov Model (HDP-HMM), also called
infinite HMM, or nonparametric Bayesian HMM. The associate editor coordinating the review of this manuscript and
approving it for publication was Xiaofei Wang
. This model mimics human cognition very well (in terms
of segmentation of the series, recognition of different states, 16771 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs etc.). These models are used to segment audio sequences for
which they give very good results for speaker recognition [6]. These recent techniques are more complex than standard
HMMs but they are worth the effort. the time-dependency of such a Markov model can be used
to reduce the frequency of measurements in routing over-
lays. This article expands on the statistical details behind
the model, describes two new applications (CAIDA MANIC
measurements, and anomaly detection), and introduces a
RIPE Atlas API for time series segmentation as a service. II. A REMINDER ON MIXTURE MODELS AND HIDDEN
MARKOV MODELS In the next two sections our goal is to help the reader under-
stand the HDP-HMM model, starting from simpler and more
popular models such as mixtures or HMMs. B. STATE OF THE ART Network delay modeling and prediction is a well-studied
problem. Some of the simplest models assume independent
observations and can be used to detect anomalous delay val-
ues. They, however, cannot predict the delay or find recurring
patterns in a delay series since they do not account for time
dependencies. Such models include the Pareto distribution [9]
and mixtures of Weibull [10] or Normal distributions [11]. More complex time series models have been used for
short-term delay predictions (from seconds to minutes), with
applications such as telerobotics. These include autoregres-
sive models [12], [13] and deep neural networks [14], [15]. Unfortunately, their parameters are more difficult to interpret
and they do not provide a segmentation of the data. Readers who are less interested in the description of the
Bayesian nonparametric context might wish to skip most
of sections II and III, and read their summaries instead
(subsections II-E and III-F). HMMs are another kind of time-series model that can
model different delay distributions and the dynamics between
them. In [16], a discrete-time HMM is used to model packet
losses, while in [17], a continuous-time HMM is used to
model both packet losses and delays. In [18], a HMM is used
to model inter-packet times and packet sizes. HMMs have few
parameters and they are easily interpretable (state transition
probabilities, means, variances, . . .). C. STRUCTURE OF THE ARTICLE The method has been implemented in RIPE Atlas to auto-
mate the processing of anchor to anchor RTT measurements,
and a Web API is available. The article introduces the API
and an accompanying notebook is provided to get started with
the API: https://github.com/maxmouchet/atlas-trends-demo. To the best of our knowledge, this is the first time that such a
method has been applied to automatically segment large delay
measurements databases, which is made possible thanks to
the robustness of this model. On the contrary, the validation
of segmentation methods based on more standard approaches
has been limited to a few subsets of time series in the
literature. The paper is structured as follows. Section II is a reminder on
mixture models (MM) and hidden Markov models (HMM). In section III we describe their nonparametric Bayesian coun-
terparts, the Dirichlet Process MM (DPMM) and the Hier-
archical Dirichlet Process HMM (HDP-HMM, or infinite
HMM). In the same section, we explain how to automatically
calibrate these models, that is how their parameters can be
inferred from measurements without human intervention. In section IV the accuracy of the model is demonstrated
on a dataset that has been labeled by humans, as well as
on some RIPE Atlas RTT time series where we discuss the
matching between routing configurations (from traceroutes)
and states learned by the statistical model. We also briefly
address the analysis of some CAIDA MANIC measurements. In Section V we introduce a Web API that makes it possible
to request the HDP-HMM analysis of anchor to anchor RTT
measurements in RIPE Atlas. We also demonstrate that ana-
lyzing the frequency of state changes in RTT time series over
Atlas allows a very precise detection of the moment of occur-
rence of events affecting large infrastructures of the Internet
(such as IXPs). In Section VI we conclude and present our
vision of the research axes to be developed in the future. A. SCOPE OF THE PAPER We also demonstrate the genericity of the HDP-HMM model,
which can fit any measured RTT time series much more
accurately than classical mixture and hidden Markov models. The goal of the article is to recall the major principles of
infinite HMMs and apply this theory to network measurement
data. Whereas [6]–[8] are written for statisticians, we want to
make the theory accessible to a wider audience and show the
potential of this model for automating the analysis of a wide
variety of delay time series. A. A TAXONOMY OF STATISTICAL MODELS However, standard HMMs require the use of heuristics
to determine the number of hidden states. To remedy this
problem, we use a nonparametric HMM for which the number
of hidden states is inferred from the data. A nonparametric
mixture model has been used in the past to model the delay
of a set of hosts measured over disjoint time intervals [19]. In comparison, our model is a nonparametric HMM and con-
cerns the delay between two hosts over a large and continuous
time interval, from a few hours to a few weeks. We start by providing a taxonomy of the different models dis-
cussed in the article. Our taxonomy takes into account three
criteria: (i) whether there is naturally a notion of ‘‘hidden
state’’ in the statistical model (ii) whether time dependency
is taken into account, and (iii) whether the number of states
is supposed to be known (and finite) or unknown (and poten-
tially infinite). The RTT is stable over long periods of time (usually a
few hours) before its distribution switches from one proba-
bility law to another (see Fig. 1). This can be explained by
reference to IP-level routing changes, congestion, and traffic We first introduced the use of the HDP-HMM for RTT
time series in [20], [21]. In these papers we explain how 16772 VOLUME 8, 2020 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs TABLE 1. Taxonomy of models. TABLE 1. Taxonomy of models. yt follows a distribution with probability density function pθk,
where θk is a parameter vector. For example, in a GMM, θk
consists of mean and variance parameters, so θk = (µk, σ 2
k )
and pθk(y) = N(y; µk, σ 2
k ) = (2πσ 2
k )−1/2 exp
−(y−µk)2
2σ 2
k
. Finally, the data distribution writes p(yt) = P
k=1:K πkpθk(yt)
where πk denotes the probability that the state of an observa-
tion is k, that is, πk = P(zt = k).
Finally, the data distribution writes p(yt) = P
k=1:K πkpθk(yt)
where πk denotes the probability that the state of an observa-
tion is k, that is, πk = P(zt = k). MM parameters φ
=
{πk, θk}k=1:K can be estimated
from measurements y1:T according to different criteria and
algorithms. C. HIDDEN MARKOV MODELS Because of the independent observations assumption, the pre-
dictive ability of MMs is limited. Knowing model parameters
and which state value zt has generated the last observation
yt does not bring any information about the next state zt+1. HMMs are a generalization of MMs that take into account
temporal dependencies among states. These temporal depen-
dencies are expressed through a Markov property assumed for
the states, written as p(zt+1|z1:t) = p(zt+1|zt). Thus, the prob-
ability distribution of the next hidden state zt+1 depends on
the current state zt only. But a classical problem in statistics with MMs or HMMs
is that the order of the model is assumed to be known
(and finite). In practice this hypothesis is unrealistic in most
applications considered. This is where models with Dirichlet
processes (DP) priors on the number of components of the
mixture, or of the HMM, find their interest. In the Dirichlet Process MM (DPMM) and the Hierarchical
Dirichlet Process HMM (HDP-HMM), the number of model
states is ‘‘infinite’’. And the order of the model can be learned
from the measured data, as is the case for the other parameters
of the model. This is an important property as it means that the
model is flexible enough to adapt to a large number of time
series, without manual human intervention (initialization of
algorithms, etc.). More formally the transition probabilities between succes-
sive states are defined via a K × K matrix 5 with entries
5ij = P(zt+1 = j|zt
= i). The model parameters are
now φ = {5, {θk}k=1:K}, the steady state probability vector
π = (π1, . . . , πK) being related to 5 through the linear
system π5 = π and πe = 1, where e = (1, . . . , 1)T . In Table 1 we have summarized which properties are
satisfied by which models. This justifies the choice of the
HDP-HMM to characterize RTTs and to automate their pro-
cessing. The MLE of HMM parameters can be estimated using a
variant of the EM algorithm known as the Baum-Welch (or
forward-backward) algorithm [24]. While easy to implement
and well-studied, this approach is prone to overfitting on
noisy data or data with few samples. Furthermore this method
requires the number K of hidden states to be known, which is
usually not the case when studying RTTs. A. A TAXONOMY OF STATISTICAL MODELS A common choice is the Maximum Likeli-
hood Estimator (MLE) which supplies the parameters that
maximize the likelihood of the observations: φMLE
=
arg maxφ p(y1, y2, . . . , yT ; φ). In general, direct maximiza-
tion of the likelihood p(y1:T ; φ) with respect to φ is infea-
sible. The Expectation-Maximization (EM) algorithm [23] is
a popular iterative two-step algorithm used to compute the
MLE for models with incomplete data, in particular mixture
models. engineering at lower layers than layer 3 [22]. Propagation
delays give a lower bound on the RTTs, and as router queue
lengths increase with the traffic, so do the observed RTTs. From a statistical point of view, it is natural to think of models
with ‘‘hidden states’’ such as MMs or HMMs. Knowing that the delay is stable over several hours means
that, if the path quality is measured at a frequency of one
‘‘ping’’ every few minutes, the delay distribution remains sta-
ble for tens or hundreds of time slots. In order to have a model
that can be used for prediction, it is necessary to account for
this temporal dependence. This is made possible by HMMs,
while mixture MMs assume independent observations. C. HIDDEN MARKOV MODELS This flexibility is obtained at the cost of a greater complex-
ity of the model of inference algorithms for parameter estima-
tion. However, we could provide an efficient implementation
for it embedded in an operational API (see Section V-B). B. MIXTURE MODELS Classical mixtures and HMMs are parametric models, mean-
ing that they have a set of parameters with fixed size. This is
a major difficulty when estimating HMM parameters as often
the number of hidden states is not known in advance. Some of the simplest statistical models that include hidden
states are mixture models. MMs are a generative statistical
model used to describe data produced by different system
states. For instance, in a Gaussian Mixture Model (GMM),
observations y1:T = (y1, y2, . . . , yT ) are assumed to be inde-
pendent and a normal distribution is associated to each hidden
state. For continuously distributed observations, condition-
ally to the underlying state zt = k ∈{1, 2, . . . , K}, where
K denotes the number of states of the model, the observation One could estimate models for different numbers of states,
but the maximum of the likelihood would increase with the
number of states as a model of order K is a degenerated
case of model of order K + 1. A classical approach consists
of penalizing the MLE optimization criterion by adding a
penalty term to the log-likelihood such as the AIC [25] or the 16773 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs BIC [26] criterions and by selecting the model that minimizes
this penalized criterion. Another approach is to use nonpara-
metric models with unbounded number of parameters. BIC [26] criterions and by selecting the model that minimizes
this penalized criterion. Another approach is to use nonpara-
metric models with unbounded number of parameters. In general, a direct maximization of the posterior likeli-
hood p(φ|y1:T ) with respect to φ is not feasible as p(φ|y1:T )
can be quite complex. Note, however, that there are situations
where the likelihood and the prior distribution are such that
posterior distribution belongs to the same family as the prior. In this case, the prior is said to be conjugate. Using conjugate
priors, when possible, often makes inference simpler. Another limitation of parametric models is that the EM
algorithm usually used to tune the parameters of the model is
sensitive to the choice of its initialization point. Appropriate
initialization strategies must be used otherwise it may con-
verge to a local but non-global maximum of the likelihood. B. MIXTURE MODELS Markov Chain Monte Carlo (MCMC) techniques, and in
particular Gibbs sampling, can be used in very general situ-
ations for inference [27]. Alternatively, variational Bayesian
methods can be considered ( [28], chap. 33). The principle of
MCMC methods is to use simulations to draw a large number
of samples φ from the posterior distribution p(φ|y1:T ). Because of these limitations, standard MMs or HMMs can-
not be used on a large scale to analyze Internet measurements. In the following section we introduce a new approach for RTT
measurement analysis, based on nonparametric Bayesian
models, and more particularly the HDP-HMM. (G(A1), . . . , G(An)) ∼Dir(αH(A1), . . . , αH(An)).
(1) (1) A more formal approach to models with an unknown num-
ber of components can be found in Bayesian statistics. The
Bayesian framework allows one to specify models with sev-
eral layers of uncertainty and infer the parameters in a system-
atic way. We will make better use of this flexibility to estimate
HMMs from RTT series where neither the number of states
nor the probability distribution in each state is known. Here Dir(α1, . . . , αn) denotes the n-variate Dirichlet distri-
bution with parameters α1:n = (α1, . . . , αn), that is to say the
probability distribution with density function: p(x1:n; α1:n) =
1
B(α)1I{1}(
X
i=1:n
xi)
Y
k=1:n
xαi−1
i
1I[0,1](xi) (2) B. DIRICHLET PROCESSES AND DP MIXTURES MMs and HMMs are interesting for characterizing time series
of RTTs. They are designed to model phenomena that change
state from time to time and in which the value of the observa-
tions, here the RTTs, noisily depends on the hidden states. One can imagine that different hidden states result from
different routing configurations, traffic engineering choices,
or link loads. However, these models are too simple to char-
acterize a large variety of RTT series and are not suitable for
automatic processing on a large scale. Modelling a HMM with an infinite number of states is gen-
erally achieved by means of a Dirichlet process (DP) prior. Modelling a HMM with an infinite number of states is gen-
erally achieved by means of a Dirichlet process (DP) prior. DPs were introduced by Ferguson [29] in 1973 and were
first applied to mixture models with an unknown number
of components in [30]. The extension to the modelling of
HMMs was first defined in 2002 in [31]. More recently this
has been formalized in the framework of hierarchical Dirich-
let processes (HDP) in [8] where HDP-HMMs have been
introduced. These models are called nonparametric Bayesian,
meaning that they are Bayesian and involve parameter spaces
of infinite dimension [32]. We propose to use a more generic model, the HDP-HMM. This model does not make assumptions about the number of
states of the system, contrary to vanilla mixtures or HMMs,
and it is possible to learn the number of states from the data
itself. Contrary to DPMMs it also takes into account time
dependency and makes it possible to account for the RTT
distribution being stable for a long period of time. A Dirichlet Process (DP) is a stochastic process G ∼
DP(α, H), the realizations of which are probability distribu-
tions. It is parameterized by a concentration parameter α and
a base distribution H. It can be seen as a process indexed by
partitions (A1, . . . , An) (n > 0) of the space E on which H is
defined, with n-variate Dirichlet random realizations: III. NONPARAMETRIC BAYESIAN APPROACH (G(A1), . . . , G(An)) ∼Dir(αH(A1), . . . , αH(An)). (1) A. BAYESIAN SETTING where 1IA(x) = 1 if x ∈A and 0 otherwise, and B(α) is a
normalization factor. In the MLE approach, estimates of the parameters are inferred
from data. In contrast, Bayesian approaches make use of
prior distributions upon the model parameters, and output a
posterior probability distribution over the model parameters. These prior distributions can account for prior knowledge of
the parameter distributions. Alternative definitions of DPs are also useful both for their
understanding and simulation. In particular it can be proved
that a Dirichlet Process G ∼DP(α, H), can also be defined
via the stick-breaking constructive approach [33]. The idea is
to build a discrete distribution by assigning probabilities πk to
samples θk drawn independently from H. As the probabilities
πk must sum to 1, a unit-length stick is divided as displayed
in Figure 2. The stick is first broken into two parts, of lengths
η1 and 1 −η1. Then the second portion, of length 1 −η1,
is broken again into two parts in proportions η2 and 1 −η2. The three resulting portions are now of lengths η1, η2(1−η1)
and (1 −η2)(1 −η1). The process of breaking the stick into
smaller parts continues indefinitely. When the dimension of the model is unknown, as for
MMs or HMMs with unknown order K, one can resort to
nonparametric Bayesian approaches, where the number of
components of the model is inferred from the data itself. Bayesian inference can be performed from the posterior
likelihood which is defined as p(φ|y1:T ) ∝p(y1:T |φ)p(φ)
where p(y1:T |φ) is the likelihood of the data y1:T , p(φ) is a
prior distribution and ∝denotes proportionality. 16774 16774 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs FIGURE 2. The stick-breaking process. FIGURE 3. The Hierarchical Dirichlet Process - Hidden Markov Model
(HDP-HMM). FIGURE 2. The stick-breaking process. The weights πk are defined as π1 = η1, π2 = η2(1 −
η1), π3
=
η3(1 −η2)(1 −η1), and in general πk
=
ηk
Q
l=1:k−1(1−ηl). The proportions ηk are independent and
ηk ∼Beta(1, α), where Beta(a, b) is the beta distribution
with parameters a and b and probability density function
xa−1(1 −x)b−11I[0,1](x). FIGURE 3. The Hierarchical Dirichlet Process - Hidden Markov Model
(HDP-HMM). The distribution with weights π = [π1, π2, . . .] is called
a Griffiths-Engen-McCloskey distribution, denoted by π ∼
GEM(α). D. INFERENCE IN DP MIXTURES G =
X
k=1:∞
πkδθk ∼DP(α, H)
(6) Inference in DPMMs is better addressed via the so-called
Polya urn representation of DPs than through stick breaking. Imagine an urn that contains black and colored balls. The
‘‘values’’ of balls are their colors. At initialization the urn
only contains α black balls. When drawing a ball from the
urn, if the ball drawn is black then a new colored ball is drawn
from a base distribution H and the black and colored balls are
put back into the urn. When the first ball drawn is not black,
it is put back into the urn together with a new one of the same
color. The labels of the infinite sequence of draws follow
a DP. (6) A. BAYESIAN SETTING Clearly, P
k=1:∞πk
=
1. We then get the
stick-breaking representation of the Dirichlet Process G: distribution pθk. This is made possible by parameterizing the
DPs Gi (i = 1, 2, . . .) by the same discrete valued base
distribution G0 G =
X
k=1:∞
πkδθk,
with π ∼GEM(α) and θk ∼H. (3) Gi =
X
k
πikδθk ∼DP(α, G0)
(7) (7) Note that the πks tend to decay to zero at geometric rate. Indeed it can easily be proven that: where G0 is modeled by a DP prior with base distribution Hλ: G0 =
X
k
βkδθk ∼DP(γ, Hλ)
(8) (8) E[πk]=E[ηk]
Y
l=1:k−1
(1−E[ηl])=
1
α+1
α
α+1
k−1
. (4) (4) This hierarchy of DPs yields the HDP-HMM process [8]. A graphical representation of the HDP-HMM is given in Fig-
ure 3, where the arrows represent the dependencies. The
HMM itself is represented by states zt and observations yt. Its parameters are (θk)k≥1 and (πi)i≥1, where pθk(yt)
=
p(yt|zt = k) and πi denotes the ith row of the transition matrix
5 of the HDP-HMM, so πij = P(zt+1 = j | zt = i). Now, suppose we want to fit a mixture model to some
observations y1:T = (y1, y2, . . . , yT ). Assume that the mixing
distributions are in the form pθ(y), where θ is a vector of
parameters and that the prior distribution over the vector
of parameters is θ
∼H. We can build a nonparametric
Bayesian generative model of observations in the form of a
Dirichlet Process Mixture model (DPMM). In this model the
distribution of observations is a mixture: j
α, γ and λ are hyper-parameters. γ and λ are the param-
eters of a Dirichlet process G0 ∼DP(γ, Hλ) that lies at the
top of the HDP hierarchy. These random dependencies and
vague priors introduce enough flexibility in the model to let
it adapt to many different time series. p(y) =
X
k=1:∞
πk pθk(y)
(5) (5) and the weights πk and parameters θk of the different compo-
nents of the mixture are defined as a Dirichlet Process: E. INFERENCE IN HDP-HMMs Inference in HDP-HMM is technically more involved than
for mixture models. We briefly summarize it here. Interested
readers can find additional information in appendices of [6]. Letting K denote the current number of states, the Gibbs
sampler should sample z1:T . Note that θ1:K can be marginal-
ized out and does not need to be sampled in Gibbs. To make
it possible, we will also have to sample the πj, which in turn
requires sampling the weights of the base distribution G0 =
P
k=1:∞βkδθk. As only (βk)k=1:K is concerned for describing
the weights of the states of the finite size data set at hand,
letting β−K = P
k=K+1:∞βk = 1 −P
k=1:K βk, we simply
sample (β1:K, β−K) that follows a Dirichlet distribution of
order K +1. The sampling of (β1:K, β−K) is described in [6]. it has been shown, in [34], that the distribution of θ′
t converges
almost surely to a DP(α,H) (when t →∞). The estimation of the parameters and states of a non-
parametric mixture model from the posterior distribution
p(z1:T , θ1:KT |y1:T ), where y1:T represent the data, can be
addressed via Gibbs sampling [7]. The principle of Gibbs
sampling [27] is to sequentially update, in turn, the values of
zt, t = 1, . . . and θk, k = 1, . . . conditionally to y1:T and to
the current value of the other parameters. It requires knowing
the distribution of each latent variable conditionally to the
observations y1:T and the other latent variables. Note also that we want to implement inference for a sticky
HDP-HMM, that is, a modified version of the HDP-HMM
that models persistency of the states by biasing the model
towards self transitions (zt−1 = j, zt = j). This is ensured
by introducing an additional parameter κ and changing the
prior upon πj: Going back to the Polya urn model, let us index by
1, . . . , Kt the distinct colors of the balls present in the urn
at time t and let zt denote the color index of the new ball. C. HIERARCHICAL DIRICHLET PROCESS HMM The idea of using a DP as a prior in mixture models has been
extended to the case of Hidden Markov Models (HMMs). In fact, for some technical reasons that we will explain,
the extension of this approach to HMM modelling involves
a hierarchy of DPs. In the Hierarchical Dirichlet Process HMM (HDP-
HMM), DPs are used as priors on the rows πi
=
(πi1, πi2, . . . , πik, . . .) of the transition matrix 5 of the hid-
den Markov chain (zt)t. This makes it possible to specify that
the number of states of the Markov chain is unknown. We are going to use this formalism in a DPMM, where zt
denotes the hidden state and yt is the observation. The Polya
urn model can be described as follows. Let us introduce θ′
t =
θzt the value of θ associated with zt. If zt = k then θ′
t = θk
and yt is distributed according to pθk(•). Given a sequence of But it is also necessary to ensure that the transition
probabilities πik, for all row i, weigh the same emission 16775 VOLUME 8, 2020 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs random variables (θ′
t)t>0 with
P(θ′
1 ∈B) = H(B),
and
P(θ′
t+1 ∈B|θ′
1:t) =
1
α + t
X
τ=1:t δθ′τ (B) + αH(B)
. (9) E. INFERENCE IN HDP-HMMs As the role of the balls can be exchanged, letting z−t =
{z1:t−1, zt+1:T } and n−t,k = #{zτ ∈z−t; zτ = k} be the
number of occurrences of the value k among z−t, it can be
shown [7] that: πj|α, β, κ ∼DP
α + κ, α(P
k βkδk) + κδj
α + κ
. (13) (13) When κ = 0 we get the standard HDP-HMM, while when
κ →∞, πj tends to only weight state j. P(dzt|z−t) =
1
α + T −1(
K−t
X
k=1
n−t,kδk(dzt) + αδK−t+1(dzt))
(10) P(dzt|z−t) =
1
α + T −1(
K−t
X
k=1
n−t,kδk(dzt) + αδK−t+1(dzt)) j
To implement the Gibbs sampler for the states z1:T let ψ =
(α, β, κ, λ), and π = (πj)j. Then P(zt|y1:T , z−t, ψ) can be
expressed by marginalizing against the πjs and θks: (10) where K−t is the number of distinct elements in z−t with
indexing set from 1 to K−t. Equation 10 can be interpreted
as follows: knowing the values of z1:t−1 and zt+1:T , the dis-
tribution of zt is a mixture of the values k ∈z−t and of a new
index value (K−t +1). The respective weights of this mixture
are
n−t,k
α+T−1 for any k ∈z−t and
α
α+T−1 for the value K−t + 1. P(zt | y1:T , z−t, ψ) ∝P(zt | z−t, ψ)p(yt | y−t, z1:T , ψ) (14) (14) Let us introduce the following notations: xi• = P
j xij and
n−t
jk denotes the number of transitions from state j to state k,
not counting the transitions zt−1 →zt or zt →zt+1. Then,
the first factor in (14) can be written as: P(dzt | z−t, ψ)
∝
XK−t
k=1
αβk + n−t
zt−1,k + κδzt−1,k
α + κ + n−t
zt−1,•
×
αβzt+1 + n−t
k,zt+1 + κδzt−1,kδk,zt+1
α + κ + n−t
k,• + δzt−1,k
δk(dzt)
+α2β−Kβzt+1
(α + κ)2
δK−t+1(dzt). ( α+
α+
It can be proven [7] that, if observations y1:T and parame-
ters θ′
−t are taken into account, then: P(dzt|z−t, y1:T , θ′
−t)
∝
XK−t
k=1 n−t,kpθk(yt)δk(dzt) + αI(yt)δK−t+1(dzt). (11)
where I(yt) = p(yt | zt = k, θ′
−t) =
R
pθ(yt)Hλ(dθ). P(dzt|z−t, y1:T , θ′
−t)
∝
XK−t
k=1 n−t,kpθk(yt)δk(dzt) + αI(yt)δK−t+1(dzt). B. CHANGE POINT DETECTION Quantifying the performance of the HDP-HMM on real RTT
time series is not easy since there is no ground truth. The
‘‘network state’’ is not known or vaguely defined. But it was
possible to compare the performance of the model in a change
point detection task where the goal is to detect significant
changes in the delay. While not the primary purpose of the
HDP-HMM, detecting change points is simply a matter of
segmenting the data and finding changes in the inferred state
sequence. This can be used to partially validate the quality of
the segmentation obtained. F. SECTION SUMMARY In this section we have introduced non-parametric Bayesian
approaches. In Bayesian statistics some of the parameters
on which the data depend are considered random. The term
‘‘non-parametric’’ means that there is a large number of
parameters that are estimated from the data. When the number of states of a mixture or a HMM is
not known in advance, it is possible to use a non-parametric
Bayesian approach using Dirichlet processes (DP) as pri-
ors. This is called the Dirichlet Process Mixture Model
(DP-MM) or the Hierarchical Dirichlet Process Hidden
Markov Model (HDP-HMM). Equivalently, the name infinite
(or non-parametric) mixture or HMM can also be used. FIGURE 4. Segmentation of RTT observations between at-vie-as1120
and sg-sin-as59253. In this work, we choose instead to use nonparametric
Dirichlet Process Mixture Models (with a Gaussian as ‘‘base’’
distribution) as emission distributions of the HDP-HMM. As such, the delay in each state is modeled by a varying num-
ber of Gaussian components. This allows us to model a wide
range of distributions, and we avoid choosing a particular
parametric emission distribution for each state of the HDP-
HMM. For each Gaussian component, we use a Normal-
Inverse-χ2 prior, which is the conjugate prior to the normal
distribution with unknown mean and variance. The use of
a nonparametric observation model reinforces the need for
Bayesian inference methods, since a more traditional MLE
approach would require several layers of penalization. Missing data, that is, states and parameters, can be esti-
mated from observations using a Gibbs sampling algorithm
which comes up to randomly simulating, in turn, the different
components of the model which are not measured directly. These components are simulated according to some condi-
tional distributions which have been specified in this section. E. INFERENCE IN HDP-HMMs (11) ×
αβzt+1 + n−t
k,zt+1 + κδzt−1,kδk,zt+1
α + κ + n−t
k,• + δzt−1,k
δk(dzt) (yt) = p(yt | zt = k, θ′
−t) =
R
pθ(yt)Hλ(dθ). here I(yt) = p(yt | zt = k, θ′
−t) =
R
pθ(yt)Hλ +α2β−Kβzt+1
(α + κ)2
δK−t+1(dzt). (15) t
R
Note that, provided I(yt) is known, the proportionality
factor in Eq. (11) can be obtained from the normalization
condition P
k P(zt
=
k
|
z−t, y1:T , θ′
−t)
=
1. If pθ
and Hλ are conjugate distributions, I(yt) can easily be cal-
culated in closed form. In other cases one can resort to
Metropolis-Hastings simulation using the prior distribution
of zt in (10) as an auxiliary distribution [7] to calculate I(yt). (15) And the second factor in (14) as: And the second factor in (14) as: p(yt | y−t, z1:T , ψ)
∝
Z
θzt
p(yt | θzt)Hλ(dθzt | {yτ; zτ = zt, τ ̸= t}). (16) (16) After sampling zt, t = 1 : T from Eq. (11), θk, k = 1, . . . can be sampled from the following distribution [7]: As far as discussed earlier, if the θks have conjugate prior
distributions, p(yt | y−t, z1:T , λ) can be calculated in closed
form. Note in addition that to avoid a particular choice of
hyperparameters (α, γ, λ) biasing the solution, they can also
be given some prior distribution. P(dθk|z1:T , y1:T , θ−k) ∝Hλ(dθk)
Y
{t;zt=k}
pθk(yt). (12) Here again simulation can be performed directly or via
Metropolis-Hastings simulation depending whether pθ and
Hλ have conjugate distributions. At the end of the process, after the z1:T have been esti-
mated, the θks can be estimated easily, e.g. by maximizing
the likelihood p({yt; zt = k} | θk). 16776 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs FIGURE 4. Segmentation of RTT observations between at-vie-as1120
and sg-sin-as59253. IV. A FIRST LOOK AT RTTS THROUGH THE HDP-HMM As stated previously, HDP-HMM is a flexible method for
inferring HMM parameters and segmenting data when the
number of latent states is unknown. This fits the problem
of segmenting RTT time series (remember that of Figure 1),
where the number of different states is not a priori known. Furthermore, it is not mandatory to make an assumption
on the type of RTT distribution in each state (Gaussian,
exponential, or other kind of parametric distributions). This
distribution can be assumed nonparametric, which introduces
even more flexibility and allows a very generic model that
adapts to a very large number of traces. pp
q
y
p
The segmentation of the series from Figure 1 using an
HDP-HMM with DP-GMM emissions is shown in Figure 4,
in which states are represented by colors. As a matter of comparison, we provide in Fig. 5 the seg-
mentation obtained with a HDP-HMM with DPMM emission
distributions, with that resulting from parametric and non-
parametric MMs and HMMs with a Gaussian observations
model. In the case of the Gaussian MM and of the HMM,
the number of latent states has been chosen by estimating
the model for a varying number of components and choos-
ing the number that minimizes the penalized log-likelihood
using the BIC criterion. As we can see the HDP-HMM
produces a segmentation close to what a human would do,
contrary to other models which generate far too many state
changes. In this section we show that the model produces realistic
segmentations from a human point of view, and that the
inferred parameters can be interpreted easily, given the appli-
cation domain. In addition, we provide two validations for
the model. We show on a labeled change point dataset that
the model performs at least as well as ad-hoc change point
detection methods. And we also show that the states inferred
from the RTT time series match well with the AS and IP paths
seen in RIPE Atlas traceroutes. A. A NONPARAMETRIC OBSERVATION MODEL Many parametric models have been proposed in the literature
to explain the distribution of the delay in computer networks
and on the Internet. For example, in [11] a Gaussian mixture
model is proposed, in [10] a Weibull mixture model, and in [9]
a Pareto distribution. In practice, however, it seems that the
distribution can be very different depending on the network
state. For example, in some states the delay can be relatively
stable with occasional spikes above a baseline, in which case
it might be modeled by an exponential distribution, while in
other states the delay can experience large variations caused
by a high volume of traffic, and might be better modeled by
a normal distribution. We have benchmarked the HDP-HMM against different
change point detection methods on a labeled dataset intro-
duced by [35]. This dataset is particularly interesting because
change points in RTT timeseries have been manually labeled
by human experts. To our knowledge, there are no other RTT
time series datasets that are both realistic and labeled. VOLUME 8, 2020 16777 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs FIGURE 5. Segmentation of a RTT time series with parametric and nonparametric mixture models and HMMs. FIGURE 6. Benchmark of the HDP-HMM against classical change point detection methods on a human-labelled change point dataset [35]. The
weighted recall gives more importance to large delay changes. FIGURE 5. Segmentation of a RTT time series with parametric and nonparametric mixture models and HMMs. metric mixture models and HMMs FIGURE 5. Segmentation of a RTT time series with parametric and nonparametric mixture models and HMMs. FIGURE 6. Benchmark of the HDP-HMM against classical change point detection methods on a human-labelled change point dataset [35]. The
weighted recall gives more importance to large delay changes. The dataset consists of 50 RTT series of varying length for
a total of 34,008 hours of observations. In [35] change point
detection is performed by minimizing Pm+1
i=1 C(yτi−1+1:τi) +
βf (m). m is the number of changes, C is a cost function that
measures the stability of the delay over a range of successive
values, and f (m) is a penalty that prevents overfitting. Differ-
ent cost functions and penalties are considered. environments such as data centers, universities, and IXPs
(Internet eXchange Points). 1RTT
measurement
results
are
available
at
https://atlas.ripe.net/
measurements/1437285. We considered the period between the 2nd
and the 9th of May 2018. A. A NONPARAMETRIC OBSERVATION MODEL Anchors tend to be located
in well-connected autonomous systems and measurements
between anchors represent more stable paths than may be
observed from probes located at the edges of the Internet. Moreover, anchors are more powerful and perform so-called
anchoring mesh measurements, where various measurements
are performed regularly between each pair of anchors. This
allows us to collect traceroute results both on the outward and
on the return path. We have compared the performance of the segmenta-
tion obtained by HDP-HMM with the best performing
changepoint detection methods of [35]. In our approach a
HDP-HMM model is learnt on each timeseries, the most
likely hidden state sequence is computed, and changepoints
are simply defined as changes in the hidden state sequence. Our dataset consists of one week of IPv4 RTT measure-
ments between all Atlas anchors and the at-vie-as1120
anchor.1 Delay is measured every four minutes using three
ICMP (Internet Control Message Protocol) pings towards the
target anchor. The minimum value of the delay is kept for each
time step. Considering the subset of anchors that were online
over the time period, we collected 301 series of 2520 data
points. The associated traceroute measurements were also
collected, both on the outward path, and on the return path. Traceroutes are performed every fifteen minutes using three
ICMP probe packets for each hop. In Figure 6 we show that the HDP-HMM performs simi-
larly to the best performing change point detection methods
of [35] in terms of precision (
# True Positive
# True Positive+# False Positive),
while
performing
better
in
terms
of
recall
(
# True Positive
# True Positive+# False Negative). This means that our model is
more sensitive to small changes in the delay without gen-
erating unnecessary false alarms. 2) INFERENCE In addition to detecting significant changes in the delay,
the HDP-HMM also provides a notion of hidden states. In this
section we validate the quality of this clustering both visually
and by studying the correlation with AS and IP paths revealed
by traceroutes. Each series were segmented using our Julia implementation
of the Gibbs sampler. It takes less than 2 seconds on a single
thread of a 2.80GHz Intel Core i7-7600U CPU to process a
2520 point time series (1 week of an Atlas RTT measurement)
with 300 iterations of the sampler. The task is highly paral-
lelizable as each time series can be processed independently. 1) DATASET RIPE Atlas offers two types of measurement sources: probes
and anchors. Probes are deployed in heterogeneous envi-
ronments while anchors are restricted to high-availability VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs FIGURE 7. Segmentation of RTT observations between at-vie-as1120 and us-bos-as26167 using an HDP-HMM with DP-GMM emissions. Each
color identifies a state or an IP path observed in the traceroute. FIGURE 8. Distribution of the number of states learned for different
timescales. Using 4 threads 300 one week long time series can be pro
FIGURE 9. Density estimation of the (standard deviation, average
duration) couple Darker colors indicate a higher density FIGURE 7. Segmentation of RTT observations between at-vie-as1120 and us-bos-as26167 using an HDP-HMM with DP-GMM emissions. Each
color identifies a state or an IP path observed in the traceroute. FIGURE 9. Density estimation of the (standard deviation, average
duration) couple. Darker colors indicate a higher density. FIGURE 8. Distribution of the number of states learned for different
timescales. FIGURE 9. Density estimation of the (standard deviation, average
duration) couple. Darker colors indicate a higher density. Using 4 threads, 300 one-week long time series can be pro-
cessed in 6 minutes. probability of self-transition. In the example of Fig. 7 the
average duration of the purple state is of 45 timesteps
(= 3 hours) and of 149.5 timesteps (= 9 hours 58 minutes)
for the green state. The standard deviation of the delay in
the purple state is of σ = 10.3 msec while the standard
deviation of the green state if of σ = 4.1 msec. States with a
high variance could possibly be explained by intra-domain
load-balancing (since Atlas ping flow ID is not constant),
congestion, or in-path devices delaying the processing of
ICMP packets. However, assessing the cause of such vari-
ations and studying the possibility of detecting them from
delay measurements is to be done in future works. Figure 8 shows the distribution of the number of states in
the resulting HMMs for different measurement timescales. It is clear that the number of states grows with the length
of the series. This is not surprising and visual inspection by
a human expert would also probably identify more states in
longer timeseries. One, three, and seven-day long series have
less than 8, 10, and 11 states respectively. 3) STATE DURATIONS VS. DELAY VARIATIONS Figure 9 displays the standard deviation of the RTT against
the average duration in a state. In the analyzed dataset,
the average state duration decreases as the RTT standard devi-
ation increases. This is not surprising as we expect Internet
paths to spend more time in stable states. An advantage of HMMs over other timeseries models (e.g. autoregressive models or neural networks) is that the param-
eters can be interpreted easily, given the application domain. In our case, the state transition matrix 5 gives us information
about the frequency of network configuration changes and
the relation between them, while the observation distributions
give us in particular the mean value of the delay and its
variance (of the delay in each configuration). 1) DATASET This confirms the
capability of the HDP-HMM to learn more complex models
as the number of RTT observations, and possibly the number
of underlying network configurations, grows. V. LARGE-SCALE MEASUREMENT ANALYSIS Internet monitoring projects such as RIPE Atlas provide
a large amount of latency information. Due to its scale,
RIPE Atlas has a good chance of providing enough infor-
mation to allow the detection of anomalous latency patterns
in important network components, such as IXPs or large
transit providers. However, detecting and characterizing these
anomalies has proven challenging (e.g. the analysis in [37]
took weeks). In this section, we start by showing the validity
of the model on a very large scale considering all the mea-
surements of the Atlas anchoring mesh. We show that real
data are as likely under the model considered as simulated
data would be. Then we introduce the trends API that we
have developed and that publicly provides an on-demand
segmentation service for Atlas RTT measurements. Finally,
we show how aggregating change points learned with the
HDP-HMM from a large number of origin-destination pairs is
a simple and elegant method for detecting and characterising
anomalies in key Internet infrastructures. FIGURE 10. Distribution of the number of states associated with a given
number of unique paths. FIGURE 10. Distribution of the number of states associated with a given
number of unique paths. In other words, only 16% of the states learned can match two
AS paths or more. States associated with more than one AS
path can be explained by delay differences too small to be
separated into two clusters. Conversely, one AS or IP path can be mapped to sev-
eral states. For example in Figure 7 we only observe the
AS path ASN MARKLEY →GTT BACKBONE →NTT
COMMUNICATIONS →ACONET SERVICES in the tracer-
outes from us-bos-as26167 to at-vie-as1120 and
ACONET SERVICES →ACONET →NEXTLAYER AS →
NTT COMMUNICATIONS →ASN MARKLEY in the reverse
traceroutes (as resolved using the RIPEstat API). In the for-
ward traceroutes we observe IP path changes every 15 min-
utes, in the GTT and NTT ASes, probably due to intra-AS
load-balancing, while in the reverse traceroutes we only
observe two different IP paths in NTT AS that are perfectly
correlated to state changes in the model. A. VALIDATION OF THE HDP-HMM MODEL AT LARGE
SCALE ON RIPE ATLAS In Section IV-B, we have shown that the HDP-HMM model
is at-least as good as classical change point detection methods
on a labelled RTT change points dataset. This however, does
not tell us whether the model fits RTT data well from a sta-
tistical point of view. In this section, we propose to compare
the likelihood of the time series (with respect to their inferred
model) with the likelihood of time series simulated according
to an HDP-HMM model. If the models fit the data well,
we can expect the likelihood of the data with respect to the
model to follow the same distribution as the likelihood of
synthetic data generated by the model. 2https://webview.orange.com 4) RELATIONSHIP WITH THE AS AND IP PATHS We hypothesized that the distribution of delay observations
is conditioned on the underlying network state, such as the
inter and intra-AS routing configuration, as well as the traffic
level. As shown in Figure 10, the majority of the states learned
over all the paths in our dataset match only one AS path and
one IP path. For example there are 595 states which always
correspond to the same AS path over the 746 states learned. On most time series we can distinguish two types of states:
those where the delay is relatively constant (such as the green
one on Fig. 7), and states where the delay is very variable
(such as the purple one). This is reflected by the variance
of the delay in the state. And the average duration of a
HMM in a state i is given by 1/(1 −πii) where πii is the 16779 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs FIGURE 10. Distribution of the number of states associated with a given
number of unique paths. In addition, we have tested the use of this segmentation
method with application-level delay measurements such as
web Page Load Times (PLTs) and obtained promising results. Figure 11 shows the PLT for the baidu.com website measured
from a probe located in Lannion, France. Measurements have
been performed with the WebView platform.2 D. CAIDA MANIC AND OTHER MEASUREMENTS a RTT difference (far - near) time series obtained with TSLP probes from the CAIDA MANIC project. Each color identifies a FIGURE 11. Segmentation of a RTT difference (far - near) time series obtained with TSLP probes from the CAIDA MANI
state. FIGURE 11. Segmentation of a RTT difference (far - near) time series obtained with TSLP probes from the CAIDA MANIC project. Each color identifies a
state. FIGURE 12. Segmentation of a PLT time series obtained from a WebView probe. Each color identifies a state. FIGURE 13. Q-Q plot of observed vs. simulated log-likelihood on 100k
time series. FIGURE 14. Distribution of observed and simulated log-likelihood on
100k time series. FIGURE 12. Segmentation of a PLT time series obtained from a WebView probe. Each color identifies a state. FIGURE 12. Segmentation of a PLT time series obtained from a WebView probe. Each color identifies a state. FIGURE 14. Distribution of observed and simulated log-likelihood on
100k time series. FIGURE 14. Distribution of observed and simulated log-likelihood on
100k time series FIGURE 13. Q-Q plot of observed vs. simulated log-likelihood on 100k
time series. FIGURE 14. Distribution of observed and simulated log-likelihood on
100k time series. FIGURE 13. Q-Q plot of observed vs. simulated log-likelihood on 100k
time series. FIGURE 13. Q-Q plot of observed vs. simulated log-likelihood on 100k
time series. FIGURE 13. Q-Q plot of observed vs. simulated log-likelihood on 100k
time series. FIGURE 14. Distribution of observed and simulated log-likelihood on
100k time series. In addition, a Neyman-Pearson (NP) test was performed
between two simple hypothesis: H0
: the time series is
distributed according to an HMM, H1
: the time series
is distributed according to an HDP-HMM. The false alarm
rate (FAR) was set to α = 5% and α = 10%. The FAR is
the probability of deciding in favor of the HDP-HMM (H1)
when the series is distributed according to an HMM (H0 is
true). When α = 5% (resp. 10%) the NP test result was
that the time series was distributed according to H1 (HDP-
HMM) in 96% (resp. 97%) of the cases. The same experiment
was performed to compare the HDP-HMM hypothesis (H1)
against the DPMM hypothesis (H0). In that case, for α = 5%
and α = 10%, the test result supported the HDP-HMM
hypothesis in 99% of the cases. consistent with what a human expert would produce
(Section IV). D. CAIDA MANIC AND OTHER MEASUREMENTS In addition, we have checked on a very large
scale (about 100k randomly chosen series among the Atlas
mesh measurements) that all these series are well modeled
by the HDP-HMM. D. CAIDA MANIC AND OTHER MEASUREMENTS In addition to RIPE Atlas delay measurements, the HDP-
HMM fits other kinds of network measurements as well. In this section we show the results obtained on delay measure-
ments from the CAIDA MANIC project [36]. The CAIDA
MANIC project uses Time Series Latency Probes (TSLP)
to measure inter-domain congestion. Once a peering link
between two ASes has been identified, ICMP probes are sent
to the near-end (i.e. the last router in the first AS) and the
far-end (i.e. the first router in the second AS) of the link. The
intuition is that if there is congestion the router queues will fill
up, and the delay between the near-end and the far-end will
increase. Using the same model as for the RIPE Atlas RTT
series, we segment the delay difference time series (far-end -
near-end) from publicly available measurements. To perform a comparison, 100k time series of one week
duration (2520 data points) from the anchoring mesh mea-
surements were considered. For each time series, the model
was learned, and their likelihood p(y|π, θ) computed with
respect to the model. In addition, for each HMM with param-
eters (π, θ), a time series y′ was sampled and its likelihood
p(y′|π, θ) computed. In Figure 11 we show the resulting segmentation for a
peering link experiencing periodic congestion. Three states
are learned. The green state, corresponding to a non-saturated
link, has a standard deviation of 0.1 ms, while the standard
deviations for the red and blue states are 7 ms and 11 ms,
respectively. The blue state seems to correspond to a state
of increased traffic, while the red state seems to correspond
to a saturated link. Because the model accounts for temporal
dependencies, it is able to clearly separate those two states
even though their distributions overleap. The distributions of the likelihood on observed and syn-
thetic time series are compared in Figures 13 (Q-Q plot)
and 14 (histograms). It can be seen that both distributions
are similar, with the simulated time series being slightly
more likely. This demonstrates that the HDP-HMM explains
well the diversity of observed trajectories in RIPE Atlas
measurements. 16780 16780 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs FIGURE 11. Segmentation of a RTT difference (far - near) time series obtained with TSLP probes from the CAIDA MANIC project. Each color identifies a
state. B. RIPE ATLAS TRENDS API In order to make our method widely accessible, we have
developed a publicly exposed Web API into RIPE Atlas. Given an origin-destination pair (measurement and probe ID)
and a time frame (start and stop time), the trends API provides
the segmentation of a RIPE Atlas delay measurement. The
API offers three endpoints, described in Table 2. The /ticks endpoint returns the minimum RTT for
a given pair with a constant time interval (duplicated
results due to probe connectivity problems are sup- Thus, we have not only visually verified on a large number
of series that the segmentation obtained with the model is 16781 VOLUME 8, 2020 M. Mouchet et al.: Large-Scale Characterization and Segmentation of Internet Path Delays With Infinite HMMs TABLE 2. Endpoints of the Atlas Trends API. FIGURE 15. RIPE Atlas Trends API sample JSON output. FIGURE 16. Change frequency on the 13th of May 2015 for the 20k pairs
that saw AMS-IX in their traceroutes the day before. FIGURE 17. Change frequency between the 9th and the 10th of
April 2018 for the 60k pairs that saw DE-CIX Frankfurt in their traceroutes
the day before. measurements are more difficult to exploit due to the anycast
nature of DNS root servers. In order to detect anomalous events in those infrastruc-
tures, we propose to aggregate the change points learned
from each time series individually, so as to obtain a state-
change frequency, which represents the number of state
changes in a given time frame over all the origin-destination
pairs considered. One problem is the selection of those
origin-destination pairs. One could imagine learning the
model for all the origin-destination pairs available in Atlas,
or a large subset, such as anchoring mesh measurements
(160k origin-destination pairs), and then looking for events
in state-change frequency. However, preliminary experimen-
tation shows that the signal obtained is too noisy and
requires a lot of manual processing to find relevant events. Instead, we propose to monitor each infrastructure individ-
ually, by considering only the origin-destination pairs for
which the infrastructure has been seen in recent traceroute
meas rements TABLE 2. Endpoints of the Atlas Trends API. FIGURE 15. RIPE Atlas Trends API sample JSON output. FIGURE 16. Change frequency on the 13th of May 2015 for the 20k pairs
that saw AMS-IX in their traceroutes the day before. FIGURE 17. B. RIPE ATLAS TRENDS API For example, the URL https://
trends.atlas.ripe.net/api/v1/trends/1437285/6222/?start=2018
-05-02&stop=2018-05-10 gives the segmentation in Figure 7
(it should take less than 10 seconds to segment one week of
data). A summary of the time series, as shown in Figure 15,
can also be requested by appending /summary to the path. Start and stop time are specified as YYYY-MM-DDTHH:MM
where THH:MM is optional and defaults to the start of the day. To validate the ability of our method to detect events,
we analyzed two events which have been discussed in the
literature (as this provides some groundtruth against which
to compare our results): AMS-IX outage in May 2015 [37],
[39], [40], and DE-CIX Frankfurt outage in April 2018 [41]. pressed, and missing results are explicitly inserted). The
/trends endpoint returns the minimum RTT and the
associated segmentation. For example, the URL https://
trends.atlas.ripe.net/api/v1/trends/1437285/6222/?start=2018
-05-02&stop=2018-05-10 gives the segmentation in Figure 7
(it should take less than 10 seconds to segment one week of
data). A summary of the time series, as shown in Figure 15,
can also be requested by appending /summary to the path. Start and stop time are specified as YYYY-MM-DDTHH:MM
where THH:MM is optional and defaults to the start of the day. In addition to this article, we provide interactive notebooks
to document and demonstrate the API, and compare various
statistical models. Links to interactive Google Colab sessions,
as well as the notebooks source and code to facilitate the
usage of the API are provided on GitHub [38]. 1) AMS-IX MAY 2015 OUTAGE According to [37], on the 13th of May 2015, AMS-IX expe-
rienced a partial outage due to a switch interface generating
looped traffic on the peering LAN. The event lasted for
seven minutes and two seconds, from 10:22:12 to 10:29:14
(UTC time) before the switch interface was disconnected. This event caused some peers located at AMS-IX to lose their
BGP session. In [37], the event was studied using traceroutes,
by looking at the percentage of paths seeing AMS-IX peering
LAN in their traceroute over time. However changes in the
IP paths often result in changes in the delay. The models
were learned, and the changepoints extracted, using the ping
measurements corresponding to the same origin-destination
pairs, provided by RIPE NCC. In addition to this article, we provide interactive notebooks
to document and demonstrate the API, and compare various
statistical models. Links to interactive Google Colab sessions,
as well as the notebooks source and code to facilitate the
usage of the API are provided on GitHub [38]. B. RIPE ATLAS TRENDS API Change frequency between the 9th and the 10th of
April 2018 for the 60k pairs that saw DE-CIX Frankfurt in their traceroutes
the day before. TABLE 2. Endpoints of the Atlas Trends API. FIGURE 15. RIPE Atlas Trends API sample JSON output. TABLE 2. Endpoints of the Atlas Trends API. FIGURE 17. Change frequency between the 9th and the 10th of
April 2018 for the 60k pairs that saw DE-CIX Frankfurt in their traceroutes
the day before. measurements are more difficult to exploit due to the anycast
nature of DNS root servers. In order to detect anomalous events in those infrastruc-
tures, we propose to aggregate the change points learned
from each time series individually, so as to obtain a state-
change frequency, which represents the number of state
changes in a given time frame over all the origin-destination
pairs considered. One problem is the selection of those
origin-destination pairs. One could imagine learning the
model for all the origin-destination pairs available in Atlas,
or a large subset, such as anchoring mesh measurements
(160k origin-destination pairs), and then looking for events
in state-change frequency. However, preliminary experimen-
tation shows that the signal obtained is too noisy and
requires a lot of manual processing to find relevant events. Instead, we propose to monitor each infrastructure individ-
ually, by considering only the origin-destination pairs for
which the infrastructure has been seen in recent traceroute
measurements. FIGURE 15. RIPE Atlas Trends API sample JSON output. FIGURE 15. RIPE Atlas Trends API sample JSON output. FIGURE 16. Change frequency on the 13th of May 2015 for the 20k pairs
that saw AMS-IX in their traceroutes the day before. FIGURE 16. Change frequency on the 13th of May 2015 for the 20k pairs
that saw AMS-IX in their traceroutes the day before. pressed, and missing results are explicitly inserted). The
/trends endpoint returns the minimum RTT and the
associated segmentation. For example, the URL https://
trends.atlas.ripe.net/api/v1/trends/1437285/6222/?start=2018
-05-02&stop=2018-05-10 gives the segmentation in Figure 7
(it should take less than 10 seconds to segment one week of
data). A summary of the time series, as shown in Figure 15,
can also be requested by appending /summary to the path. Start and stop time are specified as YYYY-MM-DDTHH:MM
where THH:MM is optional and defaults to the start of the day. pressed, and missing results are explicitly inserted). The
/trends endpoint returns the minimum RTT and the
associated segmentation. VI. CONCLUSION VI. CONCLUSION In this paper we have shown that the HDP-HMM model,
a hidden Markov chain model with a potentially infinite
number of states, is a very promising method for analyz-
ing RTT time series over the Internet on long time scales
(hours to weeks). We have reviewed the principles of this
model that produce accurate segmentation of time series and
identification of hidden states. Unlike black box approaches,
the HDP-HMM provides some explainable parameters that
can be used as input in different network management tasks
such as the choice of routes, QoS prediction, or optimization
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A novel single variant in the MEFV gene causing Mediterranean fever and Behçet’s disease: a case report
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© The Author(s). 2018 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. CASE REPORT Open Access Abstract Background: Familial Mediterranean fever is an autoinflammatory disease of unknown etiology, characterized clinically
by recurrent attacks of sudden-onset fever with arthralgia and/or thoracoabdominal pain and pathogenetically
by autosomal recessive inheritance due to a mutation in the MEFV gene. Behçet’s disease is an inflammatory
disease characterized by recurrent oral and genital aphthous ulcerations, uveitis, and skin lesions. Preliminarily,
our literature review suggested that patients with familial Mediterranean fever who also have Behçet’s disease
have only a single mutated familial Mediterranean fever gene. The MEFV gene mutation responsible for
familial Mediterranean fever is probably a susceptibility factor for Behçet’s disease, particularly for patients
with vascular involvement, and both disorders can occur concurrently in a patient, as in the present case. Case presentation: A 10-year-old girl of Moroccan origin presented to our institution for genetic consultation for
genetic testing of the MEFV gene. She had fever associated with abdominal and diffuse joint pain in addition to
headache. These symptoms have oriented pediatricians to familial Mediterranean fever. The evolution was marked by
Behçet’s syndrome symptoms. Sanger sequencing followed by complete exome sequencing analysis of the MEFV gene
for the proband mutation revealed a novel variant. We conclude that the novel single variant c.2078 T > A (p.Met693Lys)
could be responsible for the association of familial Mediterranean fever and Behçet’s disease. Conclusion: To the best of our knowledge, this is the first report of a new variant in exon 10 of the MEFV gene in
a Moroccan family. This novel variant should be listed in the MEFV sequence variant databases. rds: MEFV gene, Familial Mediterranean fever, Behçet’s disease, FMF-BD coexistence, Case report amyloidosis, which can be prevented by the daily and
lifelong administration of colchicine therapy [3]. Behçet’s
disease (BD) is a complex chronic and relapsing inflam-
matory disorder extensively described in young adults
from East Asian and Mediterranean countries. Its essential
manifestations are oral and genital ulcerations, folliculitis,
erythema nodosum, and uveitis. The presence of vasculitis
worsens the prognosis, with the potential to introduce
life-threatening complications such as thrombophlebitis,
arterial aneurysms, and occlusion [4]. The etiology of
BD is unknown. A higher frequency of MEFV muta-
tions in patients with BD with respect to their ethni-
city
has
previously
been
demonstrated
[5]. Other
groups have confirmed these data and found an asso-
ciation between the presence of MEFV mutations and
the severity of vasculitis. A novel single variant in the MEFV gene
causing Mediterranean fever and Behçet’s
disease: a case report Maria Zerkaoui1,2*, Fatima Zahra Laarabi2, Yousra Ajhoun3, Bouchra Chkirate4 and Abdelaziz Sefiani1,2 Maria Zerkaoui1,2*, Fatima Zahra Laarabi2, Yousra Ajhoun3, Bouchra Chkirate4 and Abdelaziz Se Zerkaoui et al. Journal of Medical Case Reports (2018) 12:53
DOI 10.1186/s13256-017-1552-4 Zerkaoui et al. Journal of Medical Case Reports (2018) 12:53
DOI 10.1186/s13256-017-1552-4 * Correspondence: maria.zerkaoui@gmail.com
1Human Genomic Centre, Faculty of Medicine and Pharmacy, Mohammed V
University, Rabat, Morocco
2Department of Medical Genetics, National Institute of Health, Rabat,
Morocco
Full list of author information is available at the end of the article Case presentation In 2015, we received in genetic consultation a consanguin-
eous 10-year-old Moroccan girl, the second of three sib-
lings, for genetic testing of the MEFV gene (Fig. 1). At
admission, the patient had a periodic fever with abdominal
pain, mucocutaneous symptoms (pseudo erysipelas), vom-
itus, and joint pain (monoarthritis) in addition to head-
ache. Pediatricians had excluded acute rheumatic fever on
the basis of normal levels of antistreptolysin O and normal
echocardiographic results. During the treatment, the pa-
tient demonstrated an intolerance to colchicine with diar-
rhea and abdominal pain, so the response to colchicine
was not determined. We performed deoxyribonucleic acid
(DNA) analysis. Initially, we excluded recurrent mutations
in exons 2 and 10. Molecular testing of the entire MEFV
gene revealed the novel variant c.2078 T > A, p.M693K,
predicted as damaging and deleterious by the SIFT and
PROVEAN tools (Fig. 2). This result pointed to a diagno-
sis of FMF. Fig. 2 Electrophoregrams of the novel heterozygous missense mutation
at the 2078 nucleotide position of the MEFV gene. a (red arrow) Normal
sequence of unaffected mother and heterozygous mutation c.2078 T > A
(p.Met693Lys) of unaffected father. b (red arrow) Heterozygous mutation
c.2078 T > A (p.Met693Lys) of our patient, a Moroccan girl presenting with
coexistence of familial Mediterranean fever and Behçet’s disease junction and the sigmoid sinus on the left and right
sides. The patient’s clinical history had been taken over. For a few months, the patient was showing one episode
of aphthosis in the buccal mucosa as well as urogenital
ulcers. She was given a bolus of intravenous methylpred-
nisolone for 3 consecutive days, followed by oral pred-
nisone; simultaneously, we administered intravenous
anticoagulation (enoxaparin sodium) followed by oral
acenocoumarol to maintain an international normalized
ratio between 2 and 3. Resolution of the thrombus was
achieved by the time of the 6-month follow-up visit. Thrombophlebitis of the left peroneal vein visualized by
Doppler echography appeared 2 weeks after starting
medical treatment. An ophthalmologic examination re-
vealed papillary edema degree 2 with scleromalacia. We
reconsidered the diagnosis according to the established
Tel-Hashomer clinical criteria and the International
Study Group for Behçet Disease classification criteria for
diagnosis of BD [8]. According to these criteria, the
young girl had FMF-BD. We completed the genetic ana-
lysis of her parents. Her mother was normal, and her
father was heterozygous for the same mutation but
remained asymptomatic. Case presentation Meanwhile, the child’s condition had worsened 2 weeks
after her admission; high fever had persisted through the
prior week and was associated with intense headache
and abdominal pain that were relieved with opioids. This
severity is atypical in FMF. The laboratory data revealed
an elevated level of C-reactive protein, although the
levels of white blood cells were within normal range and
antiphospholipid
antibodies
were
negative. Cerebral
angiography showed cerebral venous thrombosis of the Fig. 1 Pedigree of the patient’s family Background Familial Mediterranean fever (FMF) is an ethnically re-
stricted autosomal recessive disease, usually caused by
loss-of-function mutations in the MEFV gene on the
short arm of chromosome 16 [1]. It is the most common
genetic disease in the Eastern Mediterranean Basin, af-
fecting in particular Armenians, Turks, North Africans,
Ashkenazi Jews, and Arabs. FMF is characterized by re-
current episodes of fever accompanied by peritonitis,
pleuritis, arthritis, or erysipelas-like erythema [2]. The
most severe complication is the development of renal * Correspondence: maria.zerkaoui@gmail.com
1Human Genomic Centre, Faculty of Medicine and Pharmacy, Mohammed V
University, Rabat, Morocco
2Department of Medical Genetics, National Institute of Health, Rabat,
Morocco
Full list of author information is available at the end of the article * Correspondence: maria.zerkaoui@gmail.com
1Human Genomic Centre, Faculty of Medicine and Pharmacy, Mohammed V
University, Rabat, Morocco
2Department of Medical Genetics, National Institute of Health, Rabat,
Morocco
Full list of author information is available at the end of the article Zerkaoui et al. Journal of Medical Case Reports (2018) 12:53 Page 2 of 4 Fig. 2 Electrophoregrams of the novel heterozygous missense mutation
at the 2078 nucleotide position of the MEFV gene. a (red arrow) Normal
sequence of unaffected mother and heterozygous mutation c.2078 T > A
(p.Met693Lys) of unaffected father. b (red arrow) Heterozygous mutation
c.2078 T > A (p.Met693Lys) of our patient, a Moroccan girl presenting with
coexistence of familial Mediterranean fever and Behçet’s disease The
MEFV
gene
encodes
the
pyrin/marenostrin
expressed primarily in the myeloid cell lineage, which
modulates cell susceptibility to apoptosis through the
regulation of inflammation [6]. More than 300 variants
throughout the MEFV gene have been identified, with
M694V, M680I, and E148Q being the most common [7]. We report a case of a Moroccan patient with FMF
who also had BD (FMF-BD) who had been regarded as
having FMF alone 3 years from the onset of disease. Mo-
lecular testing revealed a novel single variant that could
be responsible for the association of FMF and BD. Discussion FMF is a genetic disease characterized by attacks of fever,
arthritis, serositis, and abdominal pain. BD is an inflam-
matory disorder with a genetic basis that is characterized
by oral and genital ulcers, uveitis, pustular erythematous
cutaneous lesions, arthritis, central nervous system in-
volvement, and possible vascular manifestations such as Fig. 1 Pedigree of the patient’s family Page 3 of 4 Zerkaoui et al. Journal of Medical Case Reports (2018) 12:53 Page 3 of 4 venous thrombosis, arteritis, and aneurysms. The diagno-
sis of BD is independently associated with higher inci-
dence of FMF, especially in females, among people of
Arab descent. Watad et al.’s data implied that understating
the differentiation between FMF and BD was not evident
and clear in a real-life population of patients with BD [9]. The clinical picture of FMF is often similar to that of BD
with regard to inflammatory reactions with activation of
neutrophils and therapeutic efficacy of colchicine, and the
possible coexistence of the former should be considered,
particularly in cases with periodic fever as in our patient. Because MEFV encodes the neutrophil protein pyrin, mu-
tations in the MEFV gene may play an important role in
the pathogenesis of BD [10]. One hypothesis is that a re-
duced level of functional pyrin on the one hand, presum-
ably characterizing heterozygosity, and increased demand
for pyrin on the other hand, by activated granulocytes of
BD, may ultimately lead to FMF attacks [11]. simple loss of function of the recessive model of FMF in-
heritance. In some cases, FMF is a dominant condition
with low penetrance and variable disease expression,
presenting not only in homozygous subjects but also in
heterozygous subjects [2]. According to their phenotype classification in the pub-
lication by Balci et al. [14], patients with a classic pheno-
type who were genetically confirmed to have MEFV
mutations are defined as phenotype I. Phenotype III was
defined as those who have no clinical signs but present
with the required genotype, whereas phenotype II refers
to patients who develop amyloid A amyloidosis without
any previous attacks typical of FMF [14]. Therefore, our
patient’s father was asymptomatic but carried the same
novel variant c.2078 T > A in the MEFV gene. FMF is typically inherited in an autosomal recessive
fashion, although there have been rare reports of domin-
ant inheritance with specific heterozygous mutations
and complex alleles [15]. Conclusions We present what is, to the best of our knowledge, the first
identification of an MEFV mutation in a Moroccan family
with associated FMF and BD. To the best of our know-
ledge, this mutation has not been reported before. There-
fore, we think that the c.2078 T > A (p.M693K) mutation
should be listed in the MEFV sequence variants of FMF-
BD databases and may help to increase public awareness
of this rare association in the Moroccan population. To identify specific phenotype/genotype correlations
exactly, the novel mutation and its relationship should
be confirmed by studying homozygous individuals for
this mutation. In 2001, Livneh et al. [11] suggested that about 60% of
patients with FMF-BD have only a single mutated FMF
gene (MEFV). Moreover, in three families in that study,
mutational analysis revealed at least one heterozygous
family member who shared the noncarrier chromosome
with the FMF-BD proband but remained unaffected. This may be explained by the presence of a low-
penetrance mutation, not detected by that team, in non-
coding regions (for example, intron, promoter, enhan-
cer), which causes FMF expression only in a fraction of
carriers [11]. A significant number of patients diagnosed
with FMF show only a single mutation despite sequen-
cing of the entire MEFV genome region or other autoin-
flammatory genes, and this has led to reconsideration of Abbreviations
BD: Behçet’s disease; FMF: Familial Mediterranean fever; FMF-BD: Familial
Mediterranean fever with associated Behçet’s disease Discussion The parents of our proband
were clinically normal. Genetic analysis of the mother’s
MEFV gene was normal. The father, even if he was un-
affected, carried the proband mutation. y
y
Diagnosis of FMF remains clinical and requires informa-
tion about family history and response to colchicine. Our
patient presented with FMF symptoms of fever, abdominal
pain, joint pain, and erysipelas-like erythema. In the
literature, severe abdominal and chest pain are reported to
be the most typical symptoms, occurring in more than
90% and more than 40% of patients, respectively. Suggest-
ive of an FMF diagnosis is the presence of a recurrent
erysipelas-like rash on the skin of the inferior limbs [12]. Our patient presented with a recurrent headache, which is
an atypical symptom of FMF. She kept having headaches,
but her pediatricians considered this symptom a conse-
quence of fever. She presented with aphthosis in the
buccal and genital regions, and after investigation, we ob-
jectified cerebral and peroneal vein thrombophlebitis,
symptoms related to BD. It has been reported that the
MEFV genetic mutation responsible for FMF is probably a
susceptibility factor for BD, particularly in cases with vas-
cular involvement, and that both disorders can coexist in
a patient, as was the case in our patient [10]. MEFV muta-
tions might be a more crucial genetic factor in female pa-
tients with vascular involvement [13]. There is good evidence that position M694 of the
pyrin molecule is critically important for its normal
function. The p.M694 is located in the putative binding
site of caspase-1, and the substitution or deletion of this
residue may interfere with the inhibitory interaction
between pyrin and caspase-1 and thus promote interleukin-
1β generation [16]. This reasoning could be applied to the
p.M693K mutation of the pyrin molecule found in our pa-
tient and thus highly suggests its pathogenic effect. Our pa-
tient carried a single mutated allele of the MEFV gene that
could be responsible for the coexistence of FMF and BD. Competing interests Competing interests
The authors declare that they have no competing interests. The authors declare that they have no competing interests. 16.
Rowczenio DM, Iancu DS, Trojer H, Gilbertson JA, Gillmore JD, Wechalekar
AD, Tekman M, Stanescu HC, Kleta R, Lane T, et al. Autosomal dominant
familial Mediterranean fever in Northern European Caucasians associated
with deletion of p.M694 residue—a case series and genetic exploration.
Rheumatology (Oxford). 2017;56:209–13. Availability of data and materials
Not applicable. 13. Cattan D. MEFV mutation carriers and diseases other than familial
Mediterranean fever: proved and non-proved associations; putative
biological advantage. Curr Drug Targets Inflamm Allergy. 2005;4(1):105–12. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations. Author details
1 1Human Genomic Centre, Faculty of Medicine and Pharmacy, Mohammed V
University, Rabat, Morocco. 2Department of Medical Genetics, National
Institute of Health, Rabat, Morocco. 3Department of Ophthalmology,
Mohammed V Military Teaching Hospital, Mohammed V University, Rabat,
Morocco. 4Pediatric Department IV, Children’s Hospital, University
M h
d V R b t M Mohammed V, Rabat, Morocco. Received: 24 March 2017 Accepted: 23 December 2017 Consent for publication Written informed consent was obtained from the patient’s legal guardian(s)
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. 15.
Stoffels M, Szperl A, Simon A, Netea MG, Plantinga TS, van Deuren M,
Kamphuis S, Lachmann HJ, Cuppen E, Kloosterman WP, et al. MEFV
mutations affecting pyrin amino acid 577 cause autosomal dominant
autoinflammatory disease. Ann Rheum Dis. 2014;73(2):455–61. Israeli patients suffering from familial Mediterranean fever and Behcet’s
disease (FMF-BD). Eur J Hum Genet. 2001;9(3):191–6.
12.
Rigante D, Vitale A, Lucherini OM, Cantarini L. The hereditary autoinflammatory
disorders uncovered. Autoimmun Rev. 2014;13(9):892–900.
13.
Cattan D. MEFV mutation carriers and diseases other than familial
Mediterranean fever: proved and non-proved associations; putative
biological advantage. Curr Drug Targets Inflamm Allergy. 2005;4(1):105–12.
14.
Balci B, Tinaztepe K, Yilmaz E, Gucer S, Ozen S, Topaloglu R, Besbas N,
Ozguc M, Bakkaloglu A. MEFV gene mutations in familial Mediterranean
fever phenotype II patients with renal amyloidosis in childhood: a
retrospective clinicopathological and molecular study. Nephrol Dial
Transplant. 2002;17(11):1921–3.
15.
Stoffels M, Szperl A, Simon A, Netea MG, Plantinga TS, van Deuren M,
Kamphuis S, Lachmann HJ, Cuppen E, Kloosterman WP, et al. MEFV
mutations affecting pyrin amino acid 577 cause autosomal dominant
autoinflammatory disease. Ann Rheum Dis. 2014;73(2):455–61.
16.
Rowczenio DM, Iancu DS, Trojer H, Gilbertson JA, Gillmore JD, Wechalekar
AD, Tekman M, Stanescu HC, Kleta R, Lane T, et al. Autosomal dominant
familial Mediterranean fever in Northern European Caucasians associated
with deletion of p.M694 residue—a case series and genetic exploration.
Rheumatology (Oxford). 2017;56:209–13. Authors’ contributions 14. Balci B, Tinaztepe K, Yilmaz E, Gucer S, Ozen S, Topaloglu R, Besbas N,
Ozguc M, Bakkaloglu A. MEFV gene mutations in familial Mediterranean
fever phenotype II patients with renal amyloidosis in childhood: a
retrospective clinicopathological and molecular study. Nephrol Dial
Transplant. 2002;17(11):1921–3. MZ was responsible for data analysis, literature review, and manuscript preparation. FZL was responsible for data analysis and carried out the molecular
genetic studies. YA provided medical care. BC provided medical care. AS reviewed the manuscript. All authors read and approved the final
manuscript. Ethics approval and consent to participate
Not applicable. Funding 12. Rigante D, Vitale A, Lucherini OM, Cantarini L. The hereditary autoinflamma
disorders uncovered. Autoimmun Rev. 2014;13(9):892–900. 12. Rigante D, Vitale A, Lucherini OM, Cantarini L. The hereditary autoinflammator
disorders uncovered. Autoimmun Rev. 2014;13(9):892–900. Availability of data and materials
Not applicable. Acknowledgements
W
h
k h Acknowledgements
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Pras E, Zaks N, Padeh S, Kastner DL, et al. A single mutated MEFV allele in 11. Livneh A, Aksentijevich I, Langevitz P, Torosyan Y, G-Shoham N, Shinar Y,
Pras E, Zaks N, Padeh S, Kastner DL, et al. A single mutated MEFV allele in
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Ball Tracking and Trajectory Prediction for Table-Tennis Robots
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Sensors
| 2,020
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Received: 26 November 2019; Accepted: 30 December 2019; Published: 7 January 2020 Abstract: Sports robots have become a popular research topic in recent years. For table-tennis
robots, ball tracking and trajectory prediction are the most important technologies. Several methods
were developed in previous research efforts, and they can be divided into two categories: physical
models and machine learning. The former use algorithms that consider gravity, air resistance,
the Magnus effect, and elastic collision. However, estimating these external forces require high
sampling frequencies that can only be achieved with high-efficiency imaging equipment. This study
thus employed machine learning to learn the flight trajectories of ping-pong balls, which consist
of two parabolic trajectories: one beginning at the serving point and ending at the landing point
on the table, and the other beginning at the landing point and ending at the striking point of the
robot. We established two artificial neural networks to learn these two trajectories. We conducted
a simulation experiment using 200 real-world trajectories as training data. The mean errors of the
proposed dual-network method and a single-network model were 39.6 mm and 42.9 mm, respectively. The results indicate that the prediction performance of the proposed dual-network method is better
than that of the single-network approach. We also used the physical model to generate 330 trajectories
for training and the simulation test results show that the trained model achieved a success rate of
97% out of 30 attempts, which was higher than the success rate of 70% obtained by the physical
model. A physical experiment presented a mean error and standard deviation of 36.6 mm and
18.8 mm, respectively. The results also show that even without the time stamps, the proposed method
maintains its prediction performance with the additional advantages of 15% fewer parameters in the
overall network and 54% shorter training time. Keywords: table-tennis robots; ball tracking and trajectory prediction; artificial neural networks sensors
Article
Ball Tracking and Trajectory Prediction for
Table-Tennis Robots
Hsien-I Lin 1,*
, Zhangguo Yu 2 and Yi-Chen Huang 1
1
Graduate Institute of Automation Technology, National Taipei University of Technology, No. 1, Sec. 3,
Zhongxiao E. Rd., Taipei 10608, Taiwan; john6402j@gmail.com
2
School of Mechatronical Engineering, Beijing Institute of Technology, Beijing 100081, China; yuzg@bit.edu
*
Correspondence: sofin@ntut.edu.tw; Tel.:+886-2-27713343
Received: 26 November 2019; Accepted: 30 December 2019; Published: 7 January 2020
A physical experiment presented a mean error and standard deviation of 36.6 mm an
18.8 mm, respectively. The results also show that even without the time stamps, the proposed metho sensors sensors Ball Tracking and Trajectory Prediction for
Table-Tennis Robots Hsien-I Lin 1,*
, Zhangguo Yu 2 and Yi-Chen Huang 1 1
Graduate Institute of Automation Technology, National Taipei University of Technology, No. 1, Sec. 3,
Zhongxiao E. Rd., Taipei 10608, Taiwan; john6402j@gmail.com
2
School of Mechatronical Engineering, Beijing Institute of Technology, Beijing 100081, China; yuzg@bit.edu.cn
*
Correspondence: sofin@ntut.edu.tw; Tel.:+886-2-27713343 Sensors 2020, 20, 333; doi:10.3390/s20020333 sensors
Article
Ball Tracking and Trajectory Prediction for
Table-Tennis Robots
Hsien-I Lin 1,*
, Zhangguo Yu 2 and Yi-Chen Huang 1
1
Graduate Institute of Automation Technology, National Taipei University of Technology, No. 1, Sec. 3,
Zhongxiao E. Rd., Taipei 10608, Taiwan; john6402j@gmail.com
2
School of Mechatronical Engineering, Beijing Institute of Technology, Beijing 100081, China; yuzg@bit.edu
*
Correspondence: sofin@ntut.edu.tw; Tel.:+886-2-27713343
Received: 26 November 2019; Accepted: 30 December 2019; Published: 7 January 2020
sensors
Article
Ball Tracking and Trajectory Prediction for
Table-Tennis Robots
Hsien-I Lin 1,*
, Zhangguo Yu 2 and Yi-Chen Huang 1
1
Graduate Institute of Automation Technology, National Taipei University of Technology, No. 1, Sec. 3,
Zhongxiao E. Rd., Taipei 10608, Taiwan; john6402j@gmail.com
2
School of Mechatronical Engineering, Beijing Institute of Technology, Beijing 100081, China; yuzg@bit.edu
*
Correspondence: sofin@ntut.edu.tw; Tel.:+886-2-27713343
Received: 26 November 2019; Accepted: 30 December 2019; Published: 7 January 2020
sensors
Article
Ball Tracking and Trajectory Prediction for
Table-Tennis Robots
Hsien-I Lin 1,*
, Zhangguo Yu 2 and Yi-Chen Huang 1
1
Graduate Institute of Automation Technology, National Taipei University of Technology, No. 1, Sec. 3,
Zhongxiao E. Rd., Taipei 10608, Taiwan; john6402j@gmail.com
2
School of Mechatronical Engineering, Beijing Institute of Technology, Beijing 100081, China; yuzg@bit.edu. *
Correspondence: sofin@ntut.edu.tw; Tel.:+886-2-27713343
Received: 26 November 2019; Accepted: 30 December 2019; Published: 7 January 2020
Abstract: Sports robots have become a popular research topic in recent years. For table-tenn
robots, ball tracking and trajectory prediction are the most important technologies. Several metho
were developed in previous research efforts, and they can be divided into two categories: physic
models and machine learning. The former use algorithms that consider gravity, air resistanc
the Magnus effect, and elastic collision. However, estimating these external forces require hig
sampling frequencies that can only be achieved with high-efficiency imaging equipment. This stud
thus employed machine learning to learn the flight trajectories of ping-pong balls, which consi
of two parabolic trajectories: one beginning at the serving point and ending at the landing poi
on the table, and the other beginning at the landing point and ending at the striking point of th
robot. We established two artificial neural networks to learn these two trajectories. We conducte
a simulation experiment using 200 real-world trajectories as training data. The mean errors of th
proposed dual-network method and a single-network model were 39.6 mm and 42.9 mm, respective
The results indicate that the prediction performance of the proposed dual-network method is bett
than that of the single-network approach. We also used the physical model to generate 330 trajectori
for training and the simulation test results show that the trained model achieved a success rate
97% out of 30 attempts, which was higher than the success rate of 70% obtained by the physic
model. 1. Introduction The processing speed of the vision system is another key factor because it indirectly affects
prediction of the direction of the ball, particularly in prediction methods using physical models. Liu [8]
proposed an onboard stereo camera system to estimate ping-pong ball trajectories in real time under
the asynchronous observations from different cameras. Graphics processing units (plGPU) have
become popular computer components in the recent trend of deep learning in image classification
because they make the graphics card less dependent on the central processing unit (CPU) and improve
the performance of graphics computing. Lampert et al. [9] employed an NVIDIA GeForce GTX280
graphics card and a CUDA framework to accelerate image processing and used four cameras to
establish 3D space. Their system needed only 5 ms to process a 3D location. Furthermore, German
company Nerian launched a real-time 3D stereo vision core that uses a field-programmable gate
array (FPGA) as the hardware for parallel computation [10]. It can complete more tasks per clock
cycle, send the computation results to a personal computer, and presents good performance in object
recognition and 3D computation. Several studies used camera arrays to realize high-speed real-time
object tracking and trajectory prediction. Zhang et al. [11] used a total of three cameras, one of which
was a pan-tilt camera with a resolution of 640 × 480 pixels; its sampling frequency could reach less
than 5 ms, and it could simultaneously follow a flying ball and analyze its status. Accurate prediction of the ball trajectory is vital to the capacity of the robot to hit the ball. Existing prediction methods can be divided into two categories: physical models and machine learning. Physical models assess the external forces affecting the trajectory of the ball, such as constant gravity
and air resistance. Balls with self-rotation are subject to a lateral force perpendicular to the plane
formed by the angular velocity vector and the motion velocity vector of the rotation. This force, known
as the Magnus effect, causes deflection in the flight trajectory of the ball. Ping-pong balls are not
heavy, so the deflection is greater than that it would be with heavier objects [12,13]. Wang et al. [14]
proposed a physical model to predict ball trajectories of topspin, backspin, rightward spin, leftward
spin, and combined spin. Huang et al. [15] proposed a physical model that considers the self-rotation
of the ball. 1. Introduction Recent years have seen the gradual maturing of sensory, machine vision, and control technology
in smart robots. Several domestic and foreign studies explored the applicability of robots to sports. KUKA AG Robotics once made a commercial in which one of their robots played table tennis against
a human, and Omron once gave a demonstration with one of their suspended robotic arms playing
table tennis against a human at an automation exhibition. Table-tennis robots use a wide range
of technologies, including object recognition, object tracking, 3D reconstruction, object trajectory
prediction, robot motion planning, and system integration. This as well as the fact that they are easy to
showcase attracted the attention of many researchers. y
A ping-pong ball trajectory system combines vision, 3D space, and prediction algorithms, none of
which are dispensable. The vision system must be able to detect and position the ball [1,2]. The data
captured by cameras are two-dimensional (2D), so three-dimensional (3D) data cannot be derived by Sensors 2020, 20, 333; doi:10.3390/s20020333 www.mdpi.com/journal/sensors 2 of 23 Sensors 2020, 20, 333 simply searching for the locations of object pixels. Wei et al. [3] proposed a method that uses a single
camera to calculate object positions. In addition to using image recognition to locate the current
pixels of the ball, they also used the shadow of the flying ball on the table to triangulate the spatial
location of the ball. However, it is difficult to detect the shadow of a sphere, and the sources of light in
general environments are complex and unpredictable. Their proposed approach was only useful if
there was only a single clear light source in the environment with no sunlight or other light sources. The installation of two or more cameras can be used to establish stereopsis. Detection of an object from
multiple perspectives can increase the dimensions of the image data and enable simple calculations of
3D data. Refs. [4,5] both adopted two cameras to track the ball in their vision system. To increase the
visual coverage and accuracy of the vision system, Chen et al. [6] installed two high-speed cameras
above the robot and above the opponent, which amounted to four cameras covering the entire table. Yang et al. [7] used six cameras to cover the table (three on each side) to achieve high precision for
every possible location of the ball. 1. Introduction It uses a real-time vision system to obtain a series of trajectory points and a camera system
combining DSP and FPGA and fits the 3D data into a quadratic polynomial, which is then used to
obtain the current flying velocity. In their experiments, their proposed method had good predictive
abilities when the sampling time was 1 ms. However, if the consecutive trajectory points were not
dense enough or if the sampling frequency was too low, the accuracy of current velocity estimates is
affected. Inputting these estimates into complex formula calculations would then increase distortion
in the final results. Zhang et al. [11] used two high-resolution cameras to analyze the self-rotation of
the ball. Zhao et al. [16] used an ultrahigh-speed camera to analyze the collision between a ping-pong
ball and the table and developed a physical model for self-rotation and collision effects. Ball status
estimates can also be obtained using various filters, such a fuzzy filter [17], an extended Kalman
filter [18], and an unscented Kalman filter [19]. Other studies presented analyses of the aerodynamics
and the friction between ping-pong balls and tables [20,21]. 3 of 23 Sensors 2020, 20, 333 Machine learning has also tended towards table-tennis research in recent years. Some of the
table-tennis robots developed in these studies employed locally weighted regression algorithms to
learn hitting movements [22,23]. In machine learning algorithms, machines analyze data to find
regularities and then use the regularities to make predictions of unknown data. Zhao et al. formulated
the likelihood of ping-pong ball motion state as a Gaussian Mixture Model (GMM) [24]. Deng et al. [25]
proposed a ball-size likelihood to estimate the ball position. Payeur et al. [26] used an artificial neural
network (ANN) to learn and predict the trajectories of moving objects; however, they merely performed
simulation experiments of simple trajectories and did not develop a novel vision system or robot. Nakashima et al. Nakashima et al. [27] used a back-propagation artificial neural network (BPANN) to
learn striking points. The inputs are the initial location and velocity difference of the ball in a free-fall
model, and the outputs are the striking point and displacement between striking points estimated
using simple physics. Although their simulation results were good, the model requires 20 trajectory
points for fitting, which implies that the sampling frequency of the vision system must be high. Zhang et al. 1. Introduction [3] also made an attempt with four initial trajectory points and time stamps as the inputs
of the network and the location and velocity of the striking point as the outputs. y
g p
p
Our objective is to track and predict ping-pong ball trajectories for a robot to hit the ball. Due
to the facts that physical models for ball flight prediction require advanced vision equipment and
that most physical models are fairly complex, we adopted machine learning to achieve prediction of
ping-pong ball trajectories with limited equipment. To achieve better prediction, this study included
a ball tracking system, 3D reconstruction, and ball trajectory prediction. The novelty of this work
is that the flight trajectory between the serving point and the end of the robotic arm was viewed as
two parabolas and two separate ANNs were used to learn these two parabolas. The ball trajectory
prediction strategy proposed in this study makes the following contributions: •
We propose an ANN-based approach to learn historical trajectory data, thereby doing away with
the need for a complex flight trajectory model. •
We propose an ANN-based approach to learn historical trajectory data, thereby doing away with
the need for a complex flight trajectory model. •
We propose an ANN-based approach to learn historical trajectory data, thereby doing away w p
g
j
y
•
The proposed method can swiftly predict where the robot should strike the ball based on the
l
d
f
l
f
f
d
h
b
h
b •
The proposed method can swiftly predict where the robot should strike the ball based on the ball
location data from only a few instants after it is served, thereby giving the robot time to react. y
,
y g
g
•
The inputs of ANNs are generally accompanied by time stamp data. We verified that removing
the time stamp data reduces the parameter demand of the entire network and greatly shortens
network training time. •
The inputs of ANNs are generally accompanied by time stamp data. We verified that removing
the time stamp data reduces the parameter demand of the entire network and greatly shortens
network training time. Figure 1 shows the flow of the proposed method. There are three main parts: 3D construction,
ping-pong ball tracking, and ping-pong trajectory prediction. 3D construction and ping-pong ball tracking
are used to obtain the accurate ball current position. 1. Introduction To hit the ball, we propose the dual neural networks
to predict the ball position on the hitting plane. These three parts are explained in Section 2. Ping-Pong Ball Tracking
3D Construction
Image Projection
Calculation of 3D
Location
Ping-Pong Ball
Trajectory Prediction
Selection of Trajectory
Regression Model
Dual Neural Networks
Figure 1. Flow of the proposed method. Selection of Trajectory
Regression Model Figure 1. Flow of the proposed method. The remainder of this paper is organized as follows. Section 2 introduces the framework for 3D
reconstruction, and Section 3 explains the ball trajectory prediction method proposed in this study. Section 4 presents the experiment results, and Section 5 contains our conclusion. 4 of 23 Sensors 2020, 20, 333 2.1. Hardware Setup Figure 2 displays the 3D space system of this study. The vision system comprises three IDS color
industrial cameras with 1.3 megapixel resolution. The FPS of the cameras was set at 169, and each had
to cover most of the table surface within their field of view. The cameras were placed on both sides of
the table: one on the right (camera#1), one on the left (camera#2), and an auxiliary camera (camera#3). The farthest visible distance of the camera system was approximately 220 cm to facilitate the widest
range of tracking. In addition, we noted whether the color of the background would interfere with
tracking. The table-tennis robot was a Stäubli industrial robot arm, which has six degrees of freedom,
repeatability of ±0.02 mm, a reach of 670 mm, and a maximum speed of 8.0 m/s at the endpoint. We used the high-precision Phoenix Technologies Inc. VZ4000v 3D motion capture system, which can
achieve an accuracy of 0.015 mm within a distance of 1.2 m, to analyze 3D errors in the images. Figure 2. 3D reconstruction system of table-tennis robot. Figure 2. 3D reconstruction system of table-tennis robot. Camera synchronization is an important issue in the multi-camera vision system. When computer
CPUs use multithreading, the various threads may not be executed at the same time depending on the
resource allocation decisions of the CPU at the time. Thus, the timing at which the cameras capture
images may not be the same. We used a master camera to send image capture signals. The slave
cameras as well as the master camera wait for the signal. Figure 3 shows the synchronization process
of the camera system. To validate the synchronization process, we collected one hundred trajectories
with synchronized and unsynchronized procedures. Curve fitting was performed for each trajectory,
and then the mean-square error was analyzed. The mean-square errors with synchronized and
unsynchronized procedures were 7.7 mm and 12.1 mm, respectively. The result validated that the
synchronization procedure helped the multi-camera vision system to acquire synchronized images. Camera synchronization is an important issue in the multi-camera vision system. When computer
CPUs use multithreading, the various threads may not be executed at the same time depending on the
resource allocation decisions of the CPU at the time. Thus, the timing at which the cameras capture
images may not be the same. We used a master camera to send image capture signals. 2.1. Hardware Setup The slave
cameras as well as the master camera wait for the signal. Figure 3 shows the synchronization process
of the camera system. To validate the synchronization process, we collected one hundred trajectories
with synchronized and unsynchronized procedures. Curve fitting was performed for each trajectory,
and then the mean-square error was analyzed. The mean-square errors with synchronized and
unsynchronized procedures were 7.7 mm and 12.1 mm, respectively. The result validated that the
synchronization procedure helped the multi-camera vision system to acquire synchronized images. Figure 4 shows the control of the cameras with the sampling time of 0.007 s. Threads 1 to 3 are the
image processing flow for each of the three cameras. When the ball is getting away from camera#1
and camera#2, the 3D position error will increase. Therefore, the auxiliary camera#3 is used to reduce
the error. The timing of using camera#3 is when the x-axis position of the ball is greater than 700 mm
(the robot side is the origin), camera#1 and camera#2 are used; otherwise, camera#1 or camera#2 is
used with the auxiliary camera#3 depending on whether the ball falls to the left or right of the table. Figure 4 shows the control of the cameras with the sampling time of 0.007 s. Threads 1 to 3 are the
image processing flow for each of the three cameras. When the ball is getting away from camera#1
and camera#2, the 3D position error will increase. Therefore, the auxiliary camera#3 is used to reduce
the error. The timing of using camera#3 is when the x-axis position of the ball is greater than 700 mm
(the robot side is the origin), camera#1 and camera#2 are used; otherwise, camera#1 or camera#2 is
used with the auxiliary camera#3 depending on whether the ball falls to the left or right of the table. 5 of 23 Sensors 2020, 20, 333 Figure 3. Synchronization procedure of the multi-camera vision system. Figure 4. Control of the cameras. 2. 3D Reconstruction
We used two industrial cameras to establish a 3D space. Using the pixel locations of the ta
om two perspectives, we positioned the location of the target in the 3D space. The coordinate sys Figure 3 Synchronization procedure of the multi camera vision system Figure 3. Synchronization procedure of the multi-camera vision system. Figure 4. Control of the cameras. Figure 4. Control of the cameras. 2.2. 3D Reconstruction We used two industrial cameras to establish a 3D space. Using the pixel locations of the target
from two perspectives, we positioned the location of the target in the 3D space. The coordinate system
of the cameras was then converted into the coordinate system of the robot arm. In this way, deriving
the 3D location of an object in images from the camera system would also give us the location of
the object in the 3D space. This approach included camera calibration and triangulation. However,
the error increases when the object is further away from the camera system. This is because the pixel
features of objects further away are not as clear and the pixel resolution at this distance has reached its
limits. We will give a complete explanation in Section 2.4. 6 of 23 Sensors 2020, 20, 333 2.3. Ping-Pong Ball Tracking To track the ball, the field of view of the cameras must encompass the entire table, which includes
various complex colors and noise. To simplify image segmentation, we painted the ping-pong ball
blue to distinguish other objects and the background. A Gaussian blur is first applied to the images
captured by the cameras to remove noise and facilitate the subsequent recognition process. HSV color
space conversion is then used to reduce the impact of bright light sources, and then a threshold value
is easily set for the color of the ball to obtain a binary image. To make the target object more complete,
morphological operators erosion and dilation are applied and then the median values of the binary image
are calculated to serve as the ball position. Performing these processes on images with large fields of view
is time-consuming. To save time, we employed the region-of-interest (ROI) operation. Once the camera
tracked the ball, then only the ROI in the images is processed. The FPS was set at 169 in this study. When
an entire image with 1280 × 1024 pixels is subjected to the object recognition procedure, the average
frequency is approximately 50.7 times/s. If an ROI is adopted, then subjecting the ROI (an image of
200 × 200 pixels) to object recognition results in an estimated frequency of 514.5 times/s. Clearly, the ROI
mechanism can significantly increase the efficiency of object recognition. 2.3.1. Image Projection The camera calibration was to obtain pixel scale, intrinsic matrix, and extrinsic matrix of the
camera. Figure 5 shows that camera calibration was conducted using the checkerboard data from
30 images of a 13 × 9 checkerboard with 60 × 60 mm2 at various angles, depths, and locations. Tables 1–4 present the intrinsic and extrinsic parameters of the right and left cameras. Figure 5. Calibration using checkerboard at different positions. Table 1. Intrinsic parameters of the right camera. U Axis
V Axis
Error+
Error−
Focal Length (pix)
1043.25281
1049.54675
2.24798
2.12426
Principal Point (pix)
633.44849
561.99736
2.89133
2.42647
Pixel Error (pix)
0.26777
0.30308 Figure 5. Calibration using checkerboard at different positions. Table 1 Intrinsic parameters of the right camera Figure 5. Calibration using checkerboard at different positions. Figure 5. Calibration using checkerboard at different position
Table 1. Intrinsic parameters of the right camera. Table 1. Intrinsic parameters of the right camera. U Axis
V Axis
Error+
Error−
Focal Length (pix)
1043.25281
1049.54675
2.24798
2.12426
Principal Point (pix)
633.44849
561.99736
2.89133
2.42647
Pixel Error (pix)
0.26777
0.30308 7 of 23 Sensors 2020, 20, 333 Table 2. Intrinsic parameters of the left camera. Table 2. Intrinsic parameters of the left camera. Table 2. Intrinsic parameters of the left camera. U Axis
V Axis
Error+
Error−
Focal Length (pix)
1049.44688
1052.73077
1.52311
1.51341
Principal Point (pix)
597.44982
513.76570
2.18344
1.66587
Pixel Error (pix)
0.23623
0.26618
Table 3. Extrinsic parameters of the right camera. X Axis
Y Axis
Z Axis
Translation vector (mm)
669.420640
−371.402272
2129.031554
Rotation matrix
−0.821611
−0.568894
−0.036249
−0.262626
0.434195
−0.861686
0.505947
−0.698451
−0.506146
Pixel Error (pix)
0.21674
0.33150
Table 4. Extrinsic parameters of the left camera. X Axis
Y Axis
Z Axis
Translation vector (mm)
−90.227708
−381.155522
2279.359066
Rotation matrix
−0.850091
0.526621
0.003993
0.265893
0.435734
−0.859905
−0.454585
−0.729936
−0.510438
Pixel Error (pix)
0.15424
0.32854 Table 3. Extrinsic parameters of the right camera. 2.3.2. Calculation of 3D Location 2.3.2. Calculation of 3D Location 2.3.2. Calculation of 3D Location As shown in Figure 6, the location of the target object can be obtained when it is within view of
the two cameras. P is the location of the object, and OLe f t and ORight denote the respective origins
of the coordinate systems of the left and right cameras. Pl and Pr represent the pixel locations of the
object in the images taken by the left and right cameras. Figure 6 shows that the plane formed by
OLe f t, ORight, and P is defined as an epipolar plane. This characteristic can be used to identify the
physical relationship between the two cameras [28]. Another approach is to assume that Ml
w and Mr
w
are homogeneous matrices converting world coordinates to left and right camera coordinates using
the extrinsic parameters of the cameras, respectively. With Mr
w as an example, formula calculations
produce Mw
r , as shown in Equation (1). Multiplying Ml
w by Mw
r then gives Ml
r, as shown in Equation (2). This is the rotation and translation matrix converting the coordinates in the right camera system to
those in the left camera system. Mw
r =
"
(Rr
w)T
−(Rr
w)T(Tr
w)
01X3
1
#
(1) (1) (Mr
l ) = (Mw
l )(Mw
r )
(2) (2) Figure 6 shows the centers of the left and right cameras both pointed at target P. The vectors
pointing from the centers of the two cameras to the pixel location of the target object are defined as ˜Pl
and ˜Pr, as shown below: Figure 6 shows the centers of the left and right cameras both pointed at target P. 2.3.2. Calculation of 3D Location The vectors
pointing from the centers of the two cameras to the pixel location of the target object are defined as ˜Pl
and ˜Pr, as shown below: ˜Pl =
(x
′
l −clx)slx
(y
′
l −cly)sly
fl
(3) (3) Sensors 2020, 20, 333 8 of 23 ˜Pr =
(x
′
r −crx)srx
(y
′
r −cry)sry
fr
(4) (4) where x
′
l and y
′
l is the pixel location of the object in the left image; clx and cly is the image center of the
left camera; slx and sly are the scale coefficients of the left camera; x
′
r and y
′
r is the pixel location of the
object in the right image; crx and cry is the image center of the right camera, and srx and sry are the
scale coefficients of the right camera. where x
′
l and y
′
l is the pixel location of the object in the left image; clx and cly is the image center of the
left camera; slx and sly are the scale coefficients of the left camera; x
′
r and y
′
r is the pixel location of the
object in the right image; crx and cry is the image center of the right camera, and srx and sry are the
scale coefficients of the right camera. Figure 6. Calculation of 3D location. Figure 6. Calculation of 3D location. In the 3D space, these two vectors intersect at the location of the target object. However, this only
occurs in ideal circumstances. Figure 7 displays the more likely circumstance in which the two
vectors are skew and do not intersect. Thus, we assume that the target object is located at the middle
point of the line segment that is the shortest distance between the two vectors. Based on rigid body
transformation, vector ˜Pr of the right camera can be converted into a vector with regard to the left
camera system using Ml
r, the physical relationship between the two cameras, which is Rl
r and Tl
r. Equation (5) represents the distance to which the vector should extend to reach Pup. Here, we assume
there is an unknown coefficient b. Similarly, Equation (6) represents the distance to which the left
vector should extend to reach Pdown, where we assume there is an unknown coefficient a. 2.3.2. Calculation of 3D Location Pup = (Rl
r)b ˜Pr + Tl
r
(5) Pup = (Rl
r)b ˜Pr + Tl
r
(5)
Pdown = a ˜Pl
(6) (5) Pdown = a ˜Pl
(6) (6) To derive Pmid, we must calculate the directional vector q of the line segment. This vector can be
obtained using the cross product of the left and right vectors, as shown in Equation (7). Please note
that ˜Pr must also undergo coordinate rotation conversion Rl
r, and the unit vector of q is
q
|q|. q = ˜Pl × (Rl
r ˜Pr)
(7) (7) The length of the line segment is unknown, so we suppose that c is the coefficient of the unit
vector of q. Based on basic concepts of vectors, this line segment can be expressed as follows: The length of the line segment is unknown, so we suppose that c is the coefficient of the unit
vector of q. Based on basic concepts of vectors, this line segment can be expressed as follows: Pup = Pdown + c q
|q|
(8) (8) 9 of 23 Sensors 2020, 20, 333 Further derivation gives Further derivation gives Further derivation gives a ˜Pl + c( ˜Pl × Rl
r ˜Pr)
| ˜Pl × Rlr ˜Pr| = b(Rl
r) ˜Pr + Tl
r
(9) (9) Suppose that Suppose that A =
h ˜Pl
−Rl
r ˜Pr
q
|q| 3×3
i Equation (9) can be simplified into Equation (10): Equation (9) can be simplified into Equation (10): Equation (9) can be simplified into Equation (10): Equation (9) can be simplified into Equation (10):
a
b
c
= A−1Tl
r. (10)
a
b
= A−1Tl
r. (10) (10) We can then derive coefficients a, b, and c, and use a and c to calculate Pmid, as shown in
Equation (12): Pmid = a ˜Pl + 1
2(c q
|q|)
(11) (11) Here, Pmid is the 3D location of the target object with regard to the left camera coordinate system. Using Mw
l , we can convert the coordinates from the left camera system to the world coordinate system,
as shown in Equation (12). This matrix can be reversely obtained using extrinsic parameter Ml
w of the
left camera. Pw = Mw
l P id
(12) Pw = Mw
l Pmid
(12) (12) Pw = Mw
l Pmid
Figure 7. Two vectors in a skew relationship. Figure 7. 2.3.2. Calculation of 3D Location Two vectors in a skew relationship. 3. Ping-Pong Ball Trajectory Prediction If a table-tennis robot wants to hit the ball, it must be able to accurately predict where the ball is
flying. When people play table tennis, the ball is subjected to various external forces that impact its
flight direction and velocity, including air resistance, the Magnus effect, gravity, and the rebound force
of collision. Thus, calculating the various physical forces with precision and then predicting the flight
trajectory is difficult. In this section, we present a machine learning approach that enables robots to
predict trajectories. In this section, we demonstrate that the coefficients of a polynomial regression
model are advantageous to represent and predict ball trajectories. g
p
p
j
Figure 10 shows the flight trajectories of a ping-pong ball. The flight trajectories are divided into
two parabolas P1 and P2. The first ANN is responsible for learning the parabola P1 in the figure, with
the anterior positions as the input and the regression coefficient of P1 as the output. The second ANN
learns parabola P2, with the regression coefficient of P1 as the input and the regression coefficient of P2
as the output. The trajectory coefficients above are the parameters of their mathematical expressions. Once the vision system detects the anterior positions, the system will immediately derive the coefficient
P2 of the trajectory after the ball’s landing point on the table and then calculate a designated striking
point. Suppose fx, fy, and fz denote the mathematical regression formulas of trajectory P2 along the
x, y, and z axes. Using fx(t), the timing t of the designated striking point (x axis) is first derived. The resulting t is then substituted into fy and fz to obtain the y and z coordinates of the striking point. Since the regression model should be chosen to predict the ball trajectory, we evaluate several
models such as exponential, Fourier, Gaussian, and polynomial curve fitting methods in Section 3.1. The result shows that polynomial curve fitting is the most suitable model to predict the ball trajectory. In Section 3.2, we also theoretically verify the existence of the relationship between the polynomial
coefficients and ball trajectory. However, Section 3.2 shows that curve fitting is highly sensitive to
noise in the input data. Thus, we propose two ANNs to model the relationship in Section 3.4. Figure 10 shows the flight trajectories of a ping-pong ball. The flight trajectories are divided into
two parabolas P1 and P2. 2.4. Estimation Error Analysis 2.4. Estimation Error Analysis 2.4. Estimation Error Analysis Even using the algorithm above to calculate the 3D location of the target object, errors still exist in
the data, and they increase with the distance between the target object and the origins of the camera
coordinate systems. This section analyzes the errors in the use of the camera systems to calculate the
locations of the target object. Here, we used a PTI motion capture system to serve as the control group
for the 3D locations obtained using the three cameras. Figure 8 exhibits the 25 trackers that we used. The world coordinates of the PTI motion capture system overlapped those of the camera systems,
meaning that the camera systems and the PTI motion capture system had the same world coordinates. To calculate the errors, we employed two stereopsis groups, one using the right camera (camera#1)
and the left camera (camera#2) to calculate the 3D location of the PTI tracker and the other using the 10 of 23 Sensors 2020, 20, 333 left camera (camera#2) and the auxiliary camera (camera#3). Using cubic interpolation, we derived
a new error distribution graph. Figure 9 displays the distributions of the errors interpolated using
cubic polynomials in the two stereopsis groups. As can be seen, the errors increase with the distances
between the left and right cameras and the trackers and is highest at Tracker No. 21, where the error is
40 mm. With the left camera and the auxiliary camera, the two cameras are closer to Tracker No. 21,
so the error reduces to approximately 25 mm. Figure 8. Locations of 25 trackers. Figure 8. Locations of 25 trackers. (a)
(b)
Figure 9. Distributions of the errors (a) using the right camera (camera#1) and the left camera
(camera#2); (b) using the left camera (camera#2) and the auxiliary camera (camera#3). Figure 8. Locations of 25 trackers. (a) (b) (b) Figure 9. Distributions of the errors (a) using the right camera (camera#1) and the left camera
(camera#2); (b) using the left camera (camera#2) and the auxiliary camera (camera#3). 11 of 23 11 of 23 Sensors 2020, 20, 333 3. Ping-Pong Ball Trajectory Prediction The first ANN is responsible for learning the parabola P1 in the figure, with
the anterior positions as the input and the regression coefficient of P1 as the output. The second ANN
learns parabola P2, with the regression coefficient of P1 as the input and the regression coefficient of P2
as the output. The trajectory coefficients above are the parameters of their mathematical expressions. Once the vision system detects the anterior positions, the system will immediately derive the coefficient
P2 of the trajectory after the ball’s landing point on the table and then calculate a designated striking
point. Suppose fx, fy, and fz denote the mathematical regression formulas of trajectory P2 along the
x, y, and z axes. Using fx(t), the timing t of the designated striking point (x axis) is first derived. The resulting t is then substituted into fy and fz to obtain the y and z coordinates of the striking point. y
Since the regression model should be chosen to predict the ball trajectory, we evaluate several
models such as exponential, Fourier, Gaussian, and polynomial curve fitting methods in Section 3.1. The result shows that polynomial curve fitting is the most suitable model to predict the ball trajectory. In Section 3.2, we also theoretically verify the existence of the relationship between the polynomial
coefficients and ball trajectory. However, Section 3.2 shows that curve fitting is highly sensitive to
noise in the input data. Thus, we propose two ANNs to model the relationship in Section 3.4. X-axis
Y-axis
Z-axis
Figure 10. Diagram of trajectory prediction. Y-axis Z-axis X-axis Figure 10. Diagram of trajectory prediction. 3.1. Selection of Trajectory Regression Model 3.1. Selection of Trajectory Regression Model 3.1. Selection of Trajectory Regression Model The data of any parabolic trajectory include time stamps and location data. Thus, we must
decide which type of regression model to use to express the trajectory data. This process mainly
involves finding the relationship between a set of independent and dependent variables and then
establishing an appropriate mathematical equation, which is referred to as a regression model. Below,
we investigate which type of regression model is more suitable for the trajectory data. We can use experiment data to determine a minimal number of parameters of suitable regression
models. The experiment data comprised 10 random trajectories selected from the training data. Each flight trajectory consists of two parabolic trajectories: the first from the initial point to the landing
point and the second from the landing point to the end point. We fitted the two trajectories using four
types of regression models, namely, Gaussian, exponential, Fourier, and polynomial, and examined 12 of 23 12 of 23 Sensors 2020, 20, 333 their results. We selected 10 random trajectories from the testing data to test R2 results of these
models. The red and blue bars in Figure 11 indicate the mean R2 results. The Fourier and polynomial
regression models presented reasonable R2 results for both trajectories, and their test results were
close to each other. However, the polynomial regression model used three parameters, whereas the
Fourier regression model used four parameters. With our objective of minimizing the number of
parameters, we ultimately chose the polynomial regression model to express the flight trajectory data. The polynomial regression model for the Z axis used three parameters, which was the quadratic
polynomial. For the X and Y axes, Figure 12 shows R2 of each trajectory and the average R2 of the
10 random trajectories was 0.9903 using the first-order polynomial, which was close to 1. Thus, the X
and Y axes were represented by the first-order polynomial regression. Mean R2 of Regression Models
Figure 11. Diagram of trajectory prediction. Figure 12. R2 of the first-order polynomial regression on the X and Y axes. 3.2. Theoretical Verification of Trajectory and Polynomial Coefficients
When the two parts of the ping-pong ball trajectory can be fitted using a polynomial, it means that
the coefficients in the polynomial effectively express the trajectory. We could therefore use ANNs to
predict the polynomial coefficients of the ball trajectories. The polynomial coefficients and conversion Mean R2 of Regression Models
Figure 11. 3.1. Selection of Trajectory Regression Model . tn
1
. . . 1
t1
m
. . . tn
m
a0
a1
. . . an−1
an
= Ta
(14) (14) (15) a = T−1q
(15) a = T−1q The input and output data of ANNs must be interrelated in a certain way; otherwise, training
the model may be too time-consuming, or poor prediction results may be produced. To verify that
the use of ANNs to learn trajectories in this study is appropriate. Based on this mathematical theory,
the trajectory point data can be mapped onto polynomial coefficient data using the Vandermonde
matrix. Thus, a corresponding relationship exists between the elements of these two sets of data. When the trajectory points serve as the inputs of the ANN and the trajectory coefficients are the
outputs, then the model has good learning ability. 3.1. Selection of Trajectory Regression Model Diagram of trajectory prediction. Figure 11. Diagram of trajectory prediction. Figure 11. Diagram of trajectory prediction. g
g
j
y p
Figure 12. R2 of the first-order polynomial regression on the X and Y axes. Figure 12. R2 of the first-order polynomial regression on the X and Y axes. 3.2. Theoretical Verification of Trajectory and Polynomial Coefficients 3.2. Theoretical Verification of Trajectory and Polynomial Coefficients When the two parts of the ping-pong ball trajectory can be fitted using a polynomial, it means that
the coefficients in the polynomial effectively express the trajectory. We could therefore use ANNs to
predict the polynomial coefficients of the ball trajectories. The polynomial coefficients and conversion 13 of 23 Sensors 2020, 20, 333 relations between trajectories can be expressed using Equation (13). Suppose that (tk,qk) is several
sets of data points where k = 0, . . . , m, tk is the time value, and qk is the positional value. Here there
exists a unique polynomial q(t) using the nth order. Equation (14) establishes the relation matrix pf
vectors q and a, which is known as the Vandermonde matrix T [29]. Using the pseudo inverse matrix
of T, Equation (15) gives coefficient a where a minimum squared-error exists between the coefficient
equation and the trajectory data. q(t) = a0 + a1t1 + · · · + antn
(13)
q =
q0
q1
. . . qm−1
qm
=
1
t1
0
. . . tn
0
1
t1
1
. . . tn
1
. . . 1
t1
m
. . . tn
m
a0
a1
. . . an−1
an
= Ta
(14)
a = T−1q
(15) q(t) = a0 + a1t1 + · · · + antn
(13) (13) q(t) = a0 + a1t1 + · · · + antn
(13)
q =
q0
q1
. . . qm−1
qm
=
1
t1
0
. . . tn
0
1
t1
1
. . . tn
1
. . . 1
t1
m
. . . tn
m
a0
a1
. . . an−1
an
= Ta
(14) q =
q0
q1
. . . qm−1
qm
=
1
t1
0
. . . tn
0
1
t1
1
. . 3.3. Comparison of Artificial Neural Network and Polynomial Curve Fitting Predictions In the prediction procedure of this study, once the vision system receives the data from a few
anterior positions, the ANN of the first trajectory can then predict the first trajectory. To demonstrate
the inadequacy of curve fitting and the prediction performance of the ANN for the first trajectory,
we predicted the landing point of the first trajectory using the proposed method and a quadratic
polynomial resulting from curve fitting. We used 30 items of data to train the ANN for the first
trajectory and used the trained ANN and the ten anterior positions in the trajectory to predict the
polynomial parameters of the first trajectory. The red circles and crosses in Figure 13 show the trajectory
obtained during testing. The blue curve presents the polynomial trajectory output by the trained ANN,
and the green curve shows the subsequent trajectory trend after the data from the ten anterior positions
were received. The mean error and standard deviation of the quadratic polynomial were 19.2 mm and
11.8 mm, respectively; however, for the ANN, the mean error and standard deviation of the quadratic
polynomial were 10.8 mm and 10.0 mm, respectively. The errors in the landing point predictions of the
ANN were smaller than those of the quadratic polynomial. Once some errors exist in capturing the ten
anterior positions of the ball, Figure 13 shows that the ANN is resistant to noise; even if an input node
contains noise or its data is incomplete, the single data does not greatly impact the overall result. 14 of 23 Sensors 2020, 20, 333 Figure 13. Trajectory based on ten positions (Blue: ANN; green: quadratic polynomial resulting from
curve fitting). Figure 13. Trajectory based on ten positions (Blue: ANN; green: quadratic polynomial resulting from
curve fitting). 3.4. Two ANNs in Trajectory Prediction 3.4. Two ANNs in Trajectory Prediction Three things can be obtained from a complete flight trajectory: the trajectory before landing,
the trajectory after landing, and ten anterior positions. Figure 14 displays the procedure of generating
the required training data. First, the lowest point in the overall trajectory along the z axis is identified,
demarcating the data of the first and second trajectory. The first trajectory is between the initial point
and the lowest point, and the second trajectory is between the lowest point and the end of the trajectory. Both trajectories are then expressed using polynomial regression to extract the trajectory parameters and
the data of the ten anterior positions. The data of the ten anterior positions and the parameters of the first
trajectory then serve as the training data for the first ANN (Network Model 1), and the parameters of the
first and second trajectories serve as the training data for the second ANN (Network Model 2). We defined the 3D polynomial of the first trajectory as Equation (18), where a1, b1, a2, b2, a3, b3, c3
are the X-, Y-, and Z-axis coefficients of the first trajectory. We defined the 3D polynomial of the second
trajectory as Equation (21), where a
′
1, b
′
1, a
′
2, b
′
2, a
′
3, b
′
3, c
′
3 are the X-, Y-, and Z-axis coefficients of the
second trajectory. Equations (22) and (23) are the corresponding inputs and outputs of the two networks. 3.4. Two ANNs in Trajectory Prediction X
=
a1t + b1
(16)
Y
=
a2t + b2
(17)
Z
=
a3t2 + b3t + c3
(18)
X
=
a
′
1t + b
′
1
(19)
Y
=
a
′
2t + b
′
2
(20)
Z
=
a
′
3t2 + b
′
3t + c
′
3
(21) X
=
a1t + b1
(16)
Y
=
a2t + b2
(17)
Z
=
a3t2 + b3t + c3
(18)
X
=
a
′
1t + b
′
1
(19)
Y
=
a
′
2t + b
′
2
(20)
Z
a
′ t2 + b
′ t + c
′
(21) X
=
a1t + b1
(16)
Y
=
a2t + b2
(17)
Z
=
a3t2 + b3t + c3
(18)
X
=
a
′
1t + b
′
1
(19)
Y
=
a
′
2t + b
′
2
(20)
Z
=
a
′
3t2 + b
′
3t + c
′
3
(21)
]i=1,...,10
Network model 1
−−−−−−−−−−−−−→
[a1, b1, a2, b2, a3, b3, c3]
(22)
b2, a3, b3, c3]
Network model 1
−−−−−−−−−−−−−→
[a
′
1, b
′
1, a
′
2, b
′
2, a
′
3, b
′
3, c
′
3]
(23) X
=
a1t + b1
(16)
Y
=
a2t + b2
(17)
Z
=
a3t2 + b3t + c3
(18)
X
=
a
′ t + b
′
(19) X
=
a1t + b1
(16)
Y
=
a2t + b2
(17)
Z
=
a3t2 + b3t + c3
(18) (16)
(17)
(18) (16) (18) Z
=
a3t2 + b3t + c3
(18)
X
=
a
′
1t + b
′
1
(19)
Y
=
a
′
2t + b
′
2
(20)
Z
=
a
′
3t2 + b
′
3t + c
′
3
(21) X
=
a
′
1t + b
′
1
(19)
Y
=
a
′
2t + b
′
2
(20)
Z
=
a
′
3t2 + b
′
3t + c
′
3
(21) [Pi, ti]i=1,...,10
Network model 1
−−−−−−−−−−−−−→
[a1, b1, a2, b2, a3, b3, c3]
(22) [Pi, ti]i=1,...,10
Network model 1
−−−−−−−−−−−−−→
[a1, b1, a2, b2, a3, b3, c3] [Pi, ti]i=1,...,10
Network model 1
−−−−−−−−−−−−−→
[a1, b1, a2, b2, a3, b3, c3]
(22) (22) [a1, b1, a2, b2, a3, b3, c3]
Network model 1
−−−−−−−−−−−−−→
[a
′
1, b
′
1, a
′
2, b
′
2, a
′
3, b
′
3, c
′
3]
(23) [a1, b1, a2, b2, a3, b3, c3]
Network model 1
−−−−−−−−−−−−−→
[a
′
1, b
′
1, a
′
2, b
′
2, a
′
3, b
′
3, c
′
3]
(23) (23) 15 of 23 Sensors 2020, 20, 333 Figure 14. 3.4. Two ANNs in Trajectory Prediction Procedure of generating the required training data. Figure 14. Procedure of generating the required training data. 4. Experimental Results In this section, we present a series of experiments conducted to demonstrate the prediction
performance of the proposed method. In Section 4.1, we compare the striking point prediction
performance of the proposed dual-network method and a single ANN [3] using the experimental data. In Section 4.2, we compare the striking point prediction performance of the proposed dual-network
method and a physical model. Section 4.3 presents an experiment investigating the influence of time
data removal, and Section 4.4 analyzes the errors of the proposed method in a physical experiment. Figure 15 shows the flight trajectory data collected from human demonstrations, in which the ping-pong
ball was hit from the serving side to a robot arm on the opposite side. The first trajectories passed over
the net and landed on the table on the robot’s side. We collected data from a total of 200 trajectories,
among which 170 trajectories were used as training data and 30 trajectories comprised the testing data. Once the ANNs were trained, we used the testing data to examine their predictive abilities. We used
a circle with a diameter of 75 mm to simulate the area of the paddle and calculated the errors, standard
deviation, the number of balls that hit the range of the paddle, and the success rates. Figure 15. Flight trajectory data collected from human demonstrations. Figure 15. Flight trajectory data collected from human demonstrations. 4.1. Dual-Network vs. Single-Network Approach 4.1. Dual-Network vs. Single-Network Approach Reference [3] used a single ANN to predict the striking points. The inputs were the data of four
anterior positions, and the outputs were the striking point and the acceleration of the ball in the X,
Y, and Z directions. We conducted an experiment to compare this single-network approach with the
proposed method. The inputs of the single network increased to ten anterior positions so that they were 16 of 23 Sensors 2020, 20, 333 consistent with the inputs of the proposed method. The input, hidden, and output layers of the model
structure in the single-network approach were 40, 10, and 6. In contrast, the input, hidden, and output
layers of the model structure in our dual-network approach were 40, 10, and 7, and the second model
was set as 7-20-7. The both networks used the mean-square error as the loss function and the hyperbolic
tangent sigmoid as the activation function. The number of epochs was 10,000. The Levenberg–Marquardt
algorithm was used for optimization. Table 5 summarizes the details of the two ANNs. After applying
the trained models to the testing data, we calculated the overall mean error, standard deviation, and the
success rate of balls falling within the boundaries of the paddle. Figure 16 presents the mean errors of the
proposed method and the single-network approach (39.6 mm and 42.9 mm), as well as their success rates
(89% and 86.7%). Based on the experiment results, we can conclude that the prediction performance of
the proposed method is better than that of the single-network approach. The difference between these
two methods is whether they consider t he process of the ball landing point (including the landing on the
table). A normal ping-pong ball trajectory consists of two trajectories in which the flight process of the
ball offers data. The single-network approach only considers the data of the anterior positions and the
final striking point, so its prediction performance is limited. The average of mean error
The average of mean success rate
(a)
(b)
Figure 16. Performance results of proposed dual-network method and single-network approach:
(a) mean error; (b) mean success rate. The average of mean error
(a) The average of mean success rate
(b) (b) (a) Figure 16. Performance results of proposed dual-network method and single-network approach:
(a) mean error; (b) mean success rate. Table 5. Details of the two ANNs. 4.1. Dual-Network vs. Single-Network Approach 1st ANN
2nd ANN
Input node number
40
7
Hidden node number
10
20
Output node number
6
7
Activation fun. Hyperbolic tangent sigmoid
Hyperbolic tangent sigmoid
Loss fun. Mean-square error
Mean-square error
Epoch number
10,000
10,000
Optimizer
Levenberg–Marquardt
Levenberg–Marquardt
4 2 Physical Model Testing Table 5. Details of the two ANNs. e = Relative velocity after collision
Relative velocity before collision
(24) e = Relative velocity after collision
Relative velocity before collision
(24) (24) 2. The Y and Z positions of the testing trajectories at x = 400 mm (the striking point), i.e., Ytrue and Ztrue,
were used to calculate the errors in the final prediction results by selecting 100 random trajectories. 2. The Y and Z positions of the testing trajectories at x = 400 mm (the striking point), i.e., Ytrue and Ztrue,
were used to calculate the errors in the final prediction results by selecting 100 random trajectories. p
y
g
j
3. The data from the ten anterior positions of the testing trajectories were obtained, and then the
initial velocities in the X, Y, and Z directions, i.e., vx, vy, and vz, using polynomial regression. p
y
g
j
The data from the ten anterior positions of the testing trajectories were obtained, and then the
initial velocities in the X, Y, and Z directions, i.e., vx, vy, and vz, using polynomial regression. y
g p
y
g
4. The downward accelerate az was defined with air resistance taken into account as shown in
Equation (25), where g denotes gravitational acceleration and equals 9.81 m/s2; m is the weight
of the ping pong ball, which is approximately 2.7 × 10−3 kg; Cd is the resistance coefficient and
equals 0.5; ρa denotes air fluid density, which is 1.29 kg/m3; and A is the cross-sectional area of
the ball, which is roughly 1.3 × 10−3 m2. The mathematical formula indicates that the velocity
and acceleration of the object change with time. Here, we set sampling time Ts to be 0.005 s. y
g p
y
g
4. The downward accelerate az was defined with air resistance taken into account as shown in
Equation (25), where g denotes gravitational acceleration and equals 9.81 m/s2; m is the weight
of the ping pong ball, which is approximately 2.7 × 10−3 kg; Cd is the resistance coefficient and
equals 0.5; ρa denotes air fluid density, which is 1.29 kg/m3; and A is the cross-sectional area of
the ball, which is roughly 1.3 × 10−3 m2. The mathematical formula indicates that the velocity
and acceleration of the object change with time. Here, we set sampling time Ts to be 0.005 s. az = g −Cd · ρa · A
2m
v2
z
(25) (25) 5. e = Relative velocity after collision
Relative velocity before collision
(24) Using the physical formulas in Equations (26)–(28), we derived the next displacement (acceleration
and velocity are updated at each sampling time). 5. Using the physical formulas in Equations (26)–(28), we derived the next displacement (acceleration
and velocity are updated at each sampling time). Sz = vzTs + 1
2azT2
s
(26)
vt+1 = vt + atTs
(27) Sz = vzTs + 1
2azT2
s
(26) (26) vt+1 = vt + atTs
(27) vt+1 = vt + atTs (27) at+1 = g + Cd · ρa · A
2m
vt+1
(28) (28) 6. When the ball reached the table (Z direction), we used the mean collision coefficient obtained in
the first step to calculate the velocity of the ball after landing, as shown in Equation (29). Once the
velocity was calculated, we could continue to calculate the position of the ball in the Z direction. 6. When the ball reached the table (Z direction), we used the mean collision coefficient obtained in
the first step to calculate the velocity of the ball after landing, as shown in Equation (29). Once the
velocity was calculated, we could continue to calculate the position of the ball in the Z direction. va f ter = vbe f ore · e
(29) (29) va f ter = vbe f ore · e
(29) 7. Not considering the influences of friction between the ball and the table surface and the
self-rotation of the ball, we could calculate the displacement of the ball in the X direction at
each sampling time using Equations (30) and (31). The velocity and acceleration of the ball in this
direction also changed with time, all the way to the striking point (x = 400 mm). The timing of the
striking point, Tend, was then recorded. 7. Not considering the influences of friction between the ball and the table surface and the
self-rotation of the ball, we could calculate the displacement of the ball in the X direction at
each sampling time using Equations (30) and (31). The velocity and acceleration of the ball in this
direction also changed with time, all the way to the striking point (x = 400 mm). The timing of the
striking point, Tend, was then recorded. ax = Cd · ρa · A
2m
v2
x
(30) (30) Sx = vxTs + 1
2axT2
s
(31) (31) 8. 4.2. Physical Model Testing In this section, we use a normal physical formula to predict the striking point. The method was
basically the same: use the data of the ten anterior points, as shown in Figure 17, to calculate the initial
velocity in three dimensions and derive the striking point while taking into account gravitational
acceleration g, air resistance, and elastic collision. Elastic collision is associated with the coefficient of
restitution of the ball, which can be obtained from real-world trajectories. For the sake of accuracy,
we added 130 real-world trajectories to calculate this coefficient. The prediction process of the physical
model was as follows: 17 of 23 Sensors 2020, 20, 333 1. Using the 330 trajectories, we obtained the mean velocities before and after collision and then the
mean collision coefficient. Based on the formula for the coefficient of restitution in Equation (24),
we concluded that the mean coefficient was 0.9203. 1. Using the 330 trajectories, we obtained the mean velocities before and after collision and then the
mean collision coefficient. Based on the formula for the coefficient of restitution in Equation (24),
we concluded that the mean coefficient was 0.9203. e = Relative velocity after collision
Relative velocity before collision
(24) Finally, we used the initial velocity in the Y direction, vy, air resistance acceleration, ay in
Equation (32), and timing of striking point, Tend, to derive the striking point in the Y direction. 18 of 23 Sensors 2020, 20, 333 Using the predicted Y and Z positions of the striking point, Ypredict and Zpredict, and the actual
striking point, Ytrue and Ztrue (Step 2), we then calculated the error using Equation (33). Using the predicted Y and Z positions of the striking point, Ypredict and Zpredict, and the actual
striking point, Ytrue and Ztrue (Step 2), we then calculated the error using Equation (33). ay = Cd · ρa · A
2m
v2
y
(32) (32) Error =
q
(Ypredict −Ytrue)2 + (Zpredict −Ztrue)2
(33) (33) Figure 17. Striking point prediction using physical model. Figure 17. Striking point prediction using physical model. Using the physical prediction method above, we selected 30 testing trajectories to calculate striking
point prediction errors. The success rate of balls hitting the paddle was 70%, and Figure 18 shows
that the overall mean error and standard deviation of striking position were 57.9 mm and 30.3 mm,
respectively. The success rate of 70% of the physical model was worse than 97% of the proposed
dual-network method. In reality, the ball trajectory is affected by the friction of the table surface and
the Magnus effect, but it is difficult to obtain precise measurements of them without more advanced
vision equipment [20]. Figure 18. Striking position error of the physical model. Figure 18. Striking position error of the physical model. 19 of 23 19 of 23 Sensors 2020, 20, 333 4.3. Removal of Time Data The vision system in this study had a fixed sampling time. Based on the training data, the average
time interval was 0.0075 s and the standard deviation was 5.19 × 10−4. The inputs of the first ANN
include time stamp data, which comprises steady values. For this reason, we tried removing the
time stamps from the input and then tested the resulting performance. We used the data from
all 330 trajectories, with 30 trajectories serving as the testing data and ten training sessions each. As shown in Figure 19, there are no significant differences between the prediction results of the two
conditions. Thus, removing the time stamps from the input of the first ANN does not affect its
prediction performance. Figure 19. Prediction error of the proposed dual-network method with and without time stamps. Figure 19. Prediction error of the proposed dual-network method with and without time stamps. This approach has its advantages. It simplifies the entire network because the inputs of the
first ANN were reduced from 40 nodes to 30 nodes (see Equation (22)), which also means that the
number of parameters in the first ANN decreased. The original prediction model contained a total of
1178 parameters (of which the first ANN accounted for 871 parameters), whereas the current model
had 998 parameters (of which the first ANN accounted for 691 parameters). This represents a 15%
reduction in the overall number of parameters. The two conditions also differed in training time. Figure 20 shows the training time of the ten tests conducted for each condition. The mean training
time was 457 s for the model with the time stamps and 209 s for the model without the time stamps,
representing a 54% reduction in training time. Figure 20. Training time of the dual-network method with and without time stamps. Figure 20. Training time of the dual-network method with and without time stamps. 20 of 23 Sensors 2020, 20, 333 20 of 23 4.5. Experiment Discussion In Section 4.1, we compared the striking point prediction performance of the dual-network
method and a single ANN. In Section 4.2, we compared the striking point prediction performance
of the physical model. Table 6 summaries the prediction error among the proposed dual-network
method, single network, and physical model. The results show that the proposed method has less
trajectory error than the other methods. Table 6. Comparison of the prediction error among the proposed dual-network method, single network,
and physical model. Proposed Dual Networks
Single Network
Physical Model
Mean (mm)
39.553
42.858
57.862 4.4. Striking Point Errors To measure the prediction error of the proposed method, we conducted an experiment as follows. This experiment was done by covering the front of the paddle held by the robot arm with a piece
of white paper and then covering that with a piece of carbon paper. When the robot arm struck the
ping-pong ball with the paddle, the ball left a mark on the white paper, and the position of the robot
arm at the time was also recorded. The distance between the center of the paddle and the striking points
(block dots) indicate the prediction errors, which we measured using a vernier caliper. The area that
the robot arm could strike was 20 cm × 18 cm, which equals 360 cm2. We conducted three sets of
experiments with ten trials each, which produced a total of 30 records. Figure 21 displays the first
trials of the striking points recorded on carbon paper in our physical experiment. Figure 22 shows that
the thirty trials and the overall mean error was 36.6 mm, and the standard deviation was 18.8 mm. Figure 23 shows the striking accuracy with respect to the paddle area. The yellow circle represents
the mean of error and the green area represents a standard deviation of error. Obviously, the area of
striking error is much smaller than the paddle area, which means that the robot was able to strike
the ball. Figure 21. Striking points on carbon paper. Figure 21. Striking points on carbon paper. Figure 22. Overall striking errors. Figure 21. Striking points on carbon paper. Figure 22. Overall striking errors. Figure 22. Overall striking errors. 21 of 23 Sensors 2020, 20, 333 Figure 23. Striking accuracy with respect to the paddle area: the green area represents the area of
striking error center with the mean (yellow) and a standard deviation. Figure 23. Striking accuracy with respect to the paddle area: the green area represents the area of
striking error center with the mean (yellow) and a standard deviation. 5. Conclusions This study developed a ping-pong ball trajectory prediction system that included ball recognition,
3D positioning, and ball trajectory prediction. Trajectory prediction performance is key to whether
a table-tennis robot can hit the ping pong ball. However, most existing studies developed physical
models for prediction, which can only achieve good prediction effects if they have high-frequency
vision systems to analyze ball status in real time. Such advanced equipment is not readily accessible
and makes it difficult to conduct fundamental studies. We therefore adopted machine learning to predict
the flight trajectories of ping-pong balls, which uses historical data to learn the regularities within. There
is no need to establish a complex physical model, and fairly good prediction results can be achieved even
with general industrial cameras readily available on the market. Each complete flight trajectory consists
of a landing point on the table and two parabolic trajectories. We used two ANNs to learn the features of
these flight trajectories. The first ANN learns the first flight trajectory. Once the vision system receives the
anterior positions, it can instantly predict the first trajectory. We demonstrated that this approach was
superior to curve fitting due to the limited amount of data and noise filtering capabilities. The second
ANN learns the second flight trajectory. Once the first trajectory is known, the second flight trajectory can
be instantly predicted. The two ANNs were then combined. A comparison of the ANN and curve fitting approaches revealed that the use of data from
ten anterior positions resulted in mean errors of 10.8 mm in the prediction results of the ANN and
19.2 mm in those of the quadratic polynomial resulting from curve fitting. We conducted a simulation
experiment using 200 real-world trajectories as training data. The mean errors of the proposed 22 of 23 Sensors 2020, 20, 333 dual-network method and a single-network model were 39.6 mm and 42.9 mm, respectively, and the
mean success rates were 88.99% and 86.66%. These results indicate that the prediction performance of
the proposed dual-network method is better than that of the single-network approach. We also used
a simple physical model to predict striking points. We employed 330 real-world trajectories, and the
resulting mean error and success rate were 57.9 mm and 70%. The success rate of 70% of the physical
model was worse than 97% of the proposed dual-network method. 5. Conclusions In the proposed dual-network
method, the inputs of the first ANN include time stamps. As our vision system takes samples at fixed
time intervals, little variation exists in the time stamp data. We thus tried removing the time stamps
from the data. We used 330 trajectories for training. The mean errors of the proposed method with
and without the time stamps were 27.5 mm and 26.3 mm, and the mean success rates were 98.67%
and 97.33%. The results show that even without the time stamps, the proposed method maintains
its prediction performance with the additional advantages of 15% fewer parameters in the overall
network and 54% shorter training time. Finally, we tested the striking ability of our robot arm, which
produced a mean error and standard deviation of 36.6 mm and 18.8 mm, respectively. Author Contributions: Conceptualization, H.-I.L. and Y.-C.H.; methodology, H.-I.L. and Y.-C.H.; software, Y.-C.H.;
validation, H.-I.L. and Y.-C.H.; formal analysis, H.-I.L. and Y.-C.H.; investigation, H.-I.L. and Y.-C.H.; resources,
H.-I.L. and Z.Y.; data curation, Y.-C.H.; writing—original draft preparation, H.-I.L.; writing—review and editing,
H.-I.L.; visualization, H.-I.L.; supervision, H.-I.L. and Z.Y.; project administration, H.-I.L. and Z.Y.; funding
acquisition, H.-I.L. and Z.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Taipei University of Technology grant number NTUT-BIT-1 Funding: This research was funded by National Taipei University of Technology grant number NTUT-BIT-105-1. C
fli
f I
Th
h
d
l
fli
f i Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Shi, Q.; Li, C.; Wang, C.; Luo, H.; Huang, Q.; Fukuda, T. Design and implementation of an omnidirectional
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Scientific Publishing: Deutschland, Germany, 2018; p. 339. 11. Zhang, Y.; Zhao, Y.; Xiong, R.; Wang, Y.; Wang, J.; Chu, J. Spin observation and trajectory prediction of
a ping-pong ball. Author Contributions: Conceptualization, H.-I.L. and Y.-C.H.; methodology, H.-I.L. and Y.-C.H.; software, Y.-C.H.;
validation, H.-I.L. and Y.-C.H.; formal analysis, H.-I.L. and Y.-C.H.; investigation, H.-I.L. and Y.-C.H.; resources,
H.-I.L. and Z.Y.; data curation, Y.-C.H.; writing—original draft preparation, H.-I.L.; writing—review and editing,
H.-I.L.; visualization, H.-I.L.; supervision, H.-I.L. and Z.Y.; project administration, H.-I.L. and Z.Y.; funding
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(ICRA), Hong Kong, China, 31 May–7 June 2014; pp. 4108–4114. 11. Zhang, Y.; Zhao, Y.; Xiong, R.; Wang, Y.; Wang, J.; Chu, J. Spin observation and trajectory prediction of
a ping-pong ball. In Proceedings of the 2014 IEEE International Conference on Robotics and Automation
(ICRA), Hong Kong, China, 31 May–7 June 2014; pp. 4108–4114. 23 of 23 Sensors 2020, 20, 333 12. Zhao, Y.; Zhang, Y.; Xiong, R.; Wang, J. Optimal state estimation of spinning ping-pong ball using continuous
motion model. IEEE Trans. Instrum. Meas. 2015, 64, 2208–2216. [CrossRef] 13. Zhang, Z.; Xu, D.; Tan, M. Visual measurement and prediction of ball trajectory for table tennis robot. IEEE Trans. Instrum. Meas. 2010, 59, 3195–3205. [CrossRef] 14. Wang, P.; Zhang, Q.; Jin, Y.; Ru, F. Studies and simulations on the flight trajectories of spinning table tennis
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networks. IEEE Trans. Ind. Electron. 1995, 42, 147–158. [CrossRef] 27. Nakashima, A.; Takayanagi, K.; Hayakawa, Y. A learning method for returning ball in robotic table tennis. In Proceedings of the 2014 International Conference on Multisensor Fusion and Information Integration for
Intelligent Systems (MFI), Beijing, China, 28–29 September 2014; pp. 1–6. 28. Zhang, Z. Determining the epipolar geometry and its uncertainty: A review. Int. J. Comput. Vis. 1998,
27, 161–195. [CrossRef] 29. Biagiotti, L.; Melchiorri, C. Trajectory Planning for Automatic Machines and Robots; Springer Science & Business
Media: Berlin/Heidelberg, Germany, 2008. c⃝2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Psychological distress, depression, anxiety, and life satisfaction following COVID-19 infection: evidence from 11 UK longitudinal population studies
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The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. Psychological distress, depression, anxiety and life satisfaction following COVID-19
infection: Evidence from 11 UK longitudinal population studies Thompson , E. J., Stafford , J., Moltrecht , B., Huggins , C. F., Kwong , A. S. F., Shaw , R. J., Zaninotto , P.,
Patel, K., Silverwood, R. J., Mc Elroy, E., Pierce, M., Green, M. J., Bowyer , R. C. E., Maddock , J., Tilling, K.,
Katikireddi, S. V., Ploubidis, G. B., Porteous, D. J., Timpson, N., ... Patalay, P. (2022). Psychological distress,
depression, anxiety and life satisfaction following COVID-19 infection: Evidence from 11 UK longitudinal
population studies. Lancet Psychiatry, 9(11), 894-906. https://doi.org/10.1016/S2215-0366(22)00307-8 Link to publication record in Ulster University Research Portal Published in:
Lancet Psychiatry
Publication Status:
Published (in print/issue): 30/11/2022
DOI:
10.1016/S2215-0366(22)00307-8
Document Version
Publisher's PDF, also known as Version of record
Document Licence:
CC BY DOI:
10.1016/S2215-0366(22)00307-8 Document Version
Publisher's PDF, also known as Version of record Summary Summary
Background Evidence on associations between COVID-19 illness and mental health is mixed. We aimed to examine
whether COVID-19 is associated with deterioration in mental health while considering pre-pandemic mental health,
time since infection, subgroup differences, and confirmation of infection via self-reported test and serology data. Methods We obtained data from 11 UK longitudinal studies with repeated measures of mental health (psychological
distress, depression, anxiety, and life satisfaction; mental health scales were standardised within each study across
time) and COVID-19 status between April, 2020, and April, 2021. We included participants with information available
on at least one mental health outcome measure and self-reported COVID-19 status (suspected or test-confirmed)
during the pandemic, and a subset with serology-confirmed COVID-19. Furthermore, only participants who had
available data on a minimum set of covariates, including age, sex, and pre-pandemic mental health were included. We
investigated associations between having ever had COVID-19 and mental health outcomes using generalised
estimating equations. We examined whether associations varied by age, sex, ethnicity, education, and pre-pandemic
mental health, whether the strength of the association varied according to time since infection, and whether
associations differed between self-reported versus confirmed (by test or serology) infection. Findings Between 21 Dec, 2021, and July 11, 2022, we analysed data from 54 442 participants (ranging from a minimum
age of 16 years in one study to a maximum category of 90 years and older in another; including 33 200 [61·0%] women
and 21 242 [39·0%] men) from 11 longitudinal UK studies. Of 40 819 participants with available ethnicity data,
36 802 (90·2%) were White. Pooled estimates of standardised differences in outcomes suggested associations between
COVID-19 and subsequent psychological distress (0·10 [95% CI 0·06 to 0·13], I²=42·8%), depression (0·08
[0·05 to 0·10], I²=20·8%), anxiety (0·08 [0·05 to 0·10], I²=0·0%), and lower life satisfaction (–0·06 [–0·08 to –0·04],
I²=29·2%). We found no evidence of interactions between COVID-19 and sex, education, ethnicity, or pre-pandemic
mental health. Associations did not vary substantially between time since infection of less than 4 weeks, 4–12 weeks,
and more than 12 weeks, and were present in all age groups, with some evidence of stronger effects in those aged
50 years and older. Participants who self-reported COVID-19 but had negative serology had worse mental health
outcomes for all measures than those without COVID-19 based on serology and self-report. Summary Participants who had
positive serology but did not self-report COVID-19 did not show association with mental health outcomes. Interpretation Self-reporting COVID-19 was longitudinally associated with deterioration in mental health and life
satisfaction. Our findings emphasise the need for greater post-infection mental health service provision, given the
substantial prevalence of COVID-19 in the UK and worldwide. Funding UK Medical Research Council and UK National Institute for Health and Care Research. Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. www.thelancet.com/psychiatry Vol 9 November 2022 Lancet Psychiatry 2022;
9: 894–906
*Joint first authors
Department of Twin Research
and Genetic Epidemiology,
School of Life Course and
Population Sciences, King’s
College London, London, UK
(E J Thompson PhD,
R C E Bowyer PhD,
C J Steves PhD); MRC Unit for
Lifelong Health and Ageing
(J Stafford PhD, K Patel PhD,
J Maddock PhD,
N Chaturvedi MD,
P Patalay PhD), Centre for
Longitudinal Studies
(B Moltrecht PhD,
R J Silverwood PhD,
G B Ploubidis PhD, P Patalay),
Department of Epidemiology
and Public Health
(P Zaninotto PhD), University
College London, London, UK;
Centre for Genomic and
Experimental Medicine
(C F Huggins PhD,
D J Porteous PhD), Division of
Psychiatry (A S F Kwong PhD),
University of Edinburgh,
Edinburgh, UK; MRC/CSO Social
& Public Health Sciences Unit,
School of Health and
Wellbeing, University of
Glasgow, Glasgow, UK
(R J Shaw PhD, M J Green PhD,
S V Katikireddi PhD); School of
Psychology, Ulster University,
Ulster, UK (E McElroy PhD);
Division of Psychology and
Mental Health, The University
of Manchester, Manchester, UK
(M Pierce PhD); Population
Health Sciences, Bristol Medical
School, University of Bristol,
Bristol, UK (K Tilling PhD,
N Timpson PhD)
Correspondence to: Correspondence to:
Dr Jean Stafford MRC Unit for
Lifelong Health and Ageing,
University College London,
London, WC1E 7HB, UK
j.stafford@ucl.ac.uk Take down policy Take down policy
The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been
made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in
the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 Articles Psychological distress, depression, anxiety, and life
satisfaction following COVID-19 infection: evidence from
11 UK longitudinal population studies Ellen J Thompson*, Jean Stafford*, Bettina Moltrecht*, Charlotte F Huggins, Alex S F Kwong, Richard J Shaw, Paola Zaninotto, Kishan Patel,
Richard J Silverwood, Eoin McElroy, Matthias Pierce, Michael J Green, Ruth C E Bowyer, Jane Maddock, Kate Tilling, S Vittal Katikireddi,
George B Ploubidis, David J Porteous, Nic Timpson, Nish Chaturvedi, Claire J Steves, Praveetha Patalay For the preregistered analyses
on the Open Science
Framework see https://osf.io/
ntmqw Introduction the mental health consequences of COVID-19 in the
general population remain poorly understood. the mental health consequences of COVID-19 in the
general population remain poorly understood. Infection with severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) can cause asymptomatic or
symptomatic COVID-19. Mental ill-health is increasingly
recognised as a potential consequence of COVID-19,
following initial evidence from case reports and studies of
other severe coronavirus infections.1 However, longitudinal
evidence in this area is limited and few studies have sought
to disentangle the effects of COVID-19 illness from the
wider mental health impacts of the pandemic.2 As such, Recent systematic reviews have yielded mixed results as
to whether COVID-19 illness is associated with
psychological distress,3,4 which might reflect a lack of high-
quality longitudinal evidence in this area. Previous
studies have been limited by small or unrepresentative
samples, cross-sectional designs, and absence of control
groups.5,6 Although several studies using routine data
reported elevated rates of psychiatric disorders following 894 Articles Research in context test-confirmed COVID-19. Participants who self-reported
suspected or test-confirmed COVID-19, including those with a
negative serology result, showed deterioration in their mental
health. However, this association was not found in participants
with positive serology who did not self-report COVID-19. These
findings raise the possibility that the effects observed are not
specific to SARS-CoV-2 infection but could still reflect the
experience of illness during this period, or be explained by other
factors. test-confirmed COVID-19. Participants who self-reported
suspected or test-confirmed COVID-19, including those with a
negative serology result, showed deterioration in their mental
health. However, this association was not found in participants
with positive serology who did not self-report COVID-19. These
findings raise the possibility that the effects observed are not
specific to SARS-CoV-2 infection but could still reflect the
experience of illness during this period, or be explained by other
factors. Added value of this study Using data from 11 UK longitudinal studies including
54 442 participants with detailed information on pre-pandemic
confounders, we found associations between COVID-19 and
subsequent psychological distress, depression, anxiety, and
lower life satisfaction. Associations were present in all age
groups but were strongest for middle-aged and older people. Effects did not attenuate over time in the 12 weeks after
infection and were found for self-reported suspected and COVID-19 illness,7–12 others have not found clear evidence
of associations13,14 and have mainly focused on more severe
COVID-19 and recorded mental health disorders.15,16
A study using data from six cohorts in Europe found
that severe acute COVID-19 illness was associated with
adverse
mental
health
outcomes.17
Additionally,
longitudinal studies in the UK have found associations
between COVID-19 and psychological distress,18–20 although
findings have been mixed across different mental health
outcomes21 and, in the COVID Symptom Study, a modest
association was found only in older participants.20 Further
longitudinal research is needed to clarify previous mixed
findings, to investigate the magnitude of any association,
and to examine whether associations are sustained in the
longer-term after infection. phase of the illness. Third, we explored whether
associations varied by age, sex, ethnicity, education, and
pre-pandemic mental health. Fourth, we examined
whether associations between COVID-19 and mental
health differed between those with suspected versus test-
confirmed COVID-19, and self-reported versus serology-
detected COVID-19. Implications of all the available evidence p
The evidence considered together indicates that COVID-19,
in both the general population and in patients admitted to
hospital, is associated with deterioration in mental health at
clinical and subclinical levels. These findings have important
implications for post-COVID-19 mental health service
provision, given the substantial global prevalence of COVID-19. Our findings also suggest that these associations might not be
specific to SARS-CoV-2, with other infections with similar
symptoms also potentially resulting in worse mental health,
and it is possible that this effect was specific to or amplified by
the wider pandemic setting. Our findings comparing self-report
and serology status highlight the potential salience of
psychosocial mechanisms, such as social isolation, loss of pay,
worry about infecting others, and the unpredictable course of
COVID-19, in underpinning some of the associations between
self-reported COVID-19 and mental health. Evidence before this study y
Although previous research suggests that SARS CoV-2 infection
might increase risk of psychological distress, findings have been
mixed and longitudinal evidence is limited. We searched
PubMed for studies published in English from Jan 1, 2020, until
April 1, 2022, using terms pertaining to “COVID-19” (focusing
specifically on “infection”) and “mental health” (including
“psychological distress,” “depression”, and “anxiety”). Most
studies were cross-sectional, lacked comparison groups, and
focused on patients admitted to hospital because of COVID-19. Several longitudinal studies, including prospective cohorts and
studies using electronic health records, found associations
between COVID-19 and psychological distress, although
findings were mixed, with some evidence of attenuating effects
over time. Further longitudinal research is needed involving
general population samples, including subclinical psychological
distress and common mental disorders, and accounting for
pre-pandemic confounders. www.thelancet.com/psychiatry Vol 9 November 2022 Articles Design and sample frame
2020 age
range, years
Pre-pandemic
survey
Details of COVID-19
surveys (response rate)
Mental health
measure
Serology data
Participants
with data
analysed, n
Age homogenous cohorts
Millennium Cohort
Study
Cohort of UK children born between
September, 2000, and January, 2002,
with regular follow-up surveys from
birth
18–20
2018
Three surveys: May 2020
(26·6%); September–October,
2020 (24·2%); February–March,
2021 (22%)
Kessler
Psychological
Distress Scale-6
987 valid samples
obtained April–June,
2021
4652
Avon Longitudinal
Study of Parents and
Children-Generation 1
Cohort of children born in southwest
England between April, 1991, and
December, 1992, with regular follow-
up surveys from birth (original young
people)
27–29
2017–18
Three surveys: April, 2020
(19%); June, 2020 (17·4%);
December, 2020 (26·4%)
Short Mood and
Feelings
Questionnaire
NA
2498
Next Steps (formerly
known as Longitudinal
Study of Young People
in England)
Sample recruited via secondary
schools in England at about age
13 years with regular follow-up
surveys
29–31
2015
Three surveys: May, 2020
(20·3%); September–October,
2020 (31·8%); February–March,
2021 (29%)
General Health
Questionnaire 12
1037 valid samples
obtained April–June,
2021
4092
British Cohort Study
1970
Cohort of all children born in Great
Britain (ie, England, Wales, and
Scotland) in 1 week in 1970, with
regular follow-up surveys from birth. Articles 50
2016
Three surveys: May, 2020
(40·4%); September–October,
2020 (43·9%); February–March,
2021 (40%)
9-item Malaise
Inventory
2074 valid samples
obtained April –June,
2021
5545
National Child
Development Study
Cohort of all children born in Great
Britain (ie, England, Wales, and
Scotland) in 1 week in 1958, with
regular follow-up surveys from birth
62
2013
Three surveys: May, 2020
(57·9%); September–October,
2020 (53·9%); February–March,
2021 (52%)
9-item Malaise
Inventory
2722 valid samples
obtained April–June,
2021
6696
National Survey of
Health and
Development
Cohort of all children born in Great
Britain (ie, England, Wales, and
Scotland) in 1 week in 1946, with
regular follow-up surveys from birth
74
2015
Three surveys: May, 2020
(68·2%); September–October,
2020 (61·5%); February–March,
2021 (89·9%)
General Health
Questionnaire 12
697 valid samples
obtained April–June,
2021
1721
Age heterogeneous studies
Understanding Society:
the UK Household
Longitudinal Survey
A nationally representative
longitudinal household panel study,
based on a clustered-stratified
probability sample of UK households,
with all adults aged 16 years or older
in selected households surveyed
annually
16–96
2018–19
Eight surveys (full or partial
interview): April, 2020
(42·0%); May, 2020 (35·1%);
June, 2020 (33·5%);
July, 2020 (32·6%); September,
2020 (30·6%); November,
2020 (28·6%); January, 2021
(28·5%); March, 2021 (30·2%)
General Health
Questionnaire 12
6006 valid samples
obtained April–June,
2021
14 154
English Longitudinal
Study of Aging
A nationally representative
population study of individuals aged
50 years and older living in England,
with biennial surveys and periodic
refreshing of the sample to maintain
representativeness
52–90+*
2018–19
Two surveys: June–July, 2020
(75%); November–December,
2020 (73%)
Centre for
Epidemiological
Studies Depression
Scale
NA
4752
Generation Scotland:
the Scottish Family
Health Study
A family-structured, population-
based Scottish cohort, with
participants aged 18–99 years
recruited between 2006 and 2011
27–100
2006–11
Three surveys: April–June, 2020
(21·3%); July–August, 2020
(15·4%); February, 2021
(14·3%)
Patient Health
Questionnaire 9
and Generalised
Anxiety Disorder
Assessment 7
NA
3937
Avon Longitudinal
Study of Parents and
Children-Generation 0
Parents of the Avon Longitudinal
Study (G1) cohort, treated as a
separate age-heterogenous study
population (original parents)
45–81
2011–13
Three surveys:
April, 2020 (12·4%); June, 2020
(12·2%); December, 2020
(14·3%)
Short Mood and
Feelings
Questionnaire
NA
3258
The UK Adult Twin
Registry
A cohort of UK volunteer adult twins
(55% monozygotic and 43%
dizygotic) who were sampled
between age 18 and 101 years
22–96
2017–18
Three surveys: July, 2020
(77·6%); November, 2020
(76·1%); March, 2021 (76%)
Hospital and
Anxiety Depression
Scale
3137 valid samples
obtained April, 2021
3137
NA=not applicable. Study design and participants y
g
The UK National Core Studies Longitudinal Health and
Wellbeing programme combines data from multiple
UK population-based longitudinal studies to support
more robust inferences that are replicable across
data sources. Coordinated analysis across different
datasets
minimises
methodological
heterogeneity
and maximises comparability, while appropriately
accounting for study designs and characteristics of
individual datasets. The analyses were preregistered
on the Open Science Framework (10.17605/OSF.IO/
KF2GA). Using data from 11 UK longitudinal studies, we aimed
to investigate mental health consequences following
COVID-19 illness up to April, 2021. First, we examined
whether
individuals
with
self-reported
COVID-19
experience higher levels of subsequent psychological
distress, depression, and anxiety, and lower life
satisfaction than those without self-reported COVID-19. Second, we examined whether associations varied
depending on how much time had passed since infection
to determine whether effects persist beyond the acute )
Data were drawn from 11 longitudinal UK population
studies that conducted surveys before and during the
COVID-19 pandemic, with the latter surveys being done
between April, 2020, and March, 2021, and serology testing 895 Articles Articles Articles *This study classified all people older than 90 years in the 90+ category to avoid disclosure of information, given the small number of participants older than 90 years. Table: 11 UK longitudinal studies 6006 valid samples
obtained April–June,
2021
14 154 of study designs, timing of the most recent pre-pandemic
and COVID-19 surveys, response rates, region within the
UK, and analytical sample sizes are shown in the table. until June, 2021. For surveys done during the COVID-19
pandemic, serology data indicating the presence or
absence of a SARS-CoV-2 infection were available. Details www.thelancet.com/psychiatry Vol 9 November 2022 896 www.thelancet.com/psychiatry Vol 9 November 2022 Articles designed to detect current symptoms of psychological
distress (ie, general anxiety and depression). Before the
pandemic, the 28-item GHQ was used, and during it the
12-item version was used, scoring each item as 0–3. BCS70
and NCDS used the 9-item version of the Malaise
Inventory, which is self-administered and used to assess
psychological distress, scoring items as yes (1) or no (0);
scores of 4 or more are indicative of probable psychiatric
distress. ELSA used the Centre for Epidemiologic Studies
Depression Scale (CES-D), an interview in which
respondents are asked whether they experienced any
depressive symptoms (eg, feeling sad or having restless
sleep) in the previous week. For the binary classification,
we considered respondents who reported four or more
depressive symptoms on the CES-D scale as having
elevated depressive symptoms. In GS, depression and
anxiety were assessed during the pandemic using the
GAD-7 and the Patient Health Questionnaire (PHQ-9). The PHQ-9 is a 9-item, validated tool for the assessment
of depressive symptoms (eg, little interest or pleasure in
doing things) in the previous 2 weeks, rated on a 4-point
Likert scale. A score of 10 or more indicates major
depression. GS also used the 28-item GHQ to assess
pre-pandemic psychological distress, and so a comparable
composite measure was created from the GAD-7 and the
PHQ-9 scales to enable evaluation of change over time. TwinsUK used Hospital Anxiety and Depression Scale
(HADS), a 14-item questionnaire used to measure the
presence of anxiety and depressive symptoms during the
previous week in non-psychiatric populations. Responses
are on a 4-point ordinal Likert scale ranging from 0 to 3,
with a total possible score of 42 (a score of ≥11 indicates
moderate to severe levels of depression and anxiety). Articles Six of the 11 studies were of birth cohorts with all
individuals of a similar age: the Millennium Cohort Study
(MCS; born 2000–02), the Avon Longitudinal Study of
Parents and Children (ALSPAC-G1, born 1990–91), Next
Steps (NS, formerly known as the Longitudinal Study of
Young People in England; born 1989–90), the 1970 British
Cohort Study (BCS70), the National Child Development
Study (NCDS; born 1958), and the National Survey of
Health and Development (NSHD; born 1946). Five studies
were age heterogeneous: Understanding Society/The UK
Household Longitudinal Study (USoc/UKHLS), the
English Longitudinal Study of Ageing (ELSA), Generation
Scotland: The Scottish Family Health Study (GS), the UK
Adult Twin Registry (TwinsUK), and the parents of the
ALSPAC-G1 birth cohort (ALSPAC-G0). We included participants in our analytical samples who
had information available on at least one mental health
outcome measure and at least one report of COVID-19
status during the pandemic. Additionally, we only
included participants who had available data on a
minimum set of covariates, namely age, sex, and pre-
pandemic mental health. Data within studies were
weighted to be representative of their target population,
accounting for sampling design, attrition up to the most
recent pre-pandemic survey, and differential non-response
to COVID-19 surveys. Weights were not available for the
GS or TwinsUK studies. Ethics statements for each of the studies and data
access details are provided in the appendix (p 5). All studies collected informed consent from their
participants. This study did not seek any additional
institutional review board approval. Outcomes We standardised continuous scales within each study
to permit comparability of mental health estimates
across studies. This standardisation was done by creating
Z scores for each continuous mental health measure
within each study. We also did analyses with dichotomous
indicators using established cut-off scores for each scale
to define high levels of psychological distress, depression,
and anxiety, and low levels of satisfaction with life. Mental
health
symptoms
(psychological
distress,
depression, anxiety, life satisfaction) were assessed using
self-report measures across multiple timepoints of the
pandemic. The studies used a variety of measures, which
are summarised in the appendix (p 6). The MCS used the
Kessler Psychological Distress Scale (K6), a six-item self-
administered questionnaire measuring psychological
distress over the previous 30 days. Each item in K6 is
related to depressive and anxiety symptoms and rated on a
5-point Likert scale, giving a score of 0–24, with a score of
>13 indicating probable psychological distress. ALSPAC
used the Short Mood and Feelings Questionnaire (SMFQ)
and the seven-item Generalised Anxiety Disorder Scale
(GAD-7). SMFQ is a 13-item questionnaire that measures
depression symptoms in the past 2 weeks, scoring each
item as 0–2, with depression rated as none (0–4), mild
(5–9), moderate (10–14), moderately severe (15–19), and
severe (20–27). GAD-7 is a validated, self-reported measure
of anxiety used widely by healthcare professionals, using a
4-point Likert scale for the previous 2 weeks, with a cut-off
point of 5 for mild anxiety, 10 for moderate, and 15 for
severe anxiety. NS and USoc used the General Health
Questionnaire (GHQ), which is self-administered and See Online for appendix www.thelancet.com/psychiatry Vol 9 November 2022 See Online for appendix Articles ran unadjusted and fully adjusted models for potential
confounders. infection at each timepoint. We also derived two variables
to examine differences between suspected and confirmed
COVID-19. First, we created a categorical variable based on
self-reported information only: no COVID-19, self-reported
suspected COVID-19 (not test-confirmed), or self-reported
test-confirmed COVID-19. Second, we used information
from a single serology timepoint (home antibody finger-
prick test results collected between April and June, 2021
[exact dates not available]) in subsamples of some studies. Using serology data combined with self-report information,
we created a categorical variable: no COVID-19, self-
reported COVID-19 with negative serology, self-reported
COVID-19 with positive serology, and positive serology in
the absence of self-reported COVID-19. Data from antibody
tests with immunoassay qualitative detection of antibodies
against SARS-CoV-2 nucleocapsid (N) protein, of which a
positive result (N-assay) is likely to identify natural
SARS-CoV-2 infection, were used for the serology. Longitudinal studies with available information on
vaccination status used a positive anti-nucleocapsid result
at any time or a positive anti-Spike result before
vaccination.22 In the UK Virus Watch prospective
community cohort, anti-N had about 80% sensitivity for
detecting prior PCR-confirmed COVID-19 infection.23 This
sensitivity remained relatively stable for at least 269 days,
although concentrations began to decline at about 120 days
post-infection. Second, we examined whether the strength of
association varied according to time since infection. We
used generalised estimating equation models to examine
whether associations between COVID-19 and subsequent
mental health varied according to time-since-infection
categories: no COVID-19, less than 4 weeks, 4–12 weeks,
or 12 weeks or more since infection. Among those with
COVID-19, we explored the relationship between
continuous time since infection in weeks and mental
health. We also ran models incorporating a quadratic
term for continuous time since infection, to test for
non-linearity. Third, we tested for interactions between COVID-19
and sex (male or female), ethnicity (White or non-White
ethnic minorities), highest educational qualification
(degree or non-degree), and pre-pandemic mental health
and life satisfaction (using the predefined cutoff points;
appendix p 6). We focused on dichotomous pre-pandemic
mental health as a potential moderator because we were
interested
in
possible
subgroup
differences
in
associations between COVID-19 and subsequent mental
health for those above and below clinical case thresholds. We stratified analyses by age in age heterogeneous
cohorts, using the following bands: 16–29 years,
30–49 years, 50–69 years, and 70 years and older. Articles Fourth, using generalised estimating equation models,
we examined whether associations between COVID-19
and subsequent mental health differed between
suspected versus confirmed infection. Initially, we
examined whether associations between COVID-19 and
mental health differed between those with self-reported
suspected COVID-19 versus self-reported and test-
confirmed
COVID-19. Additionally,
we
explored
differences in association for those with self-reported
suspected COVID-19 versus those with serology-detected
SARS-CoV-2. Given the timing of serology assessments,
which in many cohorts was alongside or after the most
recent mental health assessment and not time varying,
we examined associations with mental health at the most
recent timepoint only (using linear and modified Poisson
regression) for those who: did not have COVID-19
(reference group); self-reported COVID-19 and had
serology
evidence
of
SARS-CoV-2;
self-reported
COVID-19 but had no serology-detected SARS-CoV-2;
and did not self-report COVID-19 but had evidence of
SARS-CoV-2 infection. In an exploratory analysis, we also
tested the association between SARS-CoV-2 serology
status (positive vs negative) with most recent mental
health scores using linear and modified Poisson
regression. www.thelancet.com/psychiatry Vol 9 November 2022 Exposures Self-reported measures of COVID-19 infection, including
the date symptoms began and whether infection was test-
confirmed (via a PCR, antibody, or lateral flow test) or
suspected, were available longitudinally at multiple
timepoints during the pandemic. A subset of studies
measured serology in subsamples of their participants. We
used data from these variables to create exposure variables
to answer the different research questions. Self-reported
COVID-19 was measured in each study and at each wave,
and we used these measures to create a binary, time-
updated variable for having ever had COVID-19 (yes or no). We used information about the time since COVID-19 to
derive continuous and categorical (no COVID-19, <4 weeks,
4–12 weeks, or ≥12 weeks) variables for the time since 897 Articles Results Individual study sample
sizes ranged from 1721 in NSHD to 14 154 in USoc. Descriptive statistics for exposures, outcomes, and
covariates are presented in the appendix (pp 7–13, 15–17). cova ates a e p ese ted
t e appe d
(pp 7
3, 5
7). By the first survey timepoint (April–June, 2020),
between 5·4% (87 of 1432) of participants in NSHD and
19·3% (324 of 1678) in NS self-reported COVID-19 across
all studies (note that survey weights have been applied to
the percentages presented here). By the final timepoint for
each study (Nov 2020–April 2021), between 11·1% (173 of
1536) participants in NSHD and 45·1% (1523 of 3837) in
MCS self-reported COVID-19; these percentages are also
weighted, and inclusive of the data from the first survey
timepoint. Serology data indicated that between 33 (4·7%)
of 697 participants in NSHD and 224 (22·7%) of 987 in
MCS had positive antibody results, indicating natural
SARS-CoV-2 infection. Of those with information on
COVID-19 and serology data, between 18 (2·6%) of
697 participants in NSHD and 172 (18·1%) of 952 in MCS
had both self-reported and serology-confirmed COVID-19,
whereas those with self-reported COVID-19 but negative
serology data ranged from 59 (8·5%) in 697 NSHD to
302 (31·7%) of 952 in MCS (appendix p 12). The proportion
of people with positive serology data who did not self-
report COVID-19 ranged from 2·1% in NSHD to
17·3% in TwinsUK; the overlap between serology and test-
confirmed self-reporting ranged from 0·86% in NSHD
to 46·1% in NS (appendix p 13). g
When examining potential subgroup differences in
associations, we did not find evidence of interactions
between COVID-19 and sex, education, ethnicity, or
pre-pandemic mental health (appendix pp 27–30). Stratified analyses by age provide some suggestion that
effects might be stronger in middle-age and older-age
groups. For instance, for depression, the standardised
difference in outcome for participants with COVID-19
compared
with
those
without
COVID-19
among
individuals aged 50–69 years was 0·10 (95% CI 0·06 to
0·15), and for those aged 70 years or older was 0·10 (–0·06
to 0·25), with weaker associations in those aged 16–29 years
(0·05, –0·00 to 0·11) and those aged 30–49 years (0·04,
–0·03 to 0·10). Similar patterns were found for anxiety. For
psychological distress, there was some evidence of a
stronger association in the group aged 50–69 years (0·13,
0·10 to 0·15), whereas associations were weaker across
other age groups. Statistical analysis Where available across studies, models were adjusted for
the following covariates: sex (male or female); age
(continuous); ethnicity (self-reported and coded into White
or non-White ethnic minorities); UK country of residence
(England, Scotland, Wales, or Northern Ireland); highest
educational qualification (degree or no degree; parental
education was used for the MCS cohort, who had not all
completed their full-time education); pre-pandemic mental
health (continuous); pre-pandemic chronic illness (yes or
no); pre-pandemic disability (yes or no); pre-pandemic self-
rated health (poor, fair, or good); partnership status
(partner or no partner); occupational classification
(assessed through the National Statistics Socio-economic
Classification and coded into four categories of professional
or managerial, intermediate, lower or manual, none or
long-term unemployed; appendix p 14). All analyses were
adjusted for data collection timepoints during the
pandemic to control for overall population level changes in
mental health at different stages of the pandemic. For the analysis, first, we used generalised estimating
equations, specifying an unstructured correlation
matrix, to examine associations between having ever had
COVID-19 and mental health outcomes—psychological
distress, depression, anxiety, and life satisfaction—
accounting for correlations between repeated measures
from the same individuals. For binary mental health
outcomes, we used modified Poisson regression with
robust standard errors to calculate relative risks.24 We g
We did several sensitivity analyses. In NSHD, NCDS,
BCS70, NS, and MCS, participants who reported
“unsure” as to whether they had COVID-19 were grouped
as COVID-19 cases if they reported an estimated infection
date, or as non-cases if they did not report a date. In 898 Articles Unadjusted results of the associations between
COVID-19 and subsequent mental health are presented
in the appendix (p 21). Pooled adjusted estimates from
meta-analyses indicated that COVID-19 was associated
with an increase in subsequent psychological distress
(standardised difference in outcome between those
with and without self-reported COVID-19: 0·10 [95% CI
0·06 to 0·13], I²=42·8%; k=8), depression (0·08
[0·05 to 0·10], I²=20·8%; k=9), and anxiety (0·08
[0·05 to 0·10], I²=0·0; k=9), and negatively associated
with life satisfaction (–0·06 [–0·08 to –0·04], I²=29·2%;
k=10; figure 1A). Results were consistent for binary
outcomes in terms of effect size and direction
(psychological distress RR 1·15 [95% CI 1·05 to 1·25],
I²=88·8%; k=8), with associations also found for
depression, anxiety, and lower satisfaction with life
(figure 1B; appendix p 22). Meta-analysed coefficients
for all research questions are reported in the appendix
(pp 18–20). Results We conducted analyses between Dec 21, 2021, and
July, 11, 2022, using 11 different longitudinal studies (k),
with 54 442 participants in total (including 33 200 [61·0%]
women and 21 242 [39·0%] men). Participants ranged
from age 16 years (in the USoc study) to aged 90 years and
older (in the ELSA study). Eight of 11 studies provided data
on ethnicity (n=40 819), including 36 802 (90·2%)
participants categorised as White, 3937 (9·6%) as non-
White, and 80 (0·2%) as missing. Individual study sample
sizes ranged from 1721 in NSHD to 14 154 in USoc. Descriptive statistics for exposures, outcomes, and
covariates are presented in the appendix (pp 7–13, 15–17). By the first survey timepoint (April–June, 2020),
between 5·4% (87 of 1432) of participants in NSHD and
19·3% (324 of 1678) in NS self-reported COVID-19 across
all studies (note that survey weights have been applied to
the percentages presented here). By the final timepoint for
each study (Nov 2020–April 2021), between 11·1% (173 of
1536) participants in NSHD and 45·1% (1523 of 3837) in
MCS self-reported COVID-19; these percentages are also
weighted, and inclusive of the data from the first survey
timepoint. Serology data indicated that between 33 (4·7%)
of 697 participants in NSHD and 224 (22·7%) of 987 in
MCS had positive antibody results, indicating natural
SARS-CoV-2 infection. Of those with information on
COVID-19 and serology data, between 18 (2·6%) of
697 participants in NSHD and 172 (18·1%) of 952 in MCS
had both self-reported and serology-confirmed COVID-19,
whereas those with self-reported COVID-19 but negative
serology data ranged from 59 (8·5%) in 697 NSHD to
302 (31·7%) of 952 in MCS (appendix p 12). The proportion
of people with positive serology data who did not self-
report COVID-19 ranged from 2·1% in NSHD to
17·3% in TwinsUK; the overlap between serology and test-
confirmed self-reporting ranged from 0·86% in NSHD
to 46·1% in NS (appendix p 13). We conducted analyses between Dec 21, 2021, and
July, 11, 2022, using 11 different longitudinal studies (k),
with 54 442 participants in total (including 33 200 [61·0%]
women and 21 242 [39·0%] men). Participants ranged
from age 16 years (in the USoc study) to aged 90 years and
older (in the ELSA study). Eight of 11 studies provided data
on ethnicity (n=40 819), including 36 802 (90·2%)
participants categorised as White, 3937 (9·6%) as non-
White, and 80 (0·2%) as missing. Role of the funding source The funders of the study had no role in study design,
data collection, data analysis, data interpretation, or
writing of the report. We examined time since infection using both categorical
and continuous variables. Pooled results indicated that the
association between COVID-19 and mental health did not
differ according to time since infection for psychological
distress, with similar associations across duration
categories, although heterogeneity increased with time
since infection (figure 2). The association between
COVID-19 and mental health and time since infection
showed similar patterns for depression, anxiety, and life
satisfaction (appendix pp 23–26). For those with COVID-19,
no association was found between continuous time since
infection in weeks and all outcomes. We examined
non-linearity with a quadratic term and found no evidence
of a non-linear association. The heterogeneity (I²) between
the time-since-infection categories was 18·1%. Statistical analysis sensitivity analyses, we compared these results to
findings in which those reporting ‘‘unsure’’ were:
(1) categorised as not having had COVID-19, and (2)
retained as a separate category. We pooled estimates across studies using random
effects meta-analysis with restricted maximum likelihood. Within age heterogeneous cohorts, we pooled estimates
from age-stratified analyses using the following age bands:
16–29 years, 30–49 years, 50–69 years, and 70 years and
older, and age homogenous cohorts were grouped within
the appropriate age band. We reported heterogeneity
indices using I², and for appropriate cases, T² and
95% prediction intervals (95% PI).25 All meta-analyses
were conducted using Stata version 17. www.thelancet.com/psychiatry Vol 9 November 2022 Articles USoc/UKHLS=Understanding Society/The UK Household
Lower levels Higher levels
Lower risk Higher risk
Psychological distress
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=42·8%, p=0·093
Depression
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=20·8%, p=0·258
Anxiety
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·922
Life satisfaction
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
TwinsUK
ALSPAC−G1
I2=29·2%, p=0·176
0·01 (−0·09 to 0·11)
0·04 (−0·04 to 0·11)
0·11 (0·06 to 0·16)
0·16 (0·11 to 0·22)
0·06 (−0·08 to 0·21)
0·08 (0·04 to 0·12)
0·13 (0·05 to 0·20)
0·09 (0·03 to 0·16)
0·10 (0·06 to 0·13)
0·02 (−0·08 to 0·12)
0·02 (−0·05 to 0·09)
0·07 (0·00 to 0·13)
0·10 (0·03 to 0·16)
0·02 (−0·06 to 0·11)
0·14 (0·07 to 0·21)
0·10 (0·03 to 0·17)
0·06 (−0·00 to 0·13)
0·12 (0·05 to 0·18)
0·08 (0·05 to 0·10)
0·02 (−0·07 to 0·11)
0·06 (−0·02 to 0·14)
0·10 (0·03 to 0·16)
0·10 (0·04 to 0·16)
0·10 (−0·00 to 0·20)
0·08 (0·00 to 0·15)
0·08 (0·02 to 0·14)
0·05 (−0·02 to 0·12)
0·09 (0·01 to 0·16)
0·08 (0·05 to 0·10)
−0·02 (−0·11 to 0·08)
0·01 (−0·06 to 0·09)
−0·13 (−0·20 to −0·06)
−0·10 (−0·15 to −0·04)
−0·04 (−0·22 to 0·13)
−0·08 (−0·11 to −0·05)
−0·02 (−0·10 to 0·07)
−0·03 (−0·10 to 0·05)
−0·06 (−0·10 to −0·02)
−0·05 (−0·11 to 0·02)
−0·06 (−0·08 to −0·04)
7·48
10·58
16·05
15·74
4·14
20·53
11·57
13·90
100·00
6·33
10·94
12·65
13·59
8·11
11·25
11·76
13·49
11·89
7·13
9·60
14·16
16·18
5·54
10·18
15·31
11·72
10·18
100·00
5·34
7·53
9·12
11·66
1·78
21·99
6·14
8·54
18·23
9·66
100·00
−0·4
−0·2
0
0·2
0·4
Psychological distress
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=88·8%, p=0·000
Depression
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=76·8%, p=0·000
Anxiety
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=63·5%, p=0·005
Low life satisfaction
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=59·9%, p=0·011
0·97 (0·79 to 1·19)
1·03 (0·92 to 1·16)
1·24 (1·10 to 1·40)
1·43 (1·27 to 1·62)
1·28 (0·88 to 1·87)
1·16 (1·10 to 1·22)
1·17 (1·07 to 1·27)
1·02 (1·00 to 1·04)
1·15 (1·05 to 1·25)
0·93 (0·78 to 1·10)
0·93 (0·78 to 1·10)
1·21 (1·01 to 1·46)
1·34 (1·11 to 1·61)
1·17 (0·91 to 1·52)
1·32 (1·14 to 1·52)
1·01 (0·99 to 1·02)
1·09 (0·95 to 1·25)
1·31 (1·01 to 1·71)
1·12 (1·02 to 1·23)
1·06 (0·92 to 1·23)
1·05 (0·88 to 1·24)
1·23 (1·03 to 1·47)
1·33 (1·11 to 1·58)
1·29 (0·90 to 1·83)
1·14 (0·99 to 1·32)
1·01 (0·99 to 1·03)
1·07 (0·94 to 1·23)
1·22 (1·01 to 1·48)
1·12 (1·04 to 1·21)
0·96 (0·83 to 1·11)
0·96 (0·82 to 1·13)
1·25 (1·10 to 1·43)
1·23 (1·10 to 1·37)
0·96 (0·75 to 1·23)
1·09 (1·04 to 1·13)
0·98 (0·76 to 1·25)
1·03 (0·96 to 1·11)
1·17 (1·05 to 1·30)
1·09 (1·02 to 1·15)
8·72
13·15
13·00
12·91
4·09
16·23
14·82
17·09
100·00
10·95
10·83
10·46
10·52
7·62
12·40
17·16
12·67
7·37
100·00
11·77
10·25
9·77
9·78
3·65
11·95
21·27
12·61
8·95
100·00
9·51
8·42
10·88
12·73
4·71
19·78
4·66
16·28
13·04
100·00
0·5
1
2
A Continuous mental health outcomes
B Binary mental health outcomes
Coefficient (95% CI)
Weight (%)
RR (95% CI)
Weight (%) Figure 1: Associations between COVID−19 and continuous (A) and binary (B) mental health outcomes Figure 1: Associations between COVID−19 and continuous (A) and binary (B) mental health outcomes
Estimates from longitudinal generalised estimating equation models with ever-COVID-19 exposure and mental health outcomes for each included study and the overall pooled estimate. Articles Lower levels Higher levels
Lower risk Higher risk
Psychological distress
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=42·8%, p=0·093
Depression
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=20·8%, p=0·258
Anxiety
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·922
Life satisfaction
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
TwinsUK
ALSPAC−G1
I2=29·2%, p=0·176
0·01 (−0·09 to 0·11)
0·04 (−0·04 to 0·11)
0·11 (0·06 to 0·16)
0·16 (0·11 to 0·22)
0·06 (−0·08 to 0·21)
0·08 (0·04 to 0·12)
0·13 (0·05 to 0·20)
0·09 (0·03 to 0·16)
0·10 (0·06 to 0·13)
0·02 (−0·08 to 0·12)
0·02 (−0·05 to 0·09)
0·07 (0·00 to 0·13)
0·10 (0·03 to 0·16)
0·02 (−0·06 to 0·11)
0·14 (0·07 to 0·21)
0·10 (0·03 to 0·17)
0·06 (−0·00 to 0·13)
0·12 (0·05 to 0·18)
0·08 (0·05 to 0·10)
0·02 (−0·07 to 0·11)
0·06 (−0·02 to 0·14)
0·10 (0·03 to 0·16)
0·10 (0·04 to 0·16)
0·10 (−0·00 to 0·20)
0·08 (0·00 to 0·15)
0·08 (0·02 to 0·14)
0·05 (−0·02 to 0·12)
0·09 (0·01 to 0·16)
0·08 (0·05 to 0·10)
−0·02 (−0·11 to 0·08)
0·01 (−0·06 to 0·09)
−0·13 (−0·20 to −0·06)
−0·10 (−0·15 to −0·04)
−0·04 (−0·22 to 0·13)
−0·08 (−0·11 to −0·05)
−0·02 (−0·10 to 0·07)
−0·03 (−0·10 to 0·05)
−0·06 (−0·10 to −0·02)
−0·05 (−0·11 to 0·02)
−0·06 (−0·08 to −0·04)
7·48
10·58
16·05
15·74
4·14
20·53
11·57
13·90
100·00
6·33
10·94
12·65
13·59
8·11
11·25
11·76
13·49
11·89
7·13
9·60
14·16
16·18
5·54
10·18
15·31
11·72
10·18
100·00
5·34
7·53
9·12
11·66
1·78
21·99
6·14
8·54
18·23
9·66
100·00
−0·4
−0·2
0
0·2
0·4
Psychological distress
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=88·8%, p=0·000
Depression
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=76·8%, p=0·000
Anxiety
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=63·5%, p=0·005
Low life satisfaction
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=59·9%, p=0·011
0·97 (0·79 to 1·19)
1·03 (0·92 to 1·16)
1·24 (1·10 to 1·40)
1·43 (1·27 to 1·62)
1·28 (0·88 to 1·87)
1·16 (1·10 to 1·22)
1·17 (1·07 to 1·27)
1·02 (1·00 to 1·04)
1·15 (1·05 to 1·25)
0·93 (0·78 to 1·10)
0·93 (0·78 to 1·10)
1·21 (1·01 to 1·46)
1·34 (1·11 to 1·61)
1·17 (0·91 to 1·52)
1·32 (1·14 to 1·52)
1·01 (0·99 to 1·02)
1·09 (0·95 to 1·25)
1·31 (1·01 to 1·71)
1·12 (1·02 to 1·23)
1·06 (0·92 to 1·23)
1·05 (0·88 to 1·24)
1·23 (1·03 to 1·47)
1·33 (1·11 to 1·58)
1·29 (0·90 to 1·83)
1·14 (0·99 to 1·32)
1·01 (0·99 to 1·03)
1·07 (0·94 to 1·23)
1·22 (1·01 to 1·48)
1·12 (1·04 to 1·21)
0·96 (0·83 to 1·11)
0·96 (0·82 to 1·13)
1·25 (1·10 to 1·43)
1·23 (1·10 to 1·37)
0·96 (0·75 to 1·23)
1·09 (1·04 to 1·13)
0·98 (0·76 to 1·25)
1·03 (0·96 to 1·11)
1·17 (1·05 to 1·30)
1·09 (1·02 to 1·15)
8·72
13·15
13·00
12·91
4·09
16·23
14·82
17·09
100·00
10·95
10·83
10·46
10·52
7·62
12·40
17·16
12·67
7·37
100·00
11·77
10·25
9·77
9·78
3·65
11·95
21·27
12·61
8·95
100·00
9·51
8·42
10·88
12·73
4·71
19·78
4·66
16·28
13·04
100·00
0·5
1
2
A Continuous mental health outcomes
B Binary mental health outcomes
Coefficient (95% CI)
Weight (%)
RR (95% CI)
Weight (%) Figure 1: Associations between COVID−19 and continuous (A) and binary (B) mental health outcomes
Estimates from longitudinal generalised estimating equation models with ever-COVID-19 exposure and mental health outcomes for each included study and the overall pooled estimate. Articles ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970 British Cohort Study. ELSA=the English Longitudinal Study of
Ageing. GS=Generation Scotland: The Scottish Family Health Study. MCS=Millennium Cohort Study. NCDS=the National Child Development Study. NS=Next Steps, formerly known as the Longitudinal
Study of Young People in England. NSHD=the National Survey of Health and Development. RR=risk ratio. TwinsUK=the UK Adult Twin Registry. Articles Figure 1: Associations between COVID−19 and continuous (A) and binary (B) mental health outcomes
Estimates from longitudinal generalised estimating equation models with ever-COVID-19 exposure and mental health outcomes for each included study and the overall pooled estimate. ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970 British Cohort Study. ELSA=the English Longitudinal Study of
Ageing. GS=Generation Scotland: The Scottish Family Health Study. MCS=Millennium Cohort Study. NCDS=the National Child Development Study. NS=Next Steps, formerly known as the Longitudinal
Study of Young People in England. NSHD=the National Survey of Health and Development. RR=risk ratio. TwinsUK=the UK Adult Twin Registry. USoc/UKHLS=Understanding Society/The UK Household
Longitudinal Study. www.thelancet.com/psychiatry Vol 9 November 2022 Results Associations between COVID-19 and life
satisfaction were similar across age groups. However,
given the wide confidence intervals we cannot be confident
in these age group differences (appendix pp 31–34).f We
examined
whether
associations
differed
between participants with suspected COVID-19 versus www.thelancet.com/psychiatry Vol 9 November 2022 899 Articles Articles Articles ALSPAC-G0=the parents of the ALSPA
British Cohort Study. NCDS=the National Child Development Study. Articles NS=Next Steps, formerly know <4 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
I2=0·0%, p=0·887
4−12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
I2=21·6%, p=0·277
≥12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
I2=62·8%, p=0·029
0·09 (−0·10 to 0·29)
0·11 (−0·05 to 0·28)
0·09 (−0·05 to 0·22)
0·15 (0·03 to 0·26)
0·06 (−0·06 to 0·18)
0·10 (0·04 to 0·16)
0·06 (−0·12 to 0·25)
−0·04 (−0·21 to 0·14)
0·12 (0·03 to 0·22)
0·16 (0·08 to 0·25)
0·06 (−0·08 to 0·21)
0·10 (0·04 to 0·17)
−0·01 (−0·11 to 0·09)
0·05 (−0·04 to 0·13)
0·11 (0·05 to 0·17)
0·16 (0·10 to 0·22)
0·13 (0·05 to 0·20)
0·10 (0·04 to 0·15)
Coefficient (95% CI)
9·86
14·97
21·05
27·62
26·50
100·00
9·80
10·84
29·16
34·73
15·47
100·00
14·68
18·42
23·77
23·31
19·81
100·00
Weight (%)
−0·3
0
0·3
A Psychological distress
Lower levels Higher levels Coefficient (95% CI)
Weight (%)
<4 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=54·3%, p=0·041
4−12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=23·8%, p=0·248
≥12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·570
0·07 (−0·19 to 0·33)
0·15 (−0·04 to 0·33)
0·06 (−0·07 to 0·20)
0·20 (0·03 to 0·36)
0·00 (−0·14 to 0·14)
0·11 (0·00 to 0·22)
0·34 (0·20 to 0·48)
0·13 (0·05 to 0·22)
0·12 (−0·06 to 0·31)
−0·07 (−0·20 to 0·06)
0·00 (−0·09 to 0·09)
0·09 (−0·01 to 0·20)
0·05 (−0·11 to 0·22)
−0·01 (−0·09 to 0·07)
0·10 (0·00 to 0·19)
0·03 (−0·01 to 0·08)
−0·01 (−0·11 to 0·10)
0·03 (−0·05 to 0·11)
0·08 (0·01 to 0·16)
0·09 (0·02 to 0·16)
0·10 (0·01 to 0·18)
0·05 (−0·03 to 0·12)
0·09 (0·02 to 0·17)
0·07 (0·04 to 0·10)
7·73
12·15
16·59
13·59
15·67
18·80
15·47
100·00
6·26
11·66
19·00
15·77
7·52
21·80
18·00
100·00
7·86
14·58
16·00
19·13
12·29
16·08
14·05
100·00
−0·3
0
0·3
B Depression
Coefficient (95% CI) Weight (%)
D Life satisfaction
Lower levels Higher levels D Life satisfaction Coefficient (95% CI) Weight (%)
Coefficient (95% CI) Weight (%)
C Anxiety
D Life satisfaction
<4 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·953
4−12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·936
≥12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·498
0·05 (−0·16 to 0·26)
0·02 (−0·21 to 0·26)
0·02 (−0·14 to 0·17)
0·06 (−0·11 to 0·22)
0·10 (−0·02 to 0·23)
0·06 (−0·07 to 0·18)
0·12 (−0·01 to 0·26)
0·07 (0·01 to 0·13)
0·10 (−0·09 to 0·28)
0·05 (−0·08 to 0·17)
0·12 (0·02 to 0·23)
0·07 (−0·02 to 0·16)
0·06 (−0·07 to 0·19)
0·04 (−0·06 to 0·13)
0·05 (−0·03 to 0·14)
0·07 (0·03 to 0·11)
0·01 (−0·09 to 0·10)
0·07 (−0·02 to 0·15)
0·10 (0·03 to 0·17)
0·11 (0·05 to 0·17)
0·13 (0·05 to 0·21)
0·08 (−0·00 to 0·16)
0·13 (0·03 to 0·22)
0·09 (0·06 to 0·12)
7·56
6·09
13·88
11·85
21·22
20·30
19·10
100·00
4·89
10·82
14·97
19·60
9·68
19·04
20·99
100·00
9·61
12·47
18·32
21·61
13·78
13·73
10·48
100·00
−0·3
0
0·3
<4 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=0·6%, p=0·403
4−12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=60·2%, p=0·040
≥12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=55·0%, p=0·064
0·11 (−0·12 to 0·33)
−0·04 (−0·22 to 0·14)
−0·13 (−0·32 to 0·05)
−0·11 (−0·26 to 0·05)
0·01 (−0·10 to 0·11)
−0·03 (−0·10 to 0·03)
−0·15 (−0·33 to 0·04)
0·17 (0·03 to 0·31)
−0·07 (−0·18 to 0·04)
−0·03 (−0·10 to 0·04)
−0·02 (−0·10 to 0·07)
−0·02 (−0·09 to 0·06)
−0·00 (−0·10 to 0·10)
−0·02 (−0·10 to 0·07)
−0·14 (−0·22 to −0·07)
−0·11 (−0·17 to −0·05)
−0·05 (−0·12 to 0·02)
−0·07 (−0·12 to −0·02)
9·54
14·84
14·35
20·24
41·03
100·00
11·59
16·59
20·55
26·74
24·52
100·00
15·40
18·67
21·03
23·92
20·98
100·00
−0·3
0
0·3
Lower levels Higher levels
Lower levels Higher levels
Figure 2: Association between time since COVID-19 infection and continuous mental health outcomes
Data for each included study and the overall pooled estimate. Articles Participants who self-reported having COVID-19, but had www.thelancet.com/psychiatry Vol 9 November 2022 900 Articles Coefficient (95% CI)
Weight (%)
<4 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=54·3%, p=0·041
4−12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=23·8%, p=0·248
≥12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·570
0·07 (−0·19 to 0·33)
0·15 (−0·04 to 0·33)
0·06 (−0·07 to 0·20)
0·20 (0·03 to 0·36)
0·00 (−0·14 to 0·14)
0·11 (0·00 to 0·22)
0·34 (0·20 to 0·48)
0·13 (0·05 to 0·22)
0·12 (−0·06 to 0·31)
−0·07 (−0·20 to 0·06)
0·00 (−0·09 to 0·09)
0·09 (−0·01 to 0·20)
0·05 (−0·11 to 0·22)
−0·01 (−0·09 to 0·07)
0·10 (0·00 to 0·19)
0·03 (−0·01 to 0·08)
−0·01 (−0·11 to 0·10)
0·03 (−0·05 to 0·11)
0·08 (0·01 to 0·16)
0·09 (0·02 to 0·16)
0·10 (0·01 to 0·18)
0·05 (−0·03 to 0·12)
0·09 (0·02 to 0·17)
0·07 (0·04 to 0·10)
7·73
12·15
16·59
13·59
15·67
18·80
15·47
100·00
6·26
11·66
19·00
15·77
7·52
21·80
18·00
100·00
7·86
14·58
16·00
19·13
12·29
16·08
14·05
100·00
−0·3
0
0·3
B Depression
Coefficient (95% CI) Weight (%)
D Life satisfaction
<4 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=0·6%, p=0·403
4−12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=60·2%, p=0·040
≥12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=55·0%, p=0·064
0·11 (−0·12 to 0·33)
−0·04 (−0·22 to 0·14)
−0·13 (−0·32 to 0·05)
−0·11 (−0·26 to 0·05)
0·01 (−0·10 to 0·11)
−0·03 (−0·10 to 0·03)
−0·15 (−0·33 to 0·04)
0·17 (0·03 to 0·31)
−0·07 (−0·18 to 0·04)
−0·03 (−0·10 to 0·04)
−0·02 (−0·10 to 0·07)
−0·02 (−0·09 to 0·06)
−0·00 (−0·10 to 0·10)
−0·02 (−0·10 to 0·07)
−0·14 (−0·22 to −0·07)
−0·11 (−0·17 to −0·05)
−0·05 (−0·12 to 0·02)
−0·07 (−0·12 to −0·02)
9·54
14·84
14·35
20·24
41·03
100·00
11·59
16·59
20·55
26·74
24·52
100·00
15·40
18·67
21·03
23·92
20·98
100·00
−0·3
0
0·3
Lower levels Higher levels
Lower levels Higher levels <4 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
I2=0·0%, p=0·887
4−12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
I2=21·6%, p=0·277
≥12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
I2=62·8%, p=0·029
0·09 (−0·10 to 0·29)
0·11 (−0·05 to 0·28)
0·09 (−0·05 to 0·22)
0·15 (0·03 to 0·26)
0·06 (−0·06 to 0·18)
0·10 (0·04 to 0·16)
0·06 (−0·12 to 0·25)
−0·04 (−0·21 to 0·14)
0·12 (0·03 to 0·22)
0·16 (0·08 to 0·25)
0·06 (−0·08 to 0·21)
0·10 (0·04 to 0·17)
−0·01 (−0·11 to 0·09)
0·05 (−0·04 to 0·13)
0·11 (0·05 to 0·17)
0·16 (0·10 to 0·22)
0·13 (0·05 to 0·20)
0·10 (0·04 to 0·15)
Coefficient (95% CI)
9·86
14·97
21·05
27·62
26·50
100·00
9·80
10·84
29·16
34·73
15·47
100·00
14·68
18·42
23·77
23·31
19·81
100·00
Weight (%)
−0·3
0
0·3
A Psychological distress
Coefficient (95% CI) Weight (%)
C Anxiety
<4 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·953
4−12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·936
≥12 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·498
0·05 (−0·16 to 0·26)
0·02 (−0·21 to 0·26)
0·02 (−0·14 to 0·17)
0·06 (−0·11 to 0·22)
0·10 (−0·02 to 0·23)
0·06 (−0·07 to 0·18)
0·12 (−0·01 to 0·26)
0·07 (0·01 to 0·13)
0·10 (−0·09 to 0·28)
0·05 (−0·08 to 0·17)
0·12 (0·02 to 0·23)
0·07 (−0·02 to 0·16)
0·06 (−0·07 to 0·19)
0·04 (−0·06 to 0·13)
0·05 (−0·03 to 0·14)
0·07 (0·03 to 0·11)
0·01 (−0·09 to 0·10)
0·07 (−0·02 to 0·15)
0·10 (0·03 to 0·17)
0·11 (0·05 to 0·17)
0·13 (0·05 to 0·21)
0·08 (−0·00 to 0·16)
0·13 (0·03 to 0·22)
0·09 (0·06 to 0·12)
7·56
6·09
13·88
11·85
21·22
20·30
19·10
100·00
4·89
10·82
14·97
19·60
9·68
19·04
20·99
100·00
9·61
12·47
18·32
21·61
13·78
13·73
10·48
100·00
−0·3
0
0·3
Lower levels Higher levels
Lower levels Higher levels
Figure 2: Association between time since COVID-19 infection and continuous mental health ou
Data for each included study and the overall pooled estimate. Articles ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970
British Cohort Study. NCDS=the National Child Development Study. NS=Next Steps, formerly known as the Longitudinal Study of Young People in England. TwinsUK=the UK Adult Twin Registry. D Life satisfaction Coefficient (95% CI) Weight (%) Coefficient (95% CI) Weight (%) <4 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=0·6%, p=0·403
4−12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=60·2%, p=0·040
≥12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=55·0%, p=0·064
0·11 (−0·12 to 0·33)
−0·04 (−0·22 to 0·14)
−0·13 (−0·32 to 0·05)
−0·11 (−0·26 to 0·05)
0·01 (−0·10 to 0·11)
−0·03 (−0·10 to 0·03)
−0·15 (−0·33 to 0·04)
0·17 (0·03 to 0·31)
−0·07 (−0·18 to 0·04)
−0·03 (−0·10 to 0·04)
−0·02 (−0·10 to 0·07)
−0·02 (−0·09 to 0·06)
−0·00 (−0·10 to 0·10)
−0·02 (−0·10 to 0·07)
−0·14 (−0·22 to −0·07)
−0·11 (−0·17 to −0·05)
−0·05 (−0·12 to 0·02)
−0·07 (−0·12 to −0·02)
9·54
14·84
14·35
20·24
41·03
100·00
11·59
16·59
20·55
26·74
24·52
100·00
15·40
18·67
21·03
23·92
20·98
100·00
−0·3
0
0·3
Lower levels Higher levels <4 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=0·6%, p=0·403
4−12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=60·2%, p=0·040
≥12 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=55·0%, p=0·064
0·11 (−0·12 to 0·33)
−0·04 (−0·22 to 0·14)
−0·13 (−0·32 to 0·05)
−0·11 (−0·26 to 0·05)
0·01 (−0·10 to 0·11)
−0·03 (−0·10 to 0·03)
−0·15 (−0·33 to 0·04)
0·17 (0·03 to 0·31)
−0·07 (−0·18 to 0·04)
−0·03 (−0·10 to 0·04)
−0·02 (−0·10 to 0·07)
−0·02 (−0·09 to 0·06)
−0·00 (−0·10 to 0·10)
−0·02 (−0·10 to 0·07)
−0·14 (−0·22 to −0·07)
−0·11 (−0·17 to −0·05)
−0·05 (−0·12 to 0·02)
−0·07 (−0·12 to −0·02)
9·54
14·84
14·35
20·24
41·03
100·00
11·59
16·59
20·55
26·74
24·52
100·00
15·40
18·67
21·03
23·92
20·98
100·00
−0·3
0
0·3
Lower levels Higher levels <4 weeks since infection
MCS
NS
BCS70
NCDS
ALSPAC−G1
I2=0·6%, p=0·403
4−12 weeks since infection <4 weeks since infection
MCS
NS
BCS70
NCDS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·953
4 12 weeks since infection Figure 2: Association between time since COVID-19 infection and continuous mental health outcomes
Data for each included study and the overall pooled estimate. ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970
British Cohort Study. NCDS=the National Child Development Study. Figure 2: Association between time since COVID-19 infection and continuous mental health outcomes
Data for each included study and the overall pooled estimate. ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970
British Cohort Study. NCDS=the National Child Development Study. NS=Next Steps, formerly known as the Longitudinal Study of Young People in England. TwinsUK=the UK Adult Twin Registry. Articles ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970 British Cohort Study. ELSA=the English Longitudinal Study of
Ageing. GS=Generation Scotland: The Scottish Family Health Study. MCS=Millennium Cohort Study. NCDS=the National Child Development Study. NS=Next Steps, formerly known as the Longitudinal
Study of Young People in England. NSHD=the National Survey of Health and Development. RR=risk ratio. TwinsUK=the UK Adult Twin Registry. USoc/UKHLS=Understanding Society/The UK Household
Longitudinal Study test-confirmed COVID-19, both based on self-reporting. Both suspected COVID-19 (0·09 [95% CI 0·07 to 0·11],
I²=0·0%; k=8) and test-confirmed COVID-19 (0·11
[0·02 to 0·19], I²=68·3%; k=8) were associated with
increased psychological distress, with similar patterns
for depression and anxiety, although only suspected
and not test-confirmed COVID-19 was associated with
lower life satisfaction (figure 3; appendix 35–38). negative serology, had higher levels of psychological
distress (0·11 [95% CI 0·06 to 0·16], I²=29·5%; k=7),
depression, and anxiety, and lower life satisfaction than
those without COVID-19 based on self-report and serology. Associations were not found for those who self-reported
having COVID-19 and had positive serology for
psychological distress, depression, anxiety, or life
satisfaction (figure 4). For participants who had positive
serology but did not self-report COVID-19, we did not find
associations with any of the mental health outcomes
(eg, psychological distress, –0·02 [95% CI –0·10 to 0·05], Finally, we examined whether associations varied
according to combined self-report and serology data. Articles Suspected
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=0·0%, p=0·762
Test-confirmed
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=68·3%, p=0·002
0·06 (−0·03 to 0·15)
0·07 (−0·01 to 0·15)
0·12 (0·07 to 0·17)
0·10 (0·05 to 0·16)
0·10 (−0·09 to 0·29)
0·07 (0·03 to 0·11)
0·09 (0·02 to 0·17)
0·12 (0·04 to 0·19)
0·09 (0·07 to 0·11)
−0·12 (−0·30 to 0·05)
−0·07 (−0·24 to 0·09)
0·14 (0·03 to 0·25)
0·23 (0·11 to 0·35)
0·43 (−0·09 to 0·95)
0·18 (0·09 to 0·27)
0·22 (0·04 to 0·41)
0·07 (−0·01 to 0·15)
0·11 (0·02 to 0·19)
Coefficient (95% CI)
6·31
8·29
18·32
17·25
1·36
30·87
9·14
8·47
100·00
10·94
11·81
15·50
14·78
2·38
16·74
10·33
17·54
100·00
Weight (%)
Coefficient (95% CI)
Weight (%)
Coefficient (95% CI)
Weight (%)
Coefficient (95% CI)
Weight (%)
A Psychological distress
Suspected
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·837
Test-confirmed
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=60·3%, p=0·010
0·06 (−0·04 to 0·15)
0·04 (−0·03 to 0·12)
0·09 (0·02 to 0·16)
0·07 (0·01 to 0·13)
0·02 (−0·07 to 0·11)
0·10 (0·03 to 0·18)
0·11 (0·03 to 0·20)
0·07 (0·01 to 0·14)
0·09 (0·02 to 0·17)
0·07 (0·05 to 0·10)
−0·15 (−0·33 to 0·02)
−0·00 (−0·14 to 0·13)
−0·01 (−0·12 to 0·10)
0·20 (0·04 to 0·36)
0·04 (−0·14 to 0·21)
0·27 (0·08 to 0·46)
0·08 (−0·02 to 0·18)
0·01 (−0·12 to 0·14)
0·24 (0·07 to 0·41)
0·07 (−0·01 to 0·14)
6·74
12·23
13·32
16·16
7·51
10·89
8·20
13·12
11·81
100·00
9·45
11·94
13·61
10·32
9·50
8·73
14·54
12·30
9·61
100·00
B Depression
Suspected
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·884
Test-confirmed
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·804
0·06 (−0·03 to 0·15)
0·05 (−0·03 to 0·13)
0·11 (0·05 to 0·18)
0·06 (0·01 to 0·12)
0·13 (−0·00 to 0·25)
0·06 (−0·01 to 0·14)
0·10 (0·02 to 0·18)
0·06 (−0·02 to 0·13)
0·10 (0·01 to 0·18)
0·08 (0·05 to 0·10)
−0·08 (−0·24 to 0·08)
0·04 (−0·10 to 0·18)
0·03 (−0·09 to 0·16)
0·15 (0·00 to 0·30)
0·04 (−0·12 to 0·20)
0·06 (−0·14 to 0·27)
0·05 (−0·03 to 0·13)
0·05 (−0·09 to 0·18)
0·02 (−0·12 to 0·17)
0·04 (0·00 to 0·09)
8·09
10·43
16·72
19·40
4·10
10·77
10·28
10·66
9·56
100·00
7·47
10·09
11·13
8·53
7·18
4·49
32·65
10·13
8·34
100·00
C Anxiety
Suspected
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=43·2%, p=0·079
Test-confirmed
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=23·3%, p=0·236
−0·03 (−0·12 to 0·06)
−0·02 (−0·10 to 0·06)
−0·14 (−0·20 to −0·07)
−0·07 (−0·13 to −0·01)
−0·09 (−0·31 to 0·12)
−0·09 (−0·16 to −0·03)
0·05 (−0·05 to 0·15)
−0·01 (−0·09 to 0·06)
−0·05 (−0·13 to 0·02)
−0·05 (−0·09 to −0·02)
−0·04 (−0·20 to 0·12)
0·20 (0·05 to 0·35)
−0·02 (−0·14 to 0·09)
−0·02 (−0·14 to 0·10)
−0·28 (−0·72 to 0·16)
0·02 (−0·07 to 0·10)
−0·07 (−0·22 to 0·07)
−0·05 (−0·23 to 0·14)
−0·02 (−0·15 to 0·11)
−0·00 (−0·06 to 0·05)
9·53
11·70
13·87
15·57
2·41
13·56
8·30
12·12
12·94
100·00
8·86
9·87
14·14
14·31
1·39
21·61
10·34
7·31
12·17
100·00
D Life satisfaction
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
Lower levels Higher levels
Lower levels Higher levels
Lower levels Higher levels
Lower levels Higher levels Coefficient (95% CI)
Weight (%)
Suspected
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·837
Test-confirmed
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=60·3%, p=0·010
0·06 (−0·04 to 0·15)
0·04 (−0·03 to 0·12)
0·09 (0·02 to 0·16)
0·07 (0·01 to 0·13)
0·02 (−0·07 to 0·11)
0·10 (0·03 to 0·18)
0·11 (0·03 to 0·20)
0·07 (0·01 to 0·14)
0·09 (0·02 to 0·17)
0·07 (0·05 to 0·10)
−0·15 (−0·33 to 0·02)
−0·00 (−0·14 to 0·13)
−0·01 (−0·12 to 0·10)
0·20 (0·04 to 0·36)
0·04 (−0·14 to 0·21)
0·27 (0·08 to 0·46)
0·08 (−0·02 to 0·18)
0·01 (−0·12 to 0·14)
0·24 (0·07 to 0·41)
0·07 (−0·01 to 0·14)
6·74
12·23
13·32
16·16
7·51
10·89
8·20
13·12
11·81
100·00
9·45
11·94
13·61
10·32
9·50
8·73
14·54
12·30
9·61
100·00
B Depression
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
Lower levels Higher levels Suspected
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=0·0%, p=0·762
Test-confirmed
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=68·3%, p=0·002
0·06 (−0·03 to 0·15)
0·07 (−0·01 to 0·15)
0·12 (0·07 to 0·17)
0·10 (0·05 to 0·16)
0·10 (−0·09 to 0·29)
0·07 (0·03 to 0·11)
0·09 (0·02 to 0·17)
0·12 (0·04 to 0·19)
0·09 (0·07 to 0·11)
−0·12 (−0·30 to 0·05)
−0·07 (−0·24 to 0·09)
0·14 (0·03 to 0·25)
0·23 (0·11 to 0·35)
0·43 (−0·09 to 0·95)
0·18 (0·09 to 0·27)
0·22 (0·04 to 0·41)
0·07 (−0·01 to 0·15)
0·11 (0·02 to 0·19)
Coefficient (95% CI)
6·31
8·29
18·32
17·25
1·36
30·87
9·14
8·47
100·00
10·94
11·81
15·50
14·78
2·38
16·74
10·33
17·54
100·00
Weight (%
A Psychological distress
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
Lower levels Higher levels Lower levels Higher levels Coefficient (95% CI)
Weight (%)
Suspected
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·884
Test-confirmed
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·804
0·06 (−0·03 to 0·15)
0·05 (−0·03 to 0·13)
0·11 (0·05 to 0·18)
0·06 (0·01 to 0·12)
0·13 (−0·00 to 0·25)
0·06 (−0·01 to 0·14)
0·10 (0·02 to 0·18)
0·06 (−0·02 to 0·13)
0·10 (0·01 to 0·18)
0·08 (0·05 to 0·10)
−0·08 (−0·24 to 0·08)
0·04 (−0·10 to 0·18)
0·03 (−0·09 to 0·16)
0·15 (0·00 to 0·30)
0·04 (−0·12 to 0·20)
0·06 (−0·14 to 0·27)
0·05 (−0·03 to 0·13)
0·05 (−0·09 to 0·18)
0·02 (−0·12 to 0·17)
0·04 (0·00 to 0·09)
8·09
10·43
16·72
19·40
4·10
10·77
10·28
10·66
9·56
100·00
7·47
10·09
11·13
8·53
7·18
4·49
32·65
10·13
8·34
100·00
C Anxiety
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
Lower levels Higher levels Coefficient (95% CI)
Weight (%)
Suspected
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=43·2%, p=0·079
Test-confirmed
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=23·3%, p=0·236
−0·03 (−0·12 to 0·06)
−0·02 (−0·10 to 0·06)
−0·14 (−0·20 to −0·07)
−0·07 (−0·13 to −0·01)
−0·09 (−0·31 to 0·12)
−0·09 (−0·16 to −0·03)
0·05 (−0·05 to 0·15)
−0·01 (−0·09 to 0·06)
−0·05 (−0·13 to 0·02)
−0·05 (−0·09 to −0·02)
−0·04 (−0·20 to 0·12)
0·20 (0·05 to 0·35)
−0·02 (−0·14 to 0·09)
−0·02 (−0·14 to 0·10)
−0·28 (−0·72 to 0·16)
0·02 (−0·07 to 0·10)
−0·07 (−0·22 to 0·07)
−0·05 (−0·23 to 0·14)
−0·02 (−0·15 to 0·11)
−0·00 (−0·06 to 0·05)
9·53
11·70
13·87
15·57
2·41
13·56
8·30
12·12
12·94
100·00
8·86
9·87
14·14
14·31
1·39
21·61
10·34
7·31
12·17
100·00
D Life satisfaction
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
Lower levels Higher levels versus test-confirmed COVID-19 infection and mental health outcomes Figure 3: Suspected versus test-confirmed COVID-19 infection and mental health outcomes
Data for each included study and the overall pooled estimate. Articles Suspected
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=0·0%, p=0·762
Test-confirmed
MCS
NS
BCS70
NCDS
NSHD
USoc
GS
TwinsUK
I2=68·3%, p=0·002
0·06 (−0·03 to 0·15)
0·07 (−0·01 to 0·15)
0·12 (0·07 to 0·17)
0·10 (0·05 to 0·16)
0·10 (−0·09 to 0·29)
0·07 (0·03 to 0·11)
0·09 (0·02 to 0·17)
0·12 (0·04 to 0·19)
0·09 (0·07 to 0·11)
−0·12 (−0·30 to 0·05)
−0·07 (−0·24 to 0·09)
0·14 (0·03 to 0·25)
0·23 (0·11 to 0·35)
0·43 (−0·09 to 0·95)
0·18 (0·09 to 0·27)
0·22 (0·04 to 0·41)
0·07 (−0·01 to 0·15)
0·11 (0·02 to 0·19)
Coefficient (95% CI)
6·31
8·29
18·32
17·25
1·36
30·87
9·14
8·47
100·00
10·94
11·81
15·50
14·78
2·38
16·74
10·33
17·54
100·00
Weight (%)
Coefficient (95% CI)
Weight (%)
Coefficient (95% CI)
Weight (%)
Coefficient (95% CI)
Weight (%)
A Psychological distress
Suspected
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·837
Test-confirmed
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=60·3%, p=0·010
0·06 (−0·04 to 0·15)
0·04 (−0·03 to 0·12)
0·09 (0·02 to 0·16)
0·07 (0·01 to 0·13)
0·02 (−0·07 to 0·11)
0·10 (0·03 to 0·18)
0·11 (0·03 to 0·20)
0·07 (0·01 to 0·14)
0·09 (0·02 to 0·17)
0·07 (0·05 to 0·10)
−0·15 (−0·33 to 0·02)
−0·00 (−0·14 to 0·13)
−0·01 (−0·12 to 0·10)
0·20 (0·04 to 0·36)
0·04 (−0·14 to 0·21)
0·27 (0·08 to 0·46)
0·08 (−0·02 to 0·18)
0·01 (−0·12 to 0·14)
0·24 (0·07 to 0·41)
0·07 (−0·01 to 0·14)
6·74
12·23
13·32
16·16
7·51
10·89
8·20
13·12
11·81
100·00
9·45
11·94
13·61
10·32
9·50
8·73
14·54
12·30
9·61
100·00
B Depression
Suspected
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·884
Test-confirmed
MCS
NS
BCS70
NCDS
ELSA
GS
TwinsUK
ALSPAC−G1
ALSPAC−G0
I2=0·0%, p=0·804
0·06 (−0·03 to 0·15)
0·05 (−0·03 to 0·13)
0·11 (0·05 to 0·18)
0·06 (0·01 to 0·12)
0·13 (−0·00 to 0·25)
0·06 (−0·01 to 0·14)
0·10 (0·02 to 0·18)
0·06 (−0·02 to 0·13)
0·10 (0·01 to 0·18)
0·08 (0·05 to 0·10)
−0·08 (−0·24 to 0·08)
0·04 (−0·10 to 0·18)
0·03 (−0·09 to 0·16)
0·15 (0·00 to 0·30)
0·04 (−0·12 to 0·20)
0·06 (−0·14 to 0·27)
0·05 (−0·03 to 0·13)
0·05 (−0·09 to 0·18)
0·02 (−0·12 to 0·17)
0·04 (0·00 to 0·09)
8·09
10·43
16·72
19·40
4·10
10·77
10·28
10·66
9·56
100·00
7·47
10·09
11·13
8·53
7·18
4·49
32·65
10·13
8·34
100·00
C Anxiety
Suspected
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=43·2%, p=0·079
Test-confirmed
MCS
NS
BCS70
NCDS
NSHD
USoc
ELSA
GS
ALSPAC−G1
I2=23·3%, p=0·236
−0·03 (−0·12 to 0·06)
−0·02 (−0·10 to 0·06)
−0·14 (−0·20 to −0·07)
−0·07 (−0·13 to −0·01)
−0·09 (−0·31 to 0·12)
−0·09 (−0·16 to −0·03)
0·05 (−0·05 to 0·15)
−0·01 (−0·09 to 0·06)
−0·05 (−0·13 to 0·02)
−0·05 (−0·09 to −0·02)
−0·04 (−0·20 to 0·12)
0·20 (0·05 to 0·35)
−0·02 (−0·14 to 0·09)
−0·02 (−0·14 to 0·10)
−0·28 (−0·72 to 0·16)
0·02 (−0·07 to 0·10)
−0·07 (−0·22 to 0·07)
−0·05 (−0·23 to 0·14)
−0·02 (−0·15 to 0·11)
−0·00 (−0·06 to 0·05)
9·53
11·70
13·87
15·57
2·41
13·56
8·30
12·12
12·94
100·00
8·86
9·87
14·14
14·31
1·39
21·61
10·34
7·31
12·17
100·00
D Life satisfaction
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
−0·5 −0·3
0·3
0·5
0
0·75
–0·75
Lower levels Higher levels
Lower levels Higher levels
Lower levels Higher levels
Lower levels Higher levels Figure 3: Suspected versus test-confirmed COVID-19 infection and mental health outcomes
Data for each included study and the overall pooled estimate. Articles ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970
British Cohort Study. ELSA=the English Longitudinal Study of Ageing. GS=Generation Scotland: The Scottish Family Health Study. MCS=Millennium Cohort Study. NCDS=the National Child
Development Study. NS=Next Steps, formerly known as the Longitudinal Study of Young People in England. NSHD=the National Survey of Health and Development. TwinsUK=the UK Adult Twin
Registry. USoc/UKHLS=Understanding Society/The UK Household Longitudinal Study. Articles Figure 3: Suspected versus test-confirmed COVID-19 infection and mental health outcomes
Data for each included study and the overall pooled estimate. ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970
British Cohort Study. ELSA=the English Longitudinal Study of Ageing. GS=Generation Scotland: The Scottish Family Health Study. MCS=Millennium Cohort Study. NCDS=the National Child
Development Study. NS=Next Steps, formerly known as the Longitudinal Study of Young People in England. NSHD=the National Survey of Health and Development. TwinsUK=the UK Adult Twin
Registry. USoc/UKHLS=Understanding Society/The UK Household Longitudinal Study. Articles NS=Next Steps, formerly known as the Longitudinal Study of Young People in England. TwinsUK=the UK Adult Twin Registry. Figure 2: Association between time since COVID-19 infection and continuous mental health outcomes
Data for each included study and the overall pooled estimate. ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970
British Cohort Study. NCDS=the National Child Development Study. NS=Next Steps, formerly known as the Longitudinal Study of Young People in England. TwinsUK=the UK Adult Twin Registry. Figure 2: Association between time since COVID-19 infection and continuous mental health outcomes
f
f Figure 2: Association between time since COVID-19 infection and continuous mental health outcomes
Data for each included study and the overall pooled estimate. ALSPAC-G0=the parents of the ALSPAC-G1 birth
British Cohort Study. NCDS=the National Child Development Study. NS=Next Steps, formerly known as the Lo www.thelancet.com/psychiatry Vol 9 November 2022 901 Articles Articles Articles ALSPAC-G0=the parents of the ALSPAC-G1 birth cohort. ALSPAC-G1=Avon Longitudinal Study of Parents and Children. BCS70= the 1970
British Cohort Study. ELSA=the English Longitudinal Study of Ageing. GS=Generation Scotland: The Scottish Family Health Study. MCS=Millennium Cohort Study. NCDS=the National Child
Development Study. NS=Next Steps, formerly known as the Longitudinal Study of Young People in England. NSHD=the National Survey of Health and Development. TwinsUK=the UK Adult Twin
Registry. USoc/UKHLS=Understanding Society/The UK Household Longitudinal Study. Articles www.thelancet.com/psychiatry Vol 9 November 2022 902 Articles Negative self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=0·0%, p=0·811
Positive self-report
and negative serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=29·5%, p=0·203
Positive self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=10·0%, p=0·352
0·09 (−0·32 to 0·50)
−0·03 (−0·42 to 0·35)
0·07 (−0·13 to 0·28)
−0·06 (−0·30 to 0·18)
0·22 (−0·22 to 0·66)
−0·09 (−0·28 to 0·10)
−0·04 (−0·14 to 0·07)
−0·02 (−0·10 to 0·05)
0·06 (−0·12 to 0·25)
−0·00 (−0·13 to 0·12)
0·14 (0·05 to 0·22)
0·19 (0·12 to 0·27)
0·07 (−0·16 to 0·31)
0·10 (−0·00 to 0·21)
0·08 (−0·02to 0·19)
0·11 (0·06to 0·16)
0·01 (−0·23 to 0·24)
0·04 (−0·13 to 0·21)
0·09 (−0·03 to 0·21)
0·13 (0·01 to 0·25)
0·40 (−0·03 to 0·83)
0·08 (−0·09 to 0·25)
−0·04 (−0·16 to 0·09)
0·06 (0·00 to 0·13)
Coefficient (95% CI)
3·37
3·78
13·64
9·65
2·92
15·90
50·74
100·00
6·66
12·06
20·88
24·01
4·31
16·24
15·83
100·00
6·71
11·77
22·23
22·33
2·12
12·55
22·30
100·00
Weight (%)
Coefficient (95% CI) Weight (%)
−0·75
−0·50
−0·25
0
0·25
0·50
0·75
A Psychological distress
Negative self-report
and positive serology
MCS
NS
BCS70
NCDS
TwinsUK
I2=0·0%, p=0·418
Positive self-report
and negative serology
MCS
NS
BCS70
NCDS
TwinsUK
I2=0·0%, p=0·502
Positive self-report
and positive serology
MCS
NS
BCS70
NCDS
TwinsUK
I2=9·1%, p=0·354
0·03 (−0·75 to 0·81)
0·57 (−0·16 to 1·30)
−0·22 (−0·55 to 0·11)
−0·08 (−0·41 to 0·26)
−0·02 (−0·13 to 0·08)
−0·03 (−0·13 to 0·06)
0·19 (−0·16 to 0·54)
0·10 (−0·14 to 0·34)
0·12 (−0·02 to 0·26)
0·17 (0·06 to 0·27)
0·03 (−0·07 to 0·14)
0·11 (0·04 to 0·17)
−0·07 (−0·52 to 0·38)
0·26 (−0·07 to 0·59)
−0·03 (−0·23 to 0·16)
0·10 (−0·07 to 0·27)
−0·05 (−0·17 to 0·07)
0·01 (−0·08 to 0·10)
1·46
1·67
8·15
7·88
80·83
100·00
3·16
6·70
20·17
34·59
35·37
100·00
4·06
7·44
19·36
25·11
44·04
100·00
C Anxiety
−0·75 −0·50 −0·25
0
0·25
0·50 0·75
Lower levels Higher levels
Lower levels Higher levels Negative self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=0·0%, p=0·811
Positive self-report
and negative serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=29·5%, p=0·203
Positive self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=10·0%, p=0·352
0·09 (−0·32 to 0·50)
−0·03 (−0·42 to 0·35)
0·07 (−0·13 to 0·28)
−0·06 (−0·30 to 0·18)
0·22 (−0·22 to 0·66)
−0·09 (−0·28 to 0·10)
−0·04 (−0·14 to 0·07)
−0·02 (−0·10 to 0·05)
0·06 (−0·12 to 0·25)
−0·00 (−0·13 to 0·12)
0·14 (0·05 to 0·22)
0·19 (0·12 to 0·27)
0·07 (−0·16 to 0·31)
0·10 (−0·00 to 0·21)
0·08 (−0·02to 0·19)
0·11 (0·06to 0·16)
0·01 (−0·23 to 0·24)
0·04 (−0·13 to 0·21)
0·09 (−0·03 to 0·21)
0·13 (0·01 to 0·25)
0·40 (−0·03 to 0·83)
0·08 (−0·09 to 0·25)
−0·04 (−0·16 to 0·09)
0·06 (0·00 to 0·13)
Coefficient (95% CI)
3·37
3·78
13·64
9·65
2·92
15·90
50·74
100·00
6·66
12·06
20·88
24·01
4·31
16·24
15·83
100·00
6·71
11·77
22·23
22·33
2·12
12·55
22·30
100·00
Weight (%)
−0·75
−0·50
−0·25
0
0·25
0·50
0·75
A Psychological distress
Lower levels Higher levels Negative self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=0·0%, p=0·811
Positive self-report
and negative serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=29·5%, p=0·203
Positive self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
TwinsUK
I2=10·0%, p=0·352
0·09 (−0·32 to 0·50)
−0·03 (−0·42 to 0·35)
0·07 (−0·13 to 0·28)
−0·06 (−0·30 to 0·18)
0·22 (−0·22 to 0·66)
−0·09 (−0·28 to 0·10)
−0·04 (−0·14 to 0·07)
−0·02 (−0·10 to 0·05)
0·06 (−0·12 to 0·25)
−0·00 (−0·13 to 0·12)
0·14 (0·05 to 0·22)
0·19 (0·12 to 0·27)
0·07 (−0·16 to 0·31)
0·10 (−0·00 to 0·21)
0·08 (−0·02to 0·19)
0·11 (0·06to 0·16)
0·01 (−0·23 to 0·24)
0·04 (−0·13 to 0·21)
0·09 (−0·03 to 0·21)
0·13 (0·01 to 0·25)
0·40 (−0·03 to 0·83)
0·08 (−0·09 to 0·25)
−0·04 (−0·16 to 0·09)
0·06 (0·00 to 0·13)
Coefficient (95% CI)
3·37
3·78
13·64
9·65
2·92
15·90
50·74
100·00
6·66
12·06
20·88
24·01
4·31
16·24
15·83
100·00
6·71
11·77
22·23
22·33
2·12
12·55
22·30
100·00
Weight (%)
−0·75
−0·50
−0·25
0
0·25
0·50
0·75
A Psychological distress
C
Lower levels Higher levels Coefficient (95% CI) Weight (%)
Negative self-report
and positive serology
MCS
NS
BCS70
NCDS
TwinsUK
I2=0·0%, p=0·620
Positive self-report
and negative serology
MCS
NS
BCS70
NCDS
TwinsUK
I2=0·0%, p=0·912
Positive self-report
and positive serology
MCS
NS
BCS70
NCDS
TwinsUK
I2=0·0%, p=0·673
−0·21 (−0·99 to 0·57)
0·26 (−0·39 to 0·91)
0·13 (−0·19 to 0·44)
0·05 (−0·28 to 0·39)
−0·07 (−0·19 to 0·04)
−0·03 (−0·14 to 0·07)
0·06 (−0·29 to 0·41)
0·07 (−0·15 to 0·28)
0·15 (0·02 to 0·29)
0·16 (0·05 to 0·26)
0·11 (−0·01 to 0·22)
0·13 (0·07 to 0·19)
−0·08 (−0·52 to 0·37)
0·09 (−0·20 to 0·39)
−0·00 (−0·19 to 0·19)
0·12 (−0·05 to 0·29)
−0·03 (−0·16 to 0·10)
0·02 (−0·06 to 0·11)
1·69
2·38
10·27
9·08
76·58
100·00
3·28
8·58
22·77
35·75
29·61
100·00
3·67
8·44
20·71
25·36
41·81
100·00
B Depression
−0·75 −0·50 −0·25
0
0·25
0·50
0·75
Lower levels Higher levels B Articles (6–10% change of a standard deviation for outcomes on a
continuous scale and 9–15% increased risk of clinical
caseness) have substantial implications when considered
at the population level, especially given high infection rates. I²=0·0%; k=7; figure 4; appendix pp 39–42). In a post-hoc
exploratory analysis comparing participants with positive
serology to those with negative serology, we did not find
evidence of differences in psychological distress (0·02
[95% CI –0·03 to 0·07], I²=12·8%; k=7) or other mental
health outcomes (appendix p 43). We did not observe improvement in mental health in
the immediate months after infection.3 Studies with
longer-term follow-up examining recovery in symptoms
are needed to assess the duration of symptoms
experienced after infection. Sensitivity analyses were done to examine the
potential effect of the unsure categorisation in MCS,
NS, BCS, NCDS, and NSHD. We did a sensitivity
analysis for the first research question on overall
association between ever having had COVID-19 and
mental health outcomes, grouping those reporting
unsure with those who did not report having
had COVID-19. Associations remained consistent
(psychological distress, 0·07 [95% CI –0·01 to 0·14],
I²=70·0%; appendix p 44–45). In the second sensitivity
analysis, both self-reported COVID-19 (psychological
distress, 0·10 [95% CI 0·00 to 0·19], I²=71·0%) and
unsure
COVID-19
(psychological
distress,
0·10
[0·04 to 0·15], I²=43·8%) showed associations with
poorer mental health (appendix p 46–49). Findings are
also presented for the second question (on interactions
between COVID-19 and sex, ethnicity, educational
qualification, and pre-pandemic mental health) and the
fourth question (on self-reported suspected infection vs
self-reported test-confirmed infection vs serology-
confirmed infection) with different reference groups to
provide an alternative comparison and confirm the
interpretation (appendix pp 50–61). We found that COVID-19 was associated with poorer
mental health in all age groups, with some evidence of
stronger associations for people aged 50 years and older. This result might reflect that older people are more likely
to experience more severe COVID-19 and potentially also
greater worry around infection due to their age and
higher likelihood of pre-existing health conditions. These
findings could also reflect increased risk of microvascular
or neurological changes after COVID-19, which have
been associated with depression and anxiety phenotypes
in older adults.26–28 However, we found no differences by
sex, ethnicity, education level, and previous mental health
problems. Figure 4: Suspected versus serology-confirmed COVID-19 infection and
mental health outcomes
Data for each included study and the overall pooled estimate. ALSPAC-G0=the
parents of the BCS70= the 1970 British Cohort Study. MCS=Millennium Cohort
Study. NCDS=the National Child Development Study. NS=Next Steps, formerly
known as the Longitudinal Study of Young People in England. NSHD=the National
Survey of Health and Development. TwinsUK=the UK Adult Twin Registry.
USoc/UKHLS=Understanding Society/The UK Household Longitudinal Study. Articles Previous studies have shown that overall
mental health impacts of the pandemic have been greatest
in adults aged 25–44 years, women, and those with a
degree (vs those without a degree),19 suggesting that the
mechanisms through which COVID-19 illness affects
mental health might differ from those underpinning the
wider effects of the pandemic. Our analyses benefited from the use of serology data
in addition to information on self-reported COVID-19.26
When comparing associations for subgroups on the basis
of self-report and serology status, self-reported COVID-19
illness combined with negative serology was associated
with poorer mental health, whereas no association was
found for positive serology without self-reported
COVID-19. Similarly, in a post-hoc exploratory analysis,
we did not find evidence of differences in mental health
outcomes for those with positive compared with negative
serology. These findings align with a population-based
cross-sectional study done in France, which found that
self-reported COVID-19 infection was associated with a
range of persistent physical symptoms, including
muscular pain, fatigue, headache, palpitations, dizziness,
and cough, whereas laboratory-confirmed COVID-19 was
only associated with anosmia.29 www.thelancet.com/psychiatry Vol 9 November 2022 Coefficient (95% CI) Weight (%) Lower levels Higher levels Coefficient (95% CI) Weight (%)
Negative self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
I2=0·0%, p=0·454
Positive self-report
and negative serology
MCS
NS
BCS70
NCDS
NSHD
USoc
I2=34·1%, p=0·180
Positive self-report
and positive serology
MCS
NS
BCS70
NCDS
NSHD
USoc
I2=0·0%, p=0·710
0·48 (−0·43 to 1·40)
−0·50 (−1·40 to 0·41)
0·41 (−0·08 to 0·91)
0·30 (−0·28 to 0·87)
0·21 (−0·28 to 0·69)
0·04 (−0·18 to 0·27)
0·13 (−0·04 to 0·30)
0·09 (−0·32 to 0·50)
−0·10 (−0·40 to 0·20)
−0·22 (−0·43 to −0·01)
−0·19 (−0·37 to −0·01)
0·15 (−0·11 to 0·41)
−0·18 (−0·29 to −0·07)
−0·12 (−0·23 to −0·02)
0·03 (−0·50 to 0·56)
0·10 (−0·30 to 0·51)
0·05 (−0·24 to 0·35)
−0·01 (−0·30 to 0·28)
−0·32 (−0·78 to 0·15)
−0·11 (−0·27 to 0·05)
−0·06 (−0·17 to 0·05)
3·54
3·61
12·16
8·95
12·77
58·96
100·00
5·92
10·14
17·17
20·55
12·81
33·42
100·00
4·66
7·81
14·71
15·29
6·00
51·53
100·00
D Life satisfaction
−0·75 −0·50 −0·25 0
0·25 0·50 0·75
Lower levels Higher levels D Life satisfaction Coefficient (95% CI) Weight (%) Coefficient (95% CI) Weight (%) www.thelancet.com/psychiatry Vol 9 November 2022 903 Articles Articles Discussion i Our findings indicate that self-reported COVID-19 illness
was associated with a deterioration in mental health
outcomes in the UK population. We did not find evidence
of change in this association over time during the first few
months after infection. Subgroup analysis indicated no
differences by sex, ethnicity, education, or pre-pandemic
mental health; and there was some indication of a slightly
stronger association in some older age groups. Notably, we
observed similar associations for both suspected COVID-19
and COVID-19 confirmed by test or serology data,
suggesting that the associations could relate to the mental
health impacts of illness during this period, rather than
exposure to SARS-CoV-2 specifically. Our
findings
demonstrate
associations
between
COVID-19 and deterioration in mental health, while
controlling for overall effects of timing throughout the first
year of the pandemic, adding to existing evidence, which
has been mixed to date.3,4 The effects observed Various mechanisms have been posited to underlie
associations between COVID-19 and psychological
distress, including systemic inflammation and changes in
the brain associated with COVID-19,27 and psychosocial
mechanisms including social isolation and worries about
possible outcomes and infecting others. One possible
explanation for our findings, particularly the association
with poorer mental health for those who had negative
serology but had self-reported COVID-19, is that contextual
and psychosocial aspects of COVID-19 (such as the
experience of being unwell and worrying about potential
health, social, and economic consequences) are stronger 904 Articles predictors of poor mental health outcomes than any
specific neurological consequences of SARS-CoV-2
infection. The lack of association with mental health when
serology data detected SARS-CoV-2 infections that had not
been reported (ie, cases in which participants were
unaware of the infection) support this conclusion. and the other measures of test-confirmed and serology
based infection markers are also prone to biases and
measurement issues.8,10 Further limitations are the lack of
data available to examine possible variation in associations
by COVID-19 severity, the fact that only infections in the
first year of the pandemic have been assessed, and that
longer-term follow-up is limited at present. Nonetheless, it is important to note that participants
who self-reported COVID-19 infection but had negative
serology status might have correctly identified having
had the disease, especially given the known issue of
waning of antibodies. Contributors PP, EJT, JS, BM, CJS, NC, SVK, and ASFK conceptualised the study. EJT, JS, BM, PP, RJSi, KT, MP, NC, SVK, PZ, RJSh, ASFK, CFH, MJG,
JM, NT, and RCEB created the methodology. EJT, JS, BM, CFH, ASFK,
RJSh, and PZ analysed the data. JS and EJT created the figures. EJT, JS,
BM, and PP drafted the manuscript. All authors reviewed the
manuscript. PP, CS, and NC supervised the analysts at all stages. EJT,
JS, BM, CFH, ASFK, RJSh, PZ, EM, and KP curated data. NC, PP, GBP,
SVK, CJS, NT, and DJP acquired funding. JS (NSHD), EJT (TwinsUK),
BM (MCS, NS, BCS70, NCDS), ASFK (ALSPAC), CFH (GS), RS (USoc),
and PZ (ELSA) had access to the raw data for individual studies. JS, EJT,
and BM had access to and verified the overall data for meta-analysis. All authors had final responsibility for the decision to submit for
publication. Declaration of interests The response rates for the COVID-19 surveys in many
included studies were not consistently high. However,
rich antecedent data in longitudinal prospective studies
allowed most studies to be weighted for non-response
(aiming to reduce potential bias from selection into
analysed samples).8,10 Although we were able to control for
important confounders, we cannot definitively attribute
changes in mental health to COVID-19 illness. Included
studies used different measures to assess COVID-19 and
mental health outcomes. These were carefully reviewed
and harmonised across studies, and in meta-analysis the
heterogeneity of estimates between studies was small for
most outcomes. Nonetheless, measurement error of the
exposure is a potential limitation, given that our main
exposure variables were based on self-reported COVID-19, SVK is a member of the Scientific Advisory Group on Emergencies
subgroup on ethnicity and COVID-19 and is co-chair of the Scottish
Government’s Ethnicity Reference Group on COVID-19. NC serves on
a data safety monitoring board for trials sponsored by AstraZeneca. CJS is an academic lead on KCL Zoe Global COVID symptoms study. KT has been a paid consultant for the CHDI Foundation (member of
Statistical Advisory Group) and is a member of the steering group for
Living With COVID Recovery study at UCL (unpaid). All other authors
declare no competing interests. Discussion i In addition, those who self-
reported COVID-19 probably experienced related
symptoms, whereas those with positive serology who did
not self-report symptoms were more likely to have had
mild or asymptomatic COVID-19. Participants who
suspected COVID-19 with negative serology might have
had
another
respiratory
infection
with
shared
pathophysiological pathways, potentially contributing to
adverse mental health outcomes. Those who were
particularly concerned about consequences of infection
might have been more likely to report perceived
infection. Notably, only a subset of studies and samples
had serology data, substantially reducing power in these
analyses. Additionally, only one timepoint of serology
assessment was completed after the most recent self-
report data, limiting the conclusions that can be drawn. Antibody
concentrations
following
SARS-CoV-2
infection have been found to wane over time,30 which
could have led to underestimation of COVID-19 based
on serology data. g
p
p
Our findings suggest that people who self-reported
COVID-19 in the first year of the pandemic were
subsequently more likely to experience poorer mental
health outcomes than those who did not self-report
COVID-19. Our findings involving serology-confirmed
infection, and the lack of attenuation in association
over time, suggest that these associations might not be
specific to SARS-CoV-2 infection and potentially reflect
consequences of feeling unwell, anxieties related to a
novel infection and infecting others, or other factors
such as social isolation and loss of pay. Further research
is needed to investigate these possible underlying
mechanisms and to examine whether associations
persist over longer follow-up periods. Our findings
emphasise the important population mental health
consequences of infection and disease separately from
the potential impacts of the pandemic more widely
(eg, infection control measures). Given the high
prevalence of COVID-19 in the UK and worldwide,
these
findings
have
important
public
health
implications, highlighting the need for greater post-
infection mental health support in both clinical and
community settings. Previous studies have mainly focused on severe
COVID-19 in patients admitted to hospital or severe
mental illness.8,10 Our results add to existing evidence by
capturing broader and subclinical mental health
impacts of COVID-19 illness in the general population. The use of multiple prospective longitudinal studies
allowed us to control for time period effects and
important pre-pandemic factors including mental
health, physical health, and socio-economic factors. Additionally, we were able to examine the persistence of
associations over time and differences between
perceived and test-confirmed COVID-19. www.thelancet.com/psychiatry Vol 9 November 2022 Acknowledgments 17
Magnúsdóttir I, Lovik A, Unnarsdóttir AB, et al. Acute COVID-19
severity and mental health morbidity trajectories in patient
populations of six nations: an observational study. Lancet Public Health 2022; 7: e406–16. The COVID-19 Longitudinal Health and Wellbeing National Core Study
was funded by the UK Medical Research Council (MC_PC_20059 and
MC_PC_20030) and the CONVALESCENCE study was funded by the UK The COVID-19 Longitudinal Health and Wellbeing National Core Study
was funded by the UK Medical Research Council (MC_PC_20059 and
MC_PC_20030) and the CONVALESCENCE study was funded by the UK 18
Niedzwiedz CL, Benzeval M, Hainey K, Leyland AH, Katikireddi SV. Psychological distress among people with probable COVID-19
infection: analysis of the UK Household Longitudinal Study. BJPsych Open 2021; 7: e104. National Institute for Health and Care Research (CONVALESCENCE grant
COV-LT-0009). Full funding acknowledgements for each individual study
can be found in the appendix (pp 62–63). National Institute for Health and Care Research (CONVALESCENCE grant
COV-LT-0009). Full funding acknowledgements for each individual study
can be found in the appendix (pp 62–63). National Institute for Health and Care Research (CONVALESCENCE grant
COV-LT-0009). Full funding acknowledgements for each individual study
can be found in the appendix (pp 62–63). Articles doi:10·5522/NSHD/Q10. Information regarding access to the ALSPAC
data can be found on the ALSPAC website (http://www.bristol.ac.uk/
media-library/sites/alspac/documents/researchers/data-access/ALSPAC_
Access_Policy.pdf). All USoc data are available through the UK Data
Service (SN 6614 and SN 8644). All ELSA data are available through the
UK Data Service (SN 8688 and 5050). Access to GS data is approved by the
Generation Scotland Access Committee. See https://www.ed.ac.uk/
generation-scotland/for-researchers/access or email access@
generationscotland.org for further details. The TwinsUK Resource
Executive Committee oversees management, data sharing, and
collaborations involving the TwinsUK registry (for further details see
https://twinsuk.ac.uk/resources-for-researchers/access-our-data/). doi:10·5522/NSHD/Q10. Information regarding access to the ALSPAC
data can be found on the ALSPAC website (http://www.bristol.ac.uk/
media-library/sites/alspac/documents/researchers/data-access/ALSPAC_
Access_Policy.pdf). All USoc data are available through the UK Data
Service (SN 6614 and SN 8644). All ELSA data are available through the
UK Data Service (SN 8688 and 5050). Access to GS data is approved by the
Generation Scotland Access Committee. See https://www.ed.ac.uk/
generation-scotland/for-researchers/access or email access@
generationscotland.org for further details. The TwinsUK Resource
Executive Committee oversees management, data sharing, and
collaborations involving the TwinsUK registry (for further details see
https://twinsuk.ac.uk/resources-for-researchers/access-our-data/). 12
Xie Y, Xu E, Al-Aly Z. Risks of mental health outcomes in people
with COVID-19: cohort study. BMJ 2022; 376: e068993. 13
Abel KM, Carr MJ, Ashcroft DM, et al. Association of SARS-CoV-2
infection with psychological distress, psychotropic prescribing,
fatigue, and sleep problems among UK primary care patients. JAMA Netw Open 2021; 4: e2134803. 14
Lund LC, Hallas J, Nielsen H, et al. Post-acute effects of
SARS-CoV-2 infection in individuals not requiring hospital
admission: a Danish population-based cohort study. Lancet Infect Dis 2021; 21: 1373–82. 15
Yolken R. COVID-19 and psychiatry: can electronic medical records
provide the answers? Lancet Psychiatry 2021; 8: 89–91. 16
Weich S. Mental health after COVID-19. BMJ 2022; 376: o326. References 19
Patel K, Robertson E, Kwong ASF, et al. Psychological distress
before and during the COVID-19 pandemic among adults in the
United Kingdom based on coordinated analyses of 11 longitudinal
studies. JAMA Netw Open 2022; 5: e227629. 1
Rogers JP, Chesney E, Oliver D, et al. Psychiatric and
neuropsychiatric presentations associated with severe coronavirus
infections: a systematic review and meta-analysis with comparison
to the COVID-19 pandemic. Lancet Psychiatry 2020; 7: 611–27. 20 Klaser K, Thompson EJ, Nguyen LH, et al. Anxiety and depression
symptoms after COVID-19 infection: results from the COVID
Symptom Study app. J Neurol Neurosurg Psychiatry 2021;
92: 1254–58. 2
Santomauro DF, Mantilla Herrera AM, Shadid J, et al. Global
prevalence and burden of depressive and anxiety disorders in
204 countries and territories in 2020 due to the COVID-19
pandemic. Lancet 2021; 398: 1700–12. 21
Kwong ASF, Pearson RM, Adams MJ, et al. Mental health before
and during the COVID-19 pandemic in two longitudinal
UK population cohorts. Br J Psychiatry 2021; 218: 334–43. 3
Bourmistrova NW, Solomon T, Braude P, Strawbridge R, Carter B. Long-term effects of COVID-19 on mental health: a systematic
review. J Affect Disord 2022; 299: 118–25. 3
Bourmistrova NW, Solomon T, Braude P, Strawbridge R, Carter B. Long-term effects of COVID-19 on mental health: a systematic
review. J Affect Disord 2022; 299: 118–25. 22 Centers for Disease Control and Prevention. Interim Guidelines
for COVID-19 Antibody Testing. 2021. https://www.cdc.gov/
coronavirus/2019-ncov/lab/resources/antibody-tests-guidelines. html (accessed July 6, 2021). 4
Schou TM, Joca S, Wegener G, Bay-Richter C. Psychiatric and
neuropsychiatric sequelae of COVID-19—a systematic review. Brain Behav Immun 2021; 97: 328–48. 4
Schou TM, Joca S, Wegener G, Bay-Richter C. Psychiatric and
neuropsychiatric sequelae of COVID-19—a systematic review. Brain Behav Immun 2021; 97: 328–48. 5
Rogers JP, Watson CJ, Badenoch J, et al. Neurology and
neuropsychiatry of COVID-19: a systematic review and meta-
analysis of the early literature reveals frequent CNS
manifestations and key emerging narratives. J Neurol Neurosurg Psychiatry 2021; 92: 932–41. 23 Navaratnam AD, Shrotri M, Nguyen V, et al. Nucleocapsid and
spike antibody responses post virologically confirmed SARS-CoV-2
infection: an observational analysis in the Virus Watch community
cohort. medRxiv 2022; published online Feb 2. https://doi. org/10.1101/2022.02.01.22270269 (preprint). 6
Vindegaard N, Benros ME. COVID-19 pandemic and mental health
consequences: systematic review of the current evidence. Brain Behav Immun 2020; 89: 531–42. 6
Vindegaard N, Benros ME. Data sharing Data for NCDS (SN 6137), BCS70 (SN 8547), Next Steps (SN 5545),
MCS (SN 8682) and all four COVID-19 surveys (SN 8658) are available
through the UK Data Service. NSHD data are available on request to the
NSHD Data Sharing Committee. Interested researchers can apply to
access the NSHD data via a standard application procedure. Data requests
should be submitted to mrclha.swiftinfo@ucl.ac.uk; further details can be
found at http://www.nshd.mrc.ac.uk/data.aspx. doi:10·5522/NSHD/Q101; 905 Articles Articles References COVID-19 pandemic and mental health
consequences: systematic review of the current evidence. Brain Behav Immun 2020; 89: 531–42. 24
Zou G. A modified poisson regression approach to prospective
studies with binary data. Am J Epidemiol 2004; 159: 702–06. 25 Borenstein M, Higgins JPT, Hedges LV, Rothstein HR. Basics of
meta-analysis: I² is not an absolute measure of heterogeneity. Res Synth Methods 2017; 8: 5–18. 7
Coleman B, Casiraghi E, Blau H, et al. Increased risk of psychiatric
sequelae of COVID-19 is highest early in the clinical course. medRxiv 2021; published online Dec 2, 2021. https://doi.org/10.1101/2021.11.30.21267071 (preprint). 26 Alexopoulos GS, Meyers BS, Young RC, Campbell S, Silbersweig D,
Charlson M. ‘Vascular depression’ hypothesis. Arch Gen Psychiatry
1997; 54: 915–22. 8
Oh TK, Park HY, Song IA. Risk of psychological sequelae among
coronavirus disease-2019 survivors: a nationwide cohort study in
South Korea. Depress Anxiety 2020; 38: 247–54. 8
Oh TK, Park HY, Song IA. Risk of psychological sequelae among
coronavirus disease-2019 survivors: a nationwide cohort study in
South Korea. Depress Anxiety 2020; 38: 247–54. 27
Taylor WD, Aizenstein HJ, Alexopoulos GS. The vascular
depression hypothesis: mechanisms linking vascular disease with
depression. Mol Psychiatry 2013; 18: 963–74. 9
Taquet M, Luciano S, Geddes JR, Harrison PJ. Bidirectional
associations between COVID-19 and psychiatric disorder:
retrospective cohort studies of 62 354 COVID-19 cases in the USA. Lancet Psychiatry 2021; 8: 130–40. 28 Baldwin RC. Is vascular depression a distinct sub-type of depressive
disorder? A review of causal evidence. Int J Geriatr Psychiatry 2005;
20: 1–11. 10
Taquet M, Geddes JR, Husain M, Luciano S, Harrison PJ. 6-month
neurological and psychiatric outcomes in 236 379 survivors of
COVID-19: a retrospective cohort study using electronic health
records. Lancet Psychiatry 2021; 8: 416–27. 29 Matta J, Wiernik E, Robineau O, et al. Association of self-reported
COVID-19 infection and SARS-CoV-2 serology test results with
persistent physical symptoms among French adults during the
COVID-19 pandemic. JAMA Intern Med 2022; 182: 19–25. 11
Taquet M, Dercon Q, Luciano S, Geddes JR, Husain M,
Harrison PJ. Incidence, co-occurrence, and evolution of long-
COVID features: a 6-month retrospective cohort study of
273 618 survivors of COVID-19. PLoS Med 2021; 18: e1003773. 30 Hamady A, Lee J, Loboda ZA. Waning antibody responses in
COVID-19: what can we learn from the analysis of other
coronaviruses? Infection 2022; 50: 11–25. 906 www.thelancet.com/psychiatry Vol 9 November 2022
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English
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Highly efficient lead removal from water by Nd0.90Ho0.10FeO3 nanoparticles and studying their optical and magnetic properties
|
Scientific reports
| 2,023
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cc-by
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Highly efficient lead removal
from water by Nd0.90Ho0.10FeO3
nanoparticles and studying their
optical and magnetic properties
OPEN M. M. Arman Ho-doped NdFeO3 was synthesized using the citrate method. The X-ray diffraction (XRD)
illustrated that Nd0.90Ho0.10FeO3 was crystalline at the nanoscale, with a crystallite size of 39.136
nm. The field emission scanning electron microscope (FESEM) illustrated the porous nature of
Nd0.90Ho0.10FeO3, which increases the active sites to absorb the heavy metals on the sample
surface. Energy-dispersive X-ray (EDX) data assures the prepared sample has the chemical
formula Nd0.90Ho0.10FeO3. The magnetic properties of Nd0.90Ho0.10FeO3 were determined using the
magnetization hysteresis loop and Faraday’s method. Many magnetic parameters of the sample have
been discussed, such as the coercive field, the exchange bias (Hex), and the switching field distribution
(SFD). Ho-doped NdFeO3 has an antiferromagnetic (AFM) character with an effective magnetic
moment of 3.903 B.M. The UV–visible light absorbance of Nd0.90Ho0.10FeO3 is due to the transfer of
electrons from the oxygen 2p state to the iron 3d state. Nd0.90Ho0.10FeO3 nanoparticles have an optical
direct transition with an energy gap Eg = 1.106 eV. Ho-doped NdFeO3 can adsorb many heavy metals
(Co2+, Ni2+, Pb2+, Cr6+, and Cd2+) from water. The removal efficiency is high for Pb2+ ions, which equals
72.39%. The Langmuir isotherm mode is the best-fit model for adsorbing the Pb2+ ions from water. The orthoferrites are promising materials in many applications due to their chemical stability, magnetic, mul-
tiferroic, optical, and dielectric properties1. The general formula of orthoferrites is ABO3, where A is the rare
earth elements, i.e., La3+, Nd3+, Sm3+, and Gd3+; B is the transition metal, i.e., Fe3+; and O2- is the oxygen ion. Multiferroic materials such as BiFeO3, NdFeO3, SmFeO3, and LaFeO3 have ferroelectric and magnetic orders2–5. The applications of ABO3 are spintronics, data storage media, high-frequency devices, water purification, and
photocatalysis6–10. The orthoferrites are characterized by low cost, chemical stability, easy fabrication, and many
applications11. pp
NdFeO3 belongs to the ABO3 orthoferrite materials with the space group Pbnm. The Fe3+ ions form the FeO6
octahedron. The magnetic properties of NdFeO3 originate from the Dzyaloshinskii–Moriya exchange interaction
of the antiparallel spins between Fe3+–O2-–Fe3+, Nd3+–O2-–Fe3+, and Nd3+–O2-–Nd3+1,12. The optical properties of
NdFeO3 originate from the transition of electrons from 2p to 3d orbitals13,14. P. T. H. Duyen et al.15 studied the
optical properties of Cd-doped NdFeO3, concluding that increasing the Cd concentration decreased the optical
band gap. S. A. Mir et al.16 prepared the Ni-doped NdFeO3 and studied the dielectric properties of the samples. www.nature.com/scientificreports www.nature.com/scientificreports Materials Science Lab (1), Physics Department, Faculty of Science, Cairo University, Giza, Egypt. email: mmarmsci@
gmail.com; mmarmsci@cu.edu.eg Scientific Reports | (2023) 13:16585 Materials Science Lab (1), Physics Department, Faculty of Science, Cairo University, Giza, Egypt. email: mmarmsci@
gmail.com; mmarmsci@cu.edu.eg www.nature.com/scientificreports/ the precipitation method25, flotation26, and membrane technologies27. The most effective method for removing
HMs from water is the adsorption technique, which is characterized by its simplicity and no slugs.hi The present paper describes the preparation of the Ho-doped NdFeO3 nanoparticles for the first time using a
simple and inexpensive citrate combustion method. The sample was characterized by FESEM, EDX, and elemen-
tary mapping. The optical and magnetic properties of Nd0.90Ho0.10FeO3 were studied in detail. The removal of
HMs (Co2+, Ni2+, Pb2+, Cr6+, and Cd2+) from water was studied. The Langmuir and Freundlich isotherm models
were used to study the adsorption of Pb2+ from water on Nd0.90Ho0.10FeO3. Experimental work
Materials
d
h l Neodymium nitrate, holmium nitrate, and iron nitrate were purchased from Sigma-Aldrich with a purity of
99.9%. Preparation of the Nd0.90Ho0.10FeO3 sampleh p
0 90
0
0
3
p
The citrate combustion method is characterized by controlling the metal stoichiometry, high purity, low cost,
crystallinity, effectiveness, and high yield. Figure 1 shows the flowchart of the synthesis of Nd0.90Ho0.10FeO3 using
the citrate combustion method. The (0.9 M) Nd nitrate, (0.1 M) Ho nitrates, (1 M) Fe nitrates, and (2 M) citric
acid were dissolved in distilled water. The ammonia solution was used to adjust the pH to 7. The solution was
stirred and heated on a magnetic stirrer at 80 °C for one hour, then heated at 270 °C for 3 h until the evolution
of fumes stopped. The as-prepared sample was ground using a mortar for one hour. The obtained powder was
characterized by XRD to study the crystallinity of the Ho-doped NdFeO3. Nd0.90Ho0.10FeO3 characterizations and measurementsh 0.90
0.10
3
The crystal structure of Nd0.90Ho0.10FeO3 was studied using XRD (Bruker Advance D8 diffractometer, λ = 1.5406Å)
with 2θ in the range of 20°–80°. The XRD data was indexed with the International Centre for Diffraction Data
(ICDD) card number 01-089-6644. The morphology of Nd0.90Ho0.10FeO3 was studied using FESEM (model
Quanta 250) with EDX and elemental mapping. The magnetic properties of Nd0.90Ho0.10FeO3 were studied by
two techniques. The first is measuring the magnetization of the sample using the vibrating sample magnetom-
eter (VSM; 9600-1 LDJ, USA), which uses a magnetic field up to 20 kOe at a temperature of 300 K. The second
technique is Faraday’s method, in which a small amount of Nd0.90Ho0.10FeO3 was placed in a glass that was placed
at the field gradient to measure the DC magnetic susceptibility with temperature28. The optical properties of
Nd0.90Ho0.10FeO3 were studied using a UV–visible spectrophotometer (Jasco (V-630)). Highly efficient lead removal
from water by Nd0.90Ho0.10FeO3
nanoparticles and studying their
optical and magnetic properties
OPEN g p
p p
p
p
p
p
Numerous photocatalysts, including CuS, TiO2, CuO, BaTiO3, ZnO, and others, are used today in dye
degradation17. Their large bandgap energy, quick recombination of photoinduced charge carriers, and low visible
light absorption, however, severely limit their practical applicability18. Therefore, it is essential to create photo-
catalysts with exceptional photocatalytic activity in the visible region and a small bandgap. Perovskite-oxide-
based catalysts such as NdFeO3 have lately piqued the interest of researchers due to their good photocatalytic
activity, tunable bandgap, high stability, and quick photoinduced electron/hole mobility19–21.hi y
g p
g
y
q
p
y
The metallic elements that are characterized by their high atomic weight, specific gravity, and toxicity are
called heavy metals (HMs), such as lead (Pb2+), chromium (Cr6+), nickel (Ni2+), cadmium (Cd2+), and copper
(Co2+). Cadmium, a heavy metal, damages the bones and kidneys22. Cr6+ causes hemorrhage, severe diarrhea, and
cancer in the digestive tract23. Increasing the concentration of lead in drinking water causes kidney malfunction,
brain tissue damage, and anemia24. There are many techniques used to remove heavy metals from water, such as | https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 www.nature.com/scientificreports/ The heavy metals removal from waterh The examination of the ability of Nd0.90Ho0.10FeO3 to remove heavy metals such as Co2+, Ni2+, Pb2+, Cr6+, and
Cd2+ from water. The removal efficiency is represented by the following steps: 1. The standard solutions (50 ppm) of heavy metals were prepared. g
p
3. The pH value of the solution was adjusted using the ammonia solution or diluted nitric h
p
j
g
4. The beakers were stirred on the electric shaker for 1 h at 170 rpm.i Figure 1. Flowchart for the preparation of Nd0.90Ho0.10FeO3. Figure 1. Flowchart for the preparation of Nd0.90Ho0.10FeO3. Figure 1. Flowchart for the preparation of Nd0.90Ho0.10FeO3. https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 | www.nature.com/scientificreports/ 6. Inductively coupled plasma spectrometry (ICP, Prodigy 7) was used to determine the concentration of the
heavy metals. Results and discussion Figure 2 illustrates the XRD of the Nd0.90Ho0.10FeO3 nanoparticles. The most intense peak was observed at
2θ = 32.593º which characterized the (121) plane. The sample has a single phase orthorhombic structure. The
average lattice parameters were estimated from all peaks using the following equation29: (1)
1
d2 = h2
a2 + k2
b2 + l2
c2 (1) The average lattice parameters are reported in Table 1. The crystallite unit cell volume was calculated accord-
ing to Eq. (2). The average lattice parameters are reported in Table 1. The crystallite unit cell volume was calculated accord-
ing to Eq. (2). (2)
V = abc V = abc (2) The average crystallite size (L) of the sample was estimated from all peaks using the Scherer formula, which
is represented by the following equation30: (3)
L = 0.94
βcosθ (3) where λ denotes the X-ray wavelength, β is the full width at half maximum, and θ is the Bragg angle. The value
of L is 39.136 nm, which indicates the sample was prepared at the nanoscale.h p
p p
The tolerance factor relates to the symmetry of the crystal structure and was calculated using Eq. (4). (4)
t =
rA + rO
√
2(rFe + rO) (4) where rA, rFe, and rO are the ionic radii of the A, Fe, and oxygen ions, respectively. The value of rA was calculated
rom Eq. (5). e rA, rFe, and rO are the ionic radii of the A, Fe, and oxygen ions, respectively. The value of rA was calculated
Eq. (5). (5)
rA = 0.90 r3+
Nd + 0.10 r2+
Ho (5) The value of t is one for the ideal cubic structure, while t decreases to one for the orthorhombic structure,
where the crystallite size distortion increases. For the investigated sample, the value of t is 0.8875, which indicates 20
30
40
50
60
70
80
(161)
(242)
(331)
(123)
(311)
(230)
(040)
(022)
(121)
(111)
(020)
Nd0.90Ho0.10FeO3
ICDD Card 01-089-6644
Relative intensity ( )
2 ( )
Figure 2. XRD of Nd0.90Ho0.10FeO3, ICDD card number 01-089-6644. 20
30
40
50
60
70
80
(161)
(242)
(331)
(123)
(311)
(230)
(040)
(022)
(121)
(111)
(020)
Nd0.90Ho0.10FeO3
ICDD Card 01-089-6644
Relative intensity ( )
2 ( )
Figure 2. XRD of Nd0.90Ho0.10FeO3, ICDD card number 01-089-6644. www.nature.com/scientificreports/ the orthorhombic structure of the sample. The theoretical density (Dx) was calculated according to Eq. (6) and
reported in Table 1. the orthorhombic structure of the sample. The theoretical density (Dx) was calculated according to Eq. (6) and
reported in Table 1. (6)
Dx = ZM
NV Dx = ZM
NV Dx = ZM
NV (6) where Z is the number of molecules in a unit cell (Z = 4), M refers to the molecular weight of the sample, and
N is Avogadro’s number. The substitution of Ho3+ ions at the expense of the Nd3+ ion led to an increase in the
relative density of NdFeO3. According to M.M. Arman10, the relative density of NdFeO3 is 6.33 g/cm3 and its
unit cell volume is 236.9 (Å)3, while Nd0.90Ho0.10FeO3 has a relative density of 7.05 g/cm3 and its unit cell volume
is 235.6 (Å)3. This is due to the ionic radius of Ho3+ ions (1.073 Å) being less than that of Nd3+ ions (1.163 Å)31. h where Z is the number of molecules in a unit cell (Z = 4), M refers to the molecular weight of the sample, and
N is Avogadro’s number. The substitution of Ho3+ ions at the expense of the Nd3+ ion led to an increase in the
relative density of NdFeO3. According to M.M. Arman10, the relative density of NdFeO3 is 6.33 g/cm3 and its
unit cell volume is 236.9 (Å)3, while Nd0.90Ho0.10FeO3 has a relative density of 7.05 g/cm3 and its unit cell volume
is 235.6 (Å)3. This is due to the ionic radius of Ho3+ ions (1.073 Å) being less than that of Nd3+ ions (1.163 Å)31. h h
Figure 3 illustrates the FESEM image of the Nd0.90Ho0.10FeO3 nanoparticles. The agglomerated particles are a
result of the synthesis procedure. The sample has a porous nature, which increases the surface area of Ho-doped
NdFeO3. The presence of a lot of active sites of the Nd0.90Ho0.10FeO3 increases the adsorption of HMs on the
surface of the Nd0 90Ho0 10FeO3 sample3. 0.90
0.10
3
p
Figure 4 shows the EDX of Ho-doped NdFeO3, which assures the presence of the elements Fe, Ho, Nd, and
O in the Nd0.90Ho0.10FeO3. The table inset in Fig. 4 shows the atomic percentage (at%) and weight percentage
(wt%) of the elements, which were calculated theoretically from the sample formula and experimentally from the
EDX data. Results and discussion 20
30
40
50
60
70
80
(161)
(242)
(331)
(123)
(311)
(230)
(040)
(022)
(121)
(111)
(020)
Nd0.90Ho0.10FeO3
ICDD Card 01-089-6644
Relative intensity ( )
2 ( )
Figure 2. XRD of Nd0.90Ho0.10FeO3, ICDD card number 01-089-6644. Relative intensity ( ) Figure 2. XRD of Nd0.90Ho0.10FeO3, ICDD card number 01-089-6644. 3
Vol.:(0123
Scientific Reports | (2023) 13:16585 |
https://doi.org/10.1038/s41598-023-43734-2
Table 1. The lattice parameters, the theoretical density (Dx), the crystallite size (L), and the tolerance factor for
Nd0.90Ho0.10FeO3. Sample
a (Å) (0.0001)
B (Å) (0.0001)
C (Å) (0.0001)
V (Å3) (0.1)
Dx (g/cm3) (0.0002)
L (nm) (0.5)
t (0.0002)
Nd0.90Ho0.10FeO3
5.5696
7.7527
5.4561
235.6
7.0519
39
0.8875 3
V l (
Scientific Reports | (2023) 13:16585 |
https://doi.org/10.1038/s41598-023-43734-2
Table 1. The lattice parameters, the theoretical density (Dx), the crystallite size (L), and the tolerance factor for
Nd0.90Ho0.10FeO3. Sample
a (Å) (0.0001)
B (Å) (0.0001)
C (Å) (0.0001)
V (Å3) (0.1)
Dx (g/cm3) (0.0002)
L (nm) (0.5)
t (0.0002)
Nd0.90Ho0.10FeO3
5.5696
7.7527
5.4561
235.6
7.0519
39
0.8875 https://doi.org/10.1038/s41598-023-43734-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The values of at% and wt% of the theoretical and experimental are close to each other, which indicates
that the sample was prepared in the same chemical formula, Nd0.90Ho0.10FeO3. The peak observed at 2.11 eV is
due to the gold coating of the sample before scanning. In Fig. 4, the carbon ions were observed at 0.27 eV due
to the carbon tap where the sample was put inside the FESEM. The slight difference in wt% and at% between
experimental and theoretical values is caused by the oxygen deficiency. p
y
ygi
y
Figure 5 shows the mapping of the elements in Ho-doped NdFeO3. Figure 5.a illustrates the homogeneous
distribution of the elements in the sample. Figure 5b–e shows the distribution of each element in the sample
by distinguished color. The wt% of the elements appearing in the elemental mapping was different from that
obtained from EDX analysis due to the maps having been measured for too short a time.h The magnetic properties of Nd0.90Ho0.10FeO3 were studied via the M–H hysteresis loop and Faraday’s method. The magnetic behavior of Nd0.90Ho0.10FeO3 originates from the magnetic coupling between the magnetic ions
such as Fe3+, Nd3+, and Ho3+ ions. Figure 3. FESEM images of Nd0.90Ho0.10FeO3 with different magnifications. Figure 3. FESEM images of Nd0.90Ho0.10FeO3 with different magnifications. Figure 3. FESEM images of Nd0.90Ho0.10FeO3 with different magnifications. Energy (keV)
Intensity (Counts)
Elements
Weight percentage
(wt. %)
Atomic percentage
(at. %)
From
EDX
Expected
From
EDX
Expected
O K
16.64
19.19
55.06
60
Fe K
25.04
22.32
23.73
20
Ho L
4.11
6.59
1.32
2
Nd L
54.21
51.90
19.89
18 Energy (keV)
Intensity (Counts)
Elements
Weight percentage
(wt. %)
Atomic percentage
(at. %)
From
EDX
Expected
From
EDX
Expected
O K
16.64
19.19
55.06
60
Fe K
25.04
22.32
23.73
20
Ho L
4.11
6.59
1.32
2
Nd L
54.21
51.90
19.89
18
Figure 4. EDX for the Nd0.90Ho0.10FeO3 nanoparticles. The inset table shows the at% and wt% of the Ho, O, Fe,
and Nd elements. Intensity (Counts) Figure 4. EDX for the Nd0.90Ho0.10FeO3 nanoparticles. The inset table shows the at% and wt% of the Ho, O, F
and Nd elements. Scientific Reports | (2023) 13:16585 | https://doi.org/10.1038/s41598-023-43734-2 www.nature.com/scientificreports/ nature.com/scientificreports/
Figure 5. The elemental mapping of Nd0.90Ho0.10FeO3 nanoparticles. -20000
-10000
0
10000
20000
-3
-2
-1
0
1
2
3
H (Oe)
M (emu/g)
Nd0.90Ho0.10FeO3
Figure 6. Magnetization (M–H) curve of Nd0.90Ho0.10FeO3 nanoparticles. Figure 5. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6 illustrates the magnetization hysteresis loop of Nd0.90Ho0.10FeO3, which has AFM behavior with weak
ferromagnetic (FM) components32. The values of the saturation magnetization (Ms) and coercive field (Hc) are
reported in Table 2. The value of the squareness ratio (SQR) of the sample was calculated from Eq. (7). (7)
SQR = Mr
Ms (7) The value of SQR was reported in Table 2 and indicates that the type of magnetic interaction is magneto-static
interactions33. The exchange bias (Hex) of the sample was calculated from the Eq. (8). (8)
Hex = −[Hleft + Hright]
2 (8) where Hleft and Hright are the intercepts of the MH curve with the negative and positive x-axis, respectively. The
presence of a shift in the MH loop around the origin originated from the presence of AFM ordering with (FM)
spins in the sample.h where Hleft and Hright are the intercepts of the MH curve with the negative and positive x-axis, respectively. The
presence of a shift in the MH loop around the origin originated from the presence of AFM ordering with (FM)
spins in the sample.h p
p
The anisotropy constant (K) of Nd0.90Ho0.10FeO3 was calculated from Eq. (9)34: p
p
The anisotropy constant (K) of Nd0.90Ho0.10FeO3 was calculated from Eq. (9)34: (9)
K = HC × MS
0.96 (9) where Hc is the coercive field of the sample. The value of K was reported in Table 2.i where Hc is the coercive field of the sample. The value of K was reported in Table 2.ih eld of the sample. The value of K was reported in Table ih
Figure 7 shows the dependence of dM/dH on the magnetic field for Nd0.90Ho0.10FeO3 nanoparticles. The
switching field distribution (SFD) and the rectangularity of the H-M loop (Ha) of Nd0.90Ho0.10FeO3 were calcu-
lated using the following equations9. (10)
SFD = H
HC (10)
SFD = H
HC
(11)
Ha = 2K
Ms (10) C
(11)
Ha = 2K
Ms (11) where ∆H represents the half width at the half maximum of the dM/dH peak. Table 2 contains the values of
SFD and Ha. Many researchers have studied the preparation and properties of NdFeO3. M.M. Arman10 prepared the
NdFeO3 nanoparticles using the citrate combustion method. The values of Ms and Mr of NdFeO3 are 1.05 emu/g
and 0.11 emu/g, respectively. T. www.nature.com/scientificreports/ The elemental mapping of Nd0.90Ho0.10FeO3 nanoparticles. Figure 5. The elemental mapping of Nd0.90Ho0.10FeO3 nanoparticles. Figure 5. The elemental mapping of Nd0.90Ho0.10FeO3 nanoparticles. Figure 5. The elemental mapping of Nd0.90Ho0.10FeO3 nanoparticles. -20000
-10000
0
10000
20000
-3
-2
-1
0
1
2
3
H (Oe)
M (emu/g)
Nd0.90Ho0.10FeO3
Figure 6. Magnetization (M–H) curve of Nd0.90Ho0.10FeO3 nanoparticles. Figure 6. Magnetization (M–H) curve of Nd0.90Ho0.10FeO3 nanoparticles. 5
Vol.:(0
Scientific Reports | (2023) 13:16585 |
https://doi.org/10.1038/s41598-023-43734-2
Table 2. The values of remanence magnetization (Mr), the coercive field (Hc), the exchange bias (Hex), the
anisotropy constant (K), the switching field distribution (SFD), the rectangularity of the H-M loop (Ha), and
the squareness ratio for the sample. Sample
Ms (emu/g)
Mr (emu/g)
Hc (Oe)
SQR = Mr/Ms
Hex (Oe)
K (erg/g)
SDF
Hm (Oe)
Ha (Oe)
Nd0.90Ho0.10FeO3
2.42
0.25
200
0.103
− 10.86
504.17
2.33
255
417 Table 2. The values of remanence magnetization (Mr), the coercive field (Hc), the exchange bias (Hex), the
anisotropy constant (K), the switching field distribution (SFD), the rectangularity of the H-M loop (Ha), and
the squareness ratio for the sample. Sample
Ms (emu/g)
Mr (emu/g)
Hc (Oe)
SQR = Mr/Ms
Hex (Oe)
K (erg/g)
SDF
Hm (Oe)
Ha (Oe)
Nd0.90Ho0.10FeO3
2.42
0.25
200
0.103
− 10.86
504.17
2.33
255
417 Table 2. The values of remanence magnetization (Mr), the coercive field (Hc), the exchange bias (Hex), the
anisotropy constant (K), the switching field distribution (SFD), the rectangularity of the H-M loop (Ha), and
the squareness ratio for the sample. Sample
Ms (emu/g)
Mr (emu/g)
Hc (Oe)
SQR = Mr/Ms
Hex (Oe)
K (erg/g)
SDF
Hm (Oe)
Ha (Oe)
Nd0.90Ho0.10FeO3
2.42
0.25
200
0.103
− 10.86
504.17
2.33
255
417 Table 2. The values of remanence magnetization (Mr), the coercive field (Hc), the exchange bias (Hex), the
anisotropy constant (K), the switching field distribution (SFD), the rectangularity of the H-M loop (Ha), and
the squareness ratio for the sample. Nd0.90Ho0.10FeO3
2.42
0.25
200
0.103
− 10.86
504.17
2.33
255
417 https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 | www.nature.com/scientificreports/ The value of the μeff was determined from the fol-
lowing equation: (13)
µ
2 83
√
C µeff= 2.83
√
C (13) (13)
µeff= 2.83
√
C where C denotes the reciprocal of the slope in the paramagnetic part. The values of μeff, θ, and C were reported
n Table 3. The value of TN was determined from the differentiation of χM (dχM/dT) and listed in Table 3. Figure 10 shows the dependence of the absorbance of the UV–visible light by the sample on the wavelength
of the incident photons. In the low wavelength region (λ ≤ 460 nm), the absorbance increases rapidly with λ due
to increasing the energy of the photons, which allows the electrons to be transferred from the 2p orbital oxygen
valance band (V.B.) to the 3d orbital iron conduction band (C.B.). In the high wavelength region (λ > 460 nm),
the energy of the photons is low and can’t transfer the electrons from V.B. to C.B.hfi gy
p
The optical absorption coefficient (α) is related to the quantity of UV–visible light absorption through the
material. The value of α was determined using Eq. (14)37. 500
550
600
650
700
750
800
850
900
5
10
15
20
25
30
35
40
45
T(K)
1340 Oe
M
-1 (emu/g)
-1
Figure 9. The relation of χM
-1 to the temperature at 1340 Oe. Table 3
Values of the Curie Weiss constant (θ) the Curie constant (C) the effective magnetic moment (μf )
Sample
Field (Oe)
C (emu/g.mol) K
θ (K)
μeff (B.M.)
TN (K)
Nd0.90Ho0.10FeO3
1340
1.875
824.84
3.903
895.84 500
550
600
650
700
750
800
850
900
5
10
15
20
25
30
35
40
45
T(K)
1340 Oe
M
-1 (emu/g)
-1
Figure 9. The relation of χM
-1 to the temperature at 1340 Oe. 500
550
600
650
700
750
800
850
900
5
10
15
20
25
30
35
40
45
T(K)
1340 Oe
M
-1 (emu/g)
-1h Figure 9. The relation of χM
-1 to the temperature at 1340 Oe. Figure 9. The relation of χM to the temperature at 1340 Oe. Table 3. Values of the Curie–Weiss constant (θ), the Curie constant (C), the effective magnetic moment (μeff),
and the Neel temperature (TN) of Nd0.90Ho0.10FeO3 nanoparticles. Sample
Field (Oe)
C (emu/g.mol) K
θ (K)
μeff (B.M.)
TN (K)
Nd0.90Ho0.10FeO3
1340
1.875
824.84
3.903
895.84 Table 3. www.nature.com/scientificreports/ Shalini et al.35 studied the structure and magnetic behavior of NdFeO3, which has
Ms and Mr equal to 0.521 emu/g and 0.098 emu/g, respectively. In the presence of work, the substitution of Ho3+
ions instead of Nd3+ ions increased the magnetic properties of the NdFeO3 nanoparticles. Where the effective
magnetic moment of Nd3+ is 1.14 μB while that of Ho3+ is 10.6 μB
36. The presence of Ho3+ in the NdFeO3 increases
the magnetic interactions between the magnetic ions such as Ho3+–O2-–Ho3+, Ho3+–O2-–Nd3+, and Ho3+–O2—Fe3+.h Figure 8 shows the dependence of the molar magnetic susceptibility (χM) on T(K). The behavior of χM with
temperature assures that the sample has AFM behavior. χM decreases with raising the temperature up to the
Neel temperature (TN), after which Nd0.90Ho0.10FeO3 has a paramagnetic behavior. The AFM properties originate
from the magnetic interaction between the Fe3+ ions. The relation between χM and the applied field is inversely
proportional according to the following equation: (12)
χM = M
H χM = M
H (12) -2000 -1500 -1000 -500
0
500
1000
1500
2000
0.0
2.0x10
-4
4.0x10
-4
6.0x10
-4
8.0x10
-4
1.0x10
-3
1.2x10
-3
1.4x10
-3
2Hm
dM/dH (emu/g.Oe)
H (Oe)
Figure 7. The relation between dM/dH and the magnetic field for Nd0.90Ho0.10FeO3 nanoparticles. Figure 7. The relation between dM/dH and the magnetic field for Nd0.90Ho0.10FeO3 nanoparticles. Scientific Reports | (2023) 13:16585 | https://doi.org/10.1038/s41598-023-43734-2 www.nature.com/scientificreports/ /
p
/
300
400
500
600
700
800
900
1000
0.00
0.05
0.10
0.15
0.20
T(K)
M (emu/g)
1010 Oe
1340 Oe
1660 Oe
Figure 8. The dependence of χM on the temperature for Nd0.90Ho0.10FeO3. 300
400
500
600
700
800
900
1000
0.00
0.05
0.10
0.15
0.20
T(K)
M (emu/g)
1010 Oe
1340 Oe
1660 Oe
Figure 8. The dependence of χM on the temperature for Nd0.90Ho0.10FeO3. 300
400
500
600
700
800
900
1000
0.00
0.05
0.10
0.15
0.20
T(K)
M (emu/g)
1010 Oe
1340 Oe
1660 Oe Figure 8. The dependence of χM on the temperature for Nd0.90Ho0.10FeO3. The relation between χM
-1 and the temperature is shown in Fig. 9, which applies the Curie–Weiss law. From
the slope of the paramagnetic region in Fig. 9, the values of the effective magnetic moment (μeff), the Curie–Weiss
constant (θ), and the Curie constant (C) were determined. www.nature.com/scientificreports/ Values of the Curie–Weiss constant (θ), the Curie constant (C), the effective magnetic moment (μeff),
and the Neel temperature (TN) of Nd0.90Ho0.10FeO3 nanoparticles. https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 | www.nature.com/scientificreports/
200
400
600
800
1000
0.0
0.1
0.2
0.3
Absorbance (a.u.)
(nm)
Nd0.90Ho0.10FeO3
Figure 10. The relation between the absorbance and the wavelength of the photons for Nd0.90Ho0.10FeO3. www.nature.com/scientificreports/ 200
400
600
800
1000
0.0
0.1
0.2
0.3
Absorbance (a.u.)
(nm)
Nd0.90Ho0.10FeO3
Figure 10. The relation between the absorbance and the wavelength of the photons for Nd0.90Ho0.10FeO3. 200
400
600
800
1000
0.0
0.1
0.2
0.3
Absorbance (a.u.)
(nm)
Nd0.90Ho0.10FeO3
Figure 10. The relation between the absorbance and the wavelength of the photons for Nd0.90Ho0.10FeO3. Figure 10. The relation between the absorbance and the wavelength of the photons for Nd0.90Ho0.10FeO3. (14)
α = (2.303)A
l (14) where A is the absorbance and l denotes the length of the spacemen. Figure 11 shows the relationship between
α and the wavelength. The values of α increased rapidly with increasing λ up to 460 nm, then α decreased slowly
with increasing λ. The increasing of α is due to increasing the absorption of photons at low wavelengths and
higher frequencies, while the decreasing of α is due to decreasing the absorption of photons at high wavelengths
and lower frequencies.hfi where A is the absorbance and l denotes the length of the spacemen. Figure 11 shows the relationship between
α and the wavelength. The values of α increased rapidly with increasing λ up to 460 nm, then α decreased slowly
with increasing λ. The increasing of α is due to increasing the absorption of photons at low wavelengths and
higher frequencies, while the decreasing of α is due to decreasing the absorption of photons at high wavelengths
and lower frequencies.hfi q
The optical extinction coefficient (k) denotes the losses of electromagnetic energy in Nd0.90Ho0.10FeO3 nano-
articles. k was calculated using the Eq. (15). (15)
k = α
4π (15) Figure 12 studies the dependence of k on wavelength of the photons. The increasing of k with increasing λ
of photons is due to the fact that at high λ, the energy of the photons is small and doesn’t absorb in the sample,
increasing the energy losses, so k increases.h g
gy
The Tauc plot was used to determine the optical band gap value (Eg) and the type of optical transition of
Nd0.90Ho0.10FeO3. www.nature.com/scientificreports/ Prabagar et al.21 prepared NdFeO3 using the citrate sol–gel method with an optical bandgap of 2.48 eV
and good photocatalytic activities. While introducing the Ho3+ ions in the NdFeO3 leads to a decrease in Eg to
1.106 eV due to introducing orbitals and states of Ho3+ ions in the NdFeO3 system. The author recommended
using Nd0.90Ho0.10FeO3 as a photocatalyst for organic dye degradation in water.fi g
p
y
g
y
g
Figure 14 illustrates the dependence of the removal efficiency of the HMs at Ph = 7, which was calculated
using Eq. (17). (17)
η = Ci −Cf
Ci
× 100 (17) where Cf is the final concentration while and Ci denotes the initial concentration of the HMs. The sample
Nd0.90Ho0.10FeO3 has the ability to remove a lot of HMs from water, which indicates that it has a large surface area. The adsorption of the HMs from an aqueous solution depends on many parameters, such as temperature, pH
value of the solution, contact time, ionic radii of the HMs, initial concentration of the adsorbent, and molecular
weight of the HMs. The highest efficiency of HMs from the water was observed for Pb2+ ions with η = 72.39%
due to the fact that Pb2+ ions have a higher molecular weight than the other HMs, leading to easier adsorption.hffi where Cf is the final concentration while and Ci denotes the initial concentration of the HMs. The sample
Nd0.90Ho0.10FeO3 has the ability to remove a lot of HMs from water, which indicates that it has a large surface area. The adsorption of the HMs from an aqueous solution depends on many parameters, such as temperature, pH
value of the solution, contact time, ionic radii of the HMs, initial concentration of the adsorbent, and molecular
weight of the HMs. The highest efficiency of HMs from the water was observed for Pb2+ ions with η = 72.39%
due to the fact that Pb2+ ions have a higher molecular weight than the other HMs, leading to easier adsorption.hffi The effect of the pH value of the solution on the removal efficiency (η) of Pb2+ ions was studied and illustrated
in Fig. 15. In the acidic region (pH ≤ 6), there are a lot of H+ ions in the solution, which participate with the Pb2+
ions in the adsorption on the active sites on the surface of Nd0.90Ho0.10FeO3, so η is low. www.nature.com/scientificreports/ The Tauc equation was represented by Eq. (16)37. (16)
(αhv)x = A
hv −Eg (16) where A and hν are the constant and photon energy, respectively. The (x) value was estimated to be the type of
optical transition. For the direct transition, x equals 2, while for the indirect transition, x equals 1. The Tauc plot
is represented in Fig. 13, which illustrates that the Nd0.90Ho0.10FeO3 sample has a direct transition with Eg = 1.106 where A and hν are the constant and photon energy, respectively. The (x) value was estimated to be the type of
optical transition. For the direct transition, x equals 2, while for the indirect transition, x equals 1. The Tauc plot
is represented in Fig. 13, which illustrates that the Nd0.90Ho0.10FeO3 sample has a direct transition with Eg = 1.106 200
400
600
800
1000
0.2
0.3
0.4
0.5
0.6
0.7
(cm-1)
(nm)
Nd0.90Ho0.10FeO3
Figure 11. The dependence of α and the λ of the photons. Figure 11. The dependence of α and the λ of the photons. Scientific Reports | (2023) 13:16585 | https://doi.org/10.1038/s41598-023-43734-2 www.nature.com/scientificreports/
200
400
600
800
1000
1200
0.0
1.0x10
-6
2.0x10
-6
3.0x10
-6
4.0x10
-6
5.0x10
-6
K
(nm)
Figure 12. The relation between k and the wavelength for Nd0.90Ho0.10FeO3 nanoparticles. www.nature.com/scientificreports/ 200
400
600
800
1000
1200
0.0
1.0x10
-6
2.0x10
-6
3.0x10
-6
4.0x10
-6
5.0x10
-6
K
(nm)
Figure 12. The relation between k and the wavelength for Nd0.90Ho0.10FeO3 nanoparticles. 200
400
600
800
1000
1200
0.0
1.0x10
-6
2.0x10
-6
3.0x10
-6
4.0x10
-6
5.0x10
-6
K
(nm)
Figure 12. The relation between k and the wavelength for Nd0.90Ho0.10FeO3 nanoparticles. Figure 12. The relation between k and the wavelength for Nd0.90Ho0.10FeO3 nanoparticles. 1
2
3
4
5
0
2
4
6
h )2 (cm-1.eV)2
h (eV)
Figure 13. The Tauc plot for the optical direct transition of Nd0.90Ho0.10FeO3. Figure 13. The Tauc plot for the optical direct transition of Nd0.90Ho0.10FeO3. eV. J. S. Prabagar et al.21 prepared NdFeO3 using the citrate sol–gel method with an optical bandgap of 2.48 eV
and good photocatalytic activities. While introducing the Ho3+ ions in the NdFeO3 leads to a decrease in Eg to
1.106 eV due to introducing orbitals and states of Ho3+ ions in the NdFeO3 system. The author recommended
using Nd0.90Ho0.10FeO3 as a photocatalyst for organic dye degradation in water.fi eV. J. S. www.nature.com/scientificreports/ The maximum adsorp-
tion of Pb2+ ions was observed at pH = 7, which indicates the optimum pH condition for removal of Pb2+ from
aqueous solutions using Ho-doped NdFeO3. The FESEM images of the sample illustrate the porous nature of the
surface of the sample, which increases the active sites that adsorb the HMs from the water. In the basic medium
(pH = 8), the Pb2+ ions can be precipitated as lead hydroxide, which is not favorable. https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 | www.nature.com/scientificreports/ tificreports/
Co
Cd
Pb
Cr
Ni
0
10
20
30
40
50
60
70
Efficiency %
Heavy metals
Figure 14. The removal effectiveness of Nd0.90Ho0.10FeO3 for the different heavy metals. Co
Cd
Pb
Cr
Ni
0
10
20
30
40
50
60
70
Efficiency %
Heavy metals
Figure 14. The removal effectiveness of Nd0.90Ho0.10FeO3 for the different heavy metals. Figure 14. The removal effectiveness of Nd0.90Ho0.10FeO3 for the different heavy metals. 3
4
5
6
7
8
0
10
20
30
40
50
60
70
pH
Removal efficiency%
Figure 15. The dependence of the removal efficiency of Pb2+ ions on the pH value of the solution. Figure 15. The dependence of the removal efficiency of Pb2+ ions on the pH value of the solution. The adsorption mechanism of the Pb2+ ions from the water was studied using the adsorption isotherm models. In the present work, the Langmuir and Freundlich isotherm models were used to study the adsorption of Pb2+
on the surface of the sample.h p
The Langmuir isotherm represents the adsorption of the Pb2+ ions on the surface active sites as a single layer. The assumptions of the Langmuir isotherm are that the HMS is adsorbed on discrete surface active sites, each
HM molecule adsorbs on one active site, the sample has a uniform adsorbing surface, and HM molecules don’t
interact with each other38. The following equation describes the Langmuir isotherm mode. (18)
Ce
qe
=
1
qmKL
+ Ce
qm (18) where Ce is the equilibrium Pb2+ ion concentration, qm, and KL denote the Langmuir constants. The equilibrium
dsorption capacity (qe) was calculated using Eq. (19). (19)
qe = (Ci −Ce)V
m (19) where V and m are the volume of the Pb2+ solution and the adsorbent mass, respectively. Conclusion Nd0.90Ho0.10FeO3 was prepared in an orthorhombic structure with a crystallite size of 39.136 nm. FESEM illus-
trates the agglomerated grains due to the magnetic behavior of the sample. The EDX data shows that the elements
Fe, Ho, Nd, and O are present in Ho-doped sample without any impurities. The antiferromagnetic properties of
Nd0.90Ho0.10FeO3 originate from the magnetic coupling of Fe3+–O2-–Fe3+, Nd3+–O2-–Fe3+, and Nd3+–O2-–Nd3+. The values of Ms, Mr, Hc, Hex, K, and SDF are 2.42 emu/g, 0.25 emu/g, 200 Oe, -10.86 Oe, 514.14 erg/g, and 2.33,
respectively. The sample has a direct optical transition with Eg = 1.106 eV. Nd0.90Ho0.10FeO3 is a good absorber of
UV–visible light and can be used for photocatalysis of organic dye degradation in water. The Nd0.90Ho0.10FeO3
nanoparticles have a high efficiency (72.39%) to remove the heavy metal Pb2+ from water. The experimental data
is more fitting for the Langmuir isotherm mode. www.nature.com/scientificreports/ 2.2
2.4
2.6
2.8
3.0
3.2
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
ln q
ln Ce
Equation
y = a + b*x
Weight
No Weightin
Residual
Sum of
Squares
0.01875
Pearson's r
-0.97567
Adj. R-Square
0.93991
Value
Standard Err
E2
Intercept
4.4884
0.26259
E2
Slope
-0.8395
0.09433 Figure 17. The Freundlich model fitting for Nd0.90Ho0.10FeO3. where Kf denotes the Freundlich constant. Figure 17 shows the fitting of the experimental data with the Freun-
dlich isotherm. 2 From the inset tables in Figs. 16 and 17, the values of R2 are 0.9545 and 0.9399 for the Langmuir and Freun-
dlich isotherm modes, respectively. The Langmuir isotherm mode is the best-fit model for adsorbing the Pb2+
ions from water, and the HMs form monolayer adsorption. www.nature.com/scientificreports/ Figure 16 shows the
fitting of the experimental data with the Langmuir isotherm model.h where V and m are the volume of the Pb2+ solution and the adsorbent mass, respectively. Figure 16 shows the
fitting of the experimental data with the Langmuir isotherm model.h g
g
The Freundlich isotherm describes the mass transportation of the Pb2+ ions from the aqueous solution to the
ctive sites on the porous surface of Nd0.90Ho0.10FeO3. Equation (20) represents the Freundlich isotherm model (20)
Lnqe = LnKf + 1
nLnCe (20) https://doi.org/10.1038/s41598-023-43734-2 https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 | https://doi.org/10.1038/s41598-023-43734-2 www.nature.com/scientificreports/ entificreports/
8
10
12
14
16
18
20
22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Ce/q (g/L)
Ce(mg/L)
Equation
y = a + b*x
Weight
No Weightin
Residual
Sum of
Squares
0.2357
Pearson's r
0.98162
Adj. R-Square
0.95447
Value
Standard Err
F4
Intercept
-1.6391
0.37688
F4
Slope
0.22536
0.02191
Figure 16. The Langmuir model fitting for Ho-doped NdFeO3. 2.2
2.4
2.6
2.8
3.0
3.2
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
ln q
ln Ce
Equation
y = a + b*x
Weight
No Weightin
Residual
Sum of
Squares
0.01875
Pearson's r
-0.97567
Adj. R-Square
0.93991
Value
Standard Err
E2
Intercept
4.4884
0.26259
E2
Slope
-0.8395
0.09433
Figure 17. The Freundlich model fitting for Nd0.90Ho0.10FeO3. 8
10
12
14
16
18
20
22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Ce/q (g/L)
Ce(mg/L)
Equation
y = a + b*x
Weight
No Weightin
Residual
Sum of
Squares
0.2357
Pearson's r
0.98162
Adj. R-Square
0.95447
Value
Standard Err
F4
Intercept
-1.6391
0.37688
F4
Slope
0.22536
0.02191
Figure 16. The Langmuir model fitting for Ho-doped NdFeO3. 8
10
12
14
16
18
20
22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Ce/q (g/L)
Ce(mg/L)
Equation
y = a + b*x
Weight
No Weightin
Residual
Sum of
Squares
0.2357
Pearson's r
0.98162
Adj. R-Square
0.95447
Value
Standard Err
F4
Intercept
-1.6391
0.37688
F4
Slope
0.22536
0.02191 Figure 16. The Langmuir model fitting for Ho-doped NdFeO3. 2.2
2.4
2.6
2.8
3.0
3.2
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
ln q
ln Ce
Equation
y = a + b*x
Weight
No Weightin
Residual
Sum of
Squares
0.01875
Pearson's r
-0.97567
Adj. R-Square
0.93991
Value
Standard Err
E2
Intercept
4.4884
0.26259
E2
Slope
-0.8395
0.09433
Figure 17. The Freundlich model fitting for Nd0.90Ho0.10FeO3. References 1. Khaled, M. A., Ruvalcaba, J., Córdova, T. F., El Marssi, M. & Bouyanfif, H. Spin-lattice coupling in an epitaxial NdFeO3 thin film
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14 (2010). 5. Mbamba, C. K., Batstone, D. Data availability
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h The author declares that all the data supporting the findings of this study are available in the ICDD card number
01-089-6644. https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 | www.nature.com/scientificreports/ Received: 12 July 2023; Accepted: 27 September 2023 References Arman, M. M., Ahmed, M. K. & El-Masry, M. M. Cellulose Acetate polymer spectroscopic study comprised LaFeO3 perovskite
and graphene as a UV-to-visible light converter used in several applications. J. Mol. Struct. 1281, 135153 (2023). 8. Goodfellow, H. D. & Wang, Y. (2nd Eds.). Industrial Ventilation Design Guidebook: Volume 2: Engineering Design and Applications
(Academic press, 2021). https://doi.org/10.1038/s41598-023-43734-2 Scientific Reports | (2023) 13:16585 | www.nature.com/scientificreports/ Competing interests h p
g
The authors declare no competing interests. Author contributions
h
d
f M.M. Arman put the idea of the manuscript together, did the experimental work, and then wrote the main
manuscript text. Funding Funding
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in coopera-
tion with The Egyptian Knowledge Bank (EKB). © The Author(s) 2023 Additional information
d
d
f Correspondence and requests for materials should be addressed to M.M.A. Correspondence and requests for materials should be addressed to M.M.A. Reprints and permissions information is available at www.nature.com/reprints. 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
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English
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Case-control study of ADARB1 and ADARB2 gene variants in migraine
|
The Journal of headache and pain
| 2,015
|
cc-by
| 6,298
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This may be the author’s version of a work that was submitted/accepted
for publication in the following source: Gasparini,
Claudia,
Sutherland,
Heidi,
Maher,
Bridget,
Rodriguez
Acevedo, Astrid Jannet, Khlifi, Elhame, Haupt, Larisa, & Griffiths, Lyn
(2015) Case-control study of ADARB1 and ADARB2 gene variants in migraine. Journal of Headache and Pain, 16, Article number: 31 1-8. This file was downloaded from: https://eprints.qut.edu.au/87084/ © Consult author(s) regarding copyright matters This work is covered by copyright. Unless the document is being made available under a
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that permission from the copyright owner must be obtained for all other uses. If the docu-
ment is available under a Creative Commons License (or other specified license) then refer
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this work infringes copyright please provide details by email to qut.copyright@qut.edu.au License: Creative Commons: Attribution 2.5 Notice: Please note that this document may not be the Version of Record
(i.e. published version) of the work. Author manuscript versions (as Sub-
mitted for peer review or as Accepted for publication after peer review) can
be identified by an absence of publisher branding and/or typeset appear-
ance. If there is any doubt, please refer to the published source. https://doi.org/10.1186/s10194-015-0511-y https://doi.org/10.1186/s10194-015-0511-y Case-control study of ADARB1 and ADARB2 gene variants in migraine. Claudia F Gasparini PhD1, Heidi G Sutherland PhD2, Bridget Maher PhD2,3, Astrid
Rodriguez-Acevedo, BBiol2, Elhame Khlifi, Larisa M Haupt PhD2, Lyn R Griffiths PhD2*. 1Griffith Health Institute, Griffith University Gold Coast, Parklands Drive, Southport, QLD,
4222, Australia
2UCL Institute of Neurology, Department of Clinical and Experimental Epilepsy, Queen
Square, London, WC1N 3BG, England
3Genomics Research Centre, Institute of Health and Biomedical Innovation, Queensland
University of Technology, Musk Ave, Kelvin Grove, QLD, 4059, Australia
*Corresponding Author
Professor Lyn Griffiths
Genomics Research Centre
Institute of Health and Biomedical Innovation
Queensland University of Technology
Musk Ave, Kelvin Grove, QLD, 4059, Australia. Tel: +61 7 3138 6102
Fax: +61 7 3138 6039
Email: lyn.griffiths@qut.edu.au Case-control study of ADARB1 and ADARB2 gene variants in migraine. Claudia F Gasparini PhD1, Heidi G Sutherland PhD2, Bridget Maher PhD2,3, Astrid
Rodriguez-Acevedo, BBiol2, Elhame Khlifi, Larisa M Haupt PhD2, Lyn R Griffiths PhD2*. 1Griffith Health Institute, Griffith University Gold Coast, Parklands Drive, Southport, QLD,
4222, Australia
2UCL Institute of Neurology, Department of Clinical and Experimental Epilepsy, Queen
Square, London, WC1N 3BG, England
3Genomics Research Centre, Institute of Health and Biomedical Innovation, Queensland
University of Technology, Musk Ave, Kelvin Grove, QLD, 4059, Australia
*Corresponding Author
Professor Lyn Griffiths
Genomics Research Centre
Institute of Health and Biomedical Innovation
Queensland University of Technology
Musk Ave, Kelvin Grove, QLD, 4059, Australia. Tel: +61 7 3138 6102
Fax: +61 7 3138 6039
Email: lyn.griffiths@qut.edu.au Claudia F Gasparini PhD1, Heidi G Sutherland PhD2, Bridget Maher PhD2,3, Astrid J
Rodriguez-Acevedo, BBiol2, Elhame Khlifi, Larisa M Haupt PhD2, Lyn R Griffiths PhD2*. Claudia F Gasparini PhD1, Heidi G Sutherland PhD2, Bridget Maher PhD2,3, Astrid J
Rodriguez-Acevedo, BBiol2, Elhame Khlifi, Larisa M Haupt PhD2, Lyn R Griffiths PhD2*. UCL Institute of Neurology, Department of Clinical and Experimental Epilepsy, Queen
quare, London, WC1N 3BG, England Background: Migraine causes crippling attacks of severe head pain along with associated nausea,
vomiting, photophobia and/or phonophobia. The aim of this study was to investigate single
nucleotide polymorphisms (SNPs) in the adenosine deaminase, RNA-specific, B1 (ADARB1)
and adenosine deaminase, RNA specific, B2 (ADARB2) genes in an Australian case-control
Caucasian population for association with migraine. Both candidate genes are highly
expressed in the central nervous system (CNS) and fit criteria for migraine neuropathology. SNPs in the ADARB2 gene were previously found to be positively associated with migraine
in a pedigree-based GWAS using the genetic isolate of Norfolk Island, Australia. The
ADARB1 gene was also chosen for investigation due to its important function in editing
neurotransmitter receptor transcripts. *Corresponding Author Email: lyn.griffiths@qut.edu.au 1 Methods: Four SNPs in ADARB1 and nine in ADARB2 were selected by inspecting blocks of LD in
Haploview for genotyping using either TaqMan or Sequenom assays. These SNPs were
genotyped in two-hundred and ninety one patients who satisfied the International
Classification of Headache Disorders, ICHD-II 2004 diagnostic criteria for migraine and
three-hundred and fourteen controls and PLINK was used for association testing. Results: Chi-square (χ2) analysis found no significant association between any of the SNPs tested in
the ADARB1 and ADARB2 genes in this study and the occurrence of migraine. Conclusions: In contrast to findings that SNPs in the ADARB2 gene were positively associated with
migraine in the Norfolk Island population, we find no evidence to support the involvement of
RNA editing genes in migraine susceptibility in an Australian Caucasian population. 2 2 Migraine, Migraine with aura, Migraine without aura, RNA editing, pGWAS, Norfolk Island, Key Words: Migraine, Migraine with aura, Migraine without aura, RNA editing, pGWAS, Norfolk Island, ADARB1, ADARB2, headache. ADARB1, ADARB2, headache. 3 3 Background The International Headache Society (IHS) defines migraine as a recurrent headache disorder
typified by painful attacks lasting 4-72 hours [1]. Classification criteria recognize two types
of migraine, migraine with aura (MA) and migraine without aura (MO) with the former
accompanied by visual or auditory disturbances [1]. Migraine generally affects 12% of the
Caucasian population showing up more often in females, a statistic correlated with instability
of ovarian hormones [2]. Migraine has a major impact on the wellbeing and quality of life of
sufferers and their families in part due to days missed from the workplace and higher direct
health-care costs [3]. Numerous theories and models regarding migraine mechanisms have
emerged; the most accepted opinion is that a combination of both vascular and neural events
is involved in the initiation and perpetuation of a migraine attack [4,5]. Primarily the
generation of pain is attributed to complex processes within the nervous system that activate
the trigeminovascular system consisting of the trigeminal nerve and its ramifications which
regulate cerebral blood flow and the release of inflammatory molecules. The fact that the
diameter of cranial blood vessels along with other functions is controlled by signals
transmitted by nerves is further evidence to support a neurogenic theory. The aura on the
other hand, occurs in only a minority of migraineurs and has been attributed to an
electrophysiological phenomenon, first reported in epilepsy, termed Cortical Spreading
Depression (CSD) [6]. Interactions between the environment and the genotype are important in shaping the migraine
phenotype. Population based twin studies have confirmed a genetic influence ranging from
0.34 to 0.57 [7]. Genetic studies of a rare, and more severe migraine subtype, Familial
Hemiplegic Migraine (FHM), have identified mutations in three causal genes that code for
ion channels involved in neuronal signalling and have provided hypotheses applicable to 4 common migraine [8]. Functional studies in cellular and animal models of mutant alleles
provide direct evidence for neuronal hyperexcitability as one cellular mechanism underlying
headache or aura in FHM [9]. For the most part, genes causing more common types of
migraine have been identified from neurological, vascular and hormonal pathways and in
2010 the first functional variant to show linkage to familial MA was identified in TRESK, a
potassium channel involved in neuronal excitability [10]. Background While genome wide association
studies (GWAS) have shed new light on the types of genes involved in migraine
susceptibility, many candidate gene association studies have focused on neurotransmitter-
related pathways, as these pathways are considered to play a significant role in the migraine
process. Therefore genes affecting synthesis and activity of neurotransmitters, including
RNA editing genes, are potential candidates for involvement in migraine susceptibility. Isolated founder populations offer several advantages over mainstream (outbred) populations
for genomic studies of disease, as both environmental noise and genetic heterogeneity is
reduced. We have used the population of Norfolk Island, an island off the east coast of
Australia, for genetic studies on migraine as it has well-documented family histories and an
increased prevalence of the disorder [11]. We previously identified four SNPs forming a
22kb haplotype block in ADARB2 (Table 1 and Figure 1) that were positively associated with
migraine susceptibility in a pedigree-based GWAS (pGWAS) of the population of Norfolk
Island. [11]. ADARB2 is a member of the double-stranded RNA adenosine deaminase family
of RNA-editing enzymes [12]. The enzymatic activity of ADARs leads to the chemical
modification of Adenosine-to-Inosine (A-to-I) in specific coding regions which are then
translated as guanosines by the cells translational machinery [13]. A-to-I RNA editing is a
post-transcriptional process that permanently alters the nucleotide sequence of an RNA
molecule resulting in the synthesis of proteins not encoded by the original gene sequence 5 5 [14]. This is a form of chemical recoding that changes specific amino acid residues and alters
the biological function of translated molecules, which is most clearly demonstrated by an
alteration in channel properties including the Ca2+ permeability of glutamate receptors
(GluRs) [15]. Perturbed A-to-I RNA editing has been implicated in human cancer and viral
infections and neurodegenerative/neurological diseases such as dyschromatosis symmetrica
hereditaria (DSH), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and
Huntington’s disease epilepsy, depression and schizophrenia [16,17]. In ALS inefficient
RNA editing fails to substitute an arginine for a glutamine residue in the GluR2 Q/R site of
glutamate AMPA receptors in the spinal motor neurons and is proposed as a mechanism
responsible for motor neuron death [18]. ADARB2 (also known as ADAR3) comprises 10 exons spanning a genomic region of 9.5kb on
chromosome 10p15.3 and encodes a catalytically inactive protein, expressed in brain,
amygdala and thalamus [12]. SNP selection HapMap CEU (Utah residents with Northern and Western European ancestry) SNP genotype
data was downloaded for each candidate gene and a total of 8 haplotype blocks were
identified in the ADARB1 gene and 56 haplotype blocks in the ADARB2 gene in the program
Haploview v4.2 [24]. Thirteen SNPs were selected in the ADARB1 and ADARB2 genes are
spread out across the entire length of the gene and are located in different LD blocks and
were mostly located in intronic regions of each gene (Table 3 and 4). Case-control population The study was approved by the Griffith University Ethics Committee for experimentation in
humans. The study population was composed of 291 cases and 314 controls and
demographical characteristics of the population are reported in (Table 2). Affected
individuals were diagnosed as having MA or MO by an experienced clinical neurologist
based on responses provided in a validated medical questionnaire in accordance with criteria
determined by the International Headache Society (IHS) [1]. The migraine population
consisted of individuals of Caucasian origin, recruited from the South East Queensland
Region of Australia. The control population samples were obtained via the Genomics
Research Centre Clinic, Southport and had no history of personal or familial migraine. Signed informed consent was obtained from all patients before participation in the study. Background One SNP rs2271275 confers a (Thr-Ala) amino acid change in
the protein structure and has previously been associated with early-onset obsessive-
compulsive disorder in some American families in a genome-wide linkage scan [19]. In
addition to ADARB2 SNPs, the Norfolk Island pGWAS also found two SNPs in the glutamate
receptor, metabotropic 7 (GRM7) gene and one SNP in the 5-hydroxytryptamine serotonin
receptor 7 adenylate cyclase-coupled (HTR7) gene to be associated with migraine [11]. Collectively, association of variants in these neurotransmitter-related genes ADARB2; GRM7;
HTR7 connected by a common neurological pathway supports current theories of a perturbed
serotonin and glutamate mechanism in migraine and in the Norfolk pedigree. Previous studies have reported positive associations of SNPs in various neurotransmitter-
related genes, e.g. DBH [20] and SLC6A3 [21], with migraine in a number of case-control
populations. 6 6 Considering the association of ADARB2 with migraine in the Norfolk Island pGWAS we
decided to investigate SNPs in this gene, as well as another RNA editing gene, ADARB1, in
an Australian migraine case-control population. ADARB1 encodes an RNA editing enzyme,
expressed mainly in the CNS, that is involved in the downstream regulation of
neurotransmitters [22]. The gene is of potential interest with respect to migraine susceptibility
as the glutamate and serotonin receptor gene RNAs are the predominant substrates modified
by ADARB1 adenosine deamination [22]. ADARB1 (also known as ADAR2) spans a genomic
region of 25 kb on chromosome 21q22.3 and comprises 16 exons and regulates its own
expression through self-editing [23]. In this study we genotyped four SNPs in ADARB1 and
nine SNPs in ADARB2 using either TaqMan or Sequenom assays to investigate their
involvement in migraine. 7 7 Genotyping Methods Genomic DNA was extracted from peripheral blood samples using a salting out method as
described by Miller et al., 1988 [25]. DNA was quantified and normalised to a concentration 8 of 20ng/µL for genotyping experiments. Nine SNPs were genotyped as part of a Sequenom
plex, which allows high throughput multiplexing of the assays into a single well. Not all
SNPs are compatible with the restraints required for primer design for this method, or can be
run together so therefore we also used TaqMan assays to genotype five of the SNPs. For each
SNP, Sanger sequencing of a subset of samples was performed on a ABI3500 (Life
Technologies, Carlsband, CA, USA) to confirm the genotypes. of 20ng/µL for genotyping experiments. Nine SNPs were genotyped as part of a Sequenom
plex, which allows high throughput multiplexing of the assays into a single well. Not all
SNPs are compatible with the restraints required for primer design for this method, or can be
run together so therefore we also used TaqMan assays to genotype five of the SNPs. For each
SNP, Sanger sequencing of a subset of samples was performed on a ABI3500 (Life
Technologies, Carlsband, CA, USA) to confirm the genotypes. Genotyping by Sequenom SNPs in the ADARB1 and ADARB2 genes were genotyped using the Sequenom MassARRAY
system 4 platform, and Typer 4.0 software was used to carry out all genotyping work
(MALDI-TOF; MassARRAY system, Sequenom Inc., San Diego, CA, USA). The primers
for PCR and iPLEX reactions were designed using the online Assay design suite 1.0
Sequenom software (available at: www.mysequenom.com/tools) and obtained from IDT (see
Table 4) (Integrated DNA Technologies, Carolville, Iowa, USA). PCR and extension
reactions were performed in a 96-well plate according to the manufacturer’s instructions,
using Sequenom reagents. Completed genotyping reactions were spotted in nanoliter volumes
onto a matrix arrayed silicon chip with 96 elements (Sequenom SpectroCHIP) using the
MassARRAY Nanodispenser. Spectro CHIPs were analyzed using the Bruker Autoflex
MALDI-TOF mass spectrometer and the spectra were processed using the SpectroTYPER
software (Sequenom) to yield genotypes. Genotyping by TaqMan SNPs rs2838771, rs1051367 in ADARB1 and SNPs rs2271275, rs10903467, rs11250642 in
ADARB2 were genotyped using TaqMan® SNP Genotyping Assays from Applied
Biosystems (Life Technologies, Carlsband, CA, USA). Detailed information regarding SNPs
genotyped in the ADARB1 and ADARB2 genes and a summary of assay conditions for each
SNP are listed in Table 3. The genotyping protocol for each marker was exactly the same
except for the use of the specific Primer-Probe Mix. The final optimized PCR reaction
conditions consisted of 20ng of genomic DNA template, TaqMan Universal PCR Master Mix
(1X), SNP genotyping assay probe-primer mix (20X), DNase-free water in a 5 µL reaction
volume. The PCR thermocycling conditions consisted of one cycle at 95°C for 10 min,
followed by 40 cycles at 95 °C for 15 s, 60°C for 1 min. The data were acquired during the
annealing step and analysed using the 7900 system Sequence Detection System software
Applied Biosystems (Life Technologies Corporation) in a 384-well plate format. Nuclease-
free water was used as a negative control and DNA for each genotype included as positive
controls. 9 9 Statistical Analysis Hardy-Weinberg Equilibrium (HWE) was calculated for each SNP in case and control groups
to detect deviation from the normal genotype distribution in the population. Chi-square (χ2)
analysis was performed on SNPs in HWE to determine if significant differences existed in the
genotype and allele frequencies in the migraine population versus controls. This analysis was
completed in the software PLINK v1.07 [26]. Power estimates indicated that if the SNPs
were to confer at least a two-fold increase in relative risk of migraine, the case and control
groups used in this study are of sufficient size to have approximately 80% power to detect an
association as statistically significant at the 0.05 level. 10 10 Results The aim of this study was to determine if SNPs in the ADARB1 and ADARB2 genes
contribute to migraine susceptibility in an Australian case-control cohort. In this study, the
following 4 SNPs in the ADARB1 gene: rs2838771, rs407133, rs422720, rs1051367, and the
following 8 SNPs in the ADARB2 gene: rs3793733, rs7070629, rs10903467, rs10903479,
rs11250642, rs7094094, rs10903520, rs884861 were in HWE and analysed for association. Genotypic and allelic distributions for all 12 SNPs genotyped are shown in Table 5. Minor
Allele Frequencies obtained in this study were consistent with expected allele frequencies in
HapMap–Utah residents with Northern and Western European ancestry (CEU) populations. Chi-square (χ2) analysis was performed for migraine cases versus controls. As Table 5 shows,
for the majority of SNPs genotype and allele frequencies do not differ significantly between
migraineurs and controls. An allelic p-value of 0.05 was observed for rs884861. However,
the result for rs884861 is not significant with Bonferroni correction for multiple testing which
gives a corrected p-value threshold of 0.0042. Thus overall there was no significant
association between migraine and the ADARB1 and ADARB2 SNPs investigated. The aim of this study was to determine if SNPs in the ADARB1 and ADARB2 genes
contribute to migraine susceptibility in an Australian case-control cohort. In this study, the
following 4 SNPs in the ADARB1 gene: rs2838771, rs407133, rs422720, rs1051367, and the
following 8 SNPs in the ADARB2 gene: rs3793733, rs7070629, rs10903467, rs10903479,
rs11250642, rs7094094, rs10903520, rs884861 were in HWE and analysed for association. Discussion Susceptibility to migraine is conferred by exposure to intrinsic and environmental triggering
factors and genetics. Pinpointing SNPs associated with disease is one of the goals of GWAS
and one of many approaches used to dissect the genetic basis of migraine. This study was
undertaken to follow up findings from a pGWAS we previously conducted in the Norfolk
Island pedigree which implicated four SNPs in the RNA editing gene ADARB2 in migraine,
based on statistical significance [11]. These four SNPs, and others, in the RNA editing genes
ADARB1 and ADARB2, were further investigated in an Australian migraine case-control
population because they fit criteria for migraine neuropathology, i.e. a) are expressed in the 11 brain or CNS, b) regulate neurological pathways (e.g. neurotransmitters) and c) are plausibly
related to migraine neuropathology (e.g. cellular hyperexcitability, ion channel disruption). Furthermore, retyping top-ranking SNPs from GWAS data in independent case-controls
cohorts is important to determine the validity of GWAS findings and ascertain risk within
different population groups. To date only a few association studies have investigated SNPs in the RNA editing genes
ADARB1 and ADARB2 and none have been studied in migraine previously. Interestingly, a
GWAS of US centenarians supports a role for RNA editors as important regulators of aging
in humans. SNPs in ADARB2 were found to be associated with longevity and these findings
were replicated in three independent cohorts of different genetic backgrounds [27]. Amore et
al., 2004 identified a common neutral SNP in three out of seven patients with bipolar disorder
in the ADARB1 gene but no major alteration [28]. In a separate study by Kostyrko et al., 2006
analysed the coding sequence of ADARB1 and its association with bipolar affective disorder
and did not find any mutations except one already known transition [29]. Oguro et al., 2012
identified a longevity-associated SNP in ADARB2 rs2805533, which may modulate human
longevity by regulating metabolic factors such as abdominal obesity and lipid profiles [30]. RNA editing is a physiologically important and conserved process necessary for proper
development and functioning of neuronal cells. The importance of this process is
demonstrated by mice deficient in the RNA-editing enzyme ADARB1 which die
approximately 20 days post-birth and show early onset epilepsy [31]. This phenotype results
from under editing of a critical position that determines calcium permeability in glutamate
receptors of excitatory neurons [31]. Conclusion In this study we found no evidence for SNPs in the RNA editing genes ADARB1 and
ADARB2 to be associated with increased risk of migraine in an Australian Caucasian
population. This result is in contrast to a pGWAS study of Norfolk Island which implicated a
22 kb haploblock region in ADARB2 in migraine. Genetically isolated populations may carry
unique genetic characteristics that are not always replicated in case-control cohorts. Nevertheless, both types of populations are useful to study the genetic structure underlying
disease, providing direction to the investigation of susceptibility genes and genetic pathways. Considering that common migraine is caused by interactions of multiple loci, identifying and
validating genetic factors that influence the disorder remains an important undertaking that
may lead to improved strategies for management and treatment. Discussion ADARB1 plays an important role in ensuring
neurotransmitter receptor transcripts are properly edited at respective points [14]. The 12 particular editing function of ADARB1 upon the AMPA glutamate receptor subunit (GRIA2)
pre-mRNA makes the investigation of ADARB1 in migraine interesting, because glutamate is
a major mediator in the central nervous system and its regulation has been studied as a
possible mechanism causing migraine. Although ADARB2 is catalytically inactive it has been shown in vitro that it can act as a
competitor of ADAR (also known as ADAR1) and ADARB1 by binding to the same
transcripts and thus ADARB2 can decrease the efficiency of other RNA editing enzymes by
preventing them from deaminating their substrate RNAs in regions of the brain where they
are co-expressed [32]. ADARB2 may therefore be reducing the total number of edited
transcripts via a competitive mechanism, which in turn may lead to poor ionic conductance
and synaptic dysfunction in the CNS. One example that supports this hypothesis is perturbed
A-to-I editing of the mRNA encoding the GRIA2 subunit of glutamate AMPA receptors
which manifests in the disease amyotrophic lateral sclerosis (ALS) [33]. Functional studies
are needed to further investigate the activity of ADARB2 in vivo. In this study we tested four SNPs in the ADARB1 gene and eight SNPs in the ADARB2 for
association with migraine susceptibility. Results of χ2 analysis indicated that neither genotype
nor allele frequency distributions for the migraine and control groups were significantly
different after Bonferroni correction for multiple testing. Although we were unable to
replicate our findings from the Norfolk Island population implicating four ADARB2 SNPs in
in migraine in an Australian Caucasian population, it does not exclude a role for ADARB2 in
migraine in the unique Norfolk Island population isolate. This may be because rare variants
that are over-represented in the unique pedigree structure of the Norfolk Island population
contibuted to the initial finding and that the common variants selected for this study are not 13 relevent in the case-control population analysed here. Fine-mapping and sequencing for rare
variants in the Norfolk Island samples could shed further light on this. 14 14 Authors’ Contributions CFG, EK, and HGS performed the experiments, and analysed and interpreted the data. Authors CFG, HGS, BM, AJR, EK, LMH, and LRG conceived and supervised the project
and revised the manuscript for intellectual content and final approval of the completed
manuscript. All authors read and approved the final manuscript. Funding Acknowledgement Claudia Gasparini was funded by a Griffith University Postgraduate scholarship. This
research was supported by an Australian International Science Linkages grant and by
infrastructure purchased with Australian Government EIF Super Science Funds as part of the
Therapeutic Innovation Australia - Queensland Node project. We thank the migraine patients
and their families for their participation in this research. Thank you to Rebecca Grealy and
Bishakha Roy for technical advice. List of Abbreviations ADARB1: adenosine deaminase, RNA-specific, B1: ADARB2: adenosine deaminase, RNA
specific, B2, ALS: amyotrophic lateral sclerosis, A-to-I: Adenosine-to-Inosine, CEU: Utah
residents with Northern and Western European ancestry, CNS: Central Nervous System,
CSD: Cortical Spreading Depression, DBH: Dopamine Beta Hydroxylase, DSH:
dyschromatosis symmetrica hereditaria, FHM: Familial Hemiplegic Migraine, GRIA2:
glutamate receptor, ionotropic, AMPA2, GRM7: glutamate receptor, metabotropic 7, HTR7:
5-hydroxytryptamine serotonin receptor 7 adenylate cyclase-coupled, HWE: Hardy-Weinberg
Equilibrium, IHS: International Headache Society, MA: Migraine with Aura, MO: Migraine
without Aura, LD: linkage disequilibrium, pGWAS: pedigree-based genome wide association
study, SLC6A3: solute carrier family 6 (neurotransmitter transporter), member 3, SNPs:
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Haplotype block of 4 SNPs implicated in the ADARB2 gene in the
Norfolk Island pGWAS [11]. Figure 1
Haplotype block of 4 SNPs implicated in the ADARB2 gene in the
Norfolk Island pGWAS [11]. Table 1
SNPs in ADARB2 associated with migraine in the Norfolk Island pGWAS adapted from [11]. Locus
Gene
No. SNPs in Gene
NCBI dbSNP Ref No. REFERENCES NCBI build 37.1 position (BP)
Function
Minor/Major allele
MAF
Beta
P Value
10p15
ADARB2
4
rs10903399
1227868
Downstream
C/T
0.330
0.64
7.68E−05
rs1046914
1228206
3Prime utr
G/A
0.328
0.67
3.43E−05
rs2271275
1230968
Non-synon
G/A
0.368
0.65
2.67E−05
rs883248
1250184
Intronic
G/A
0.439
0.67
3.83E−06 Table 1
SNPs in ADARB2 associated with migraine in the Norfolk Island pGWAS adapted from [11]. Table 1
SNPs in ADARB2 associated with migraine in the Norfolk Island pGWAS adapted from [11]. ARB2 associated with migraine in the Norfolk Island pGWAS adapted from [11]. Table 2
Demographical characteristics of Migraine Cases and Controls. Parameter
Cases (n = 291)
Controls (n = 314)
Age: Mean (range)
46.4 (-18-67)
44.4 (18-63)
Sex
Male
70
89
Female
221
225
MA
227
N/A
MO
64
N/A e 2
Demographical characteristics of Migraine Cases and Controls. MA - migraine with aura, MO - migraine without aura, N/A - not applicable. Table 3
TaqMan Assay and SNP information. TaqMan Assay ID
Gene
SNP ID
Position
SNP Type
Allele Change
aCEU MAF
C__15831699_10
ADARB1
rs2838771
Chr.21: 46501576
Transversion Substitution
C:G
G=0.41
C_1211569_1_
ADARB1
rs1051367
Chr.21: 46641968
Silent Mutation
A:G
G=0.47
C_15959830_10
ADARB2
rs2271275
Chr.10: 1230968
Missense Mutation
C:T
C=0.34
C_32118695_20
ADARB2
rs10903467
Chr.10: 1535739
Intron, Transition
Substitution
C:T
T=0.44
C__30856132_20
ADARB2
rs11250642
Chr.10: 1622644
Intron, Transition
Substitution
C:T
T=0.50
aCEU MAF = HapMap Caucasian Minor Allele Frequency aCEU MAF = HapMap Caucasian Minor Allele Frequency able 4
SNPs genotyped in ADARB1 and ADARB2 by Sequenom. Gene
SNP ID
Forward, Reverse and Extension Primer Sequences
Position
SNP type
Allele Change
aCEU MAF
ADARB1
rs407133
F: 5’ ACGTTGGATGCCTGCTCTGCAGTAATGAAC 3’
R: 5’ ACGTTGGATGTCCTCCTCTCTTAACTCACG 3’
E: 5’ TCGGACCAATGCTGA 3’
45396317
Intron
C:G
G 0.46
rs422720
F: 5’ ACGTTGGATGGGAAGCAGTACATGTTCATTG 3’
R: 5’ ACGTTGGATGGACTAATGCAGATGATCACC 3’
E: 5’ gggtCAGTACATGTTCATTGTAAGAATT 3’
45403382
Intron
A:C
C 0.36
ADARB2
rs3793733
F: 5’ ACGTTGGATGCAACTCCATGTCAAAAGTGC 3’
R: 5’ ACGTTGGATGATGCCAGGACTCAGGTGCTT 3’
E: 5’caggAAGCATATTGTCAACCTTCCTC 3’
1411326
Missense
G:A
A 0.01
rs7070629
F: 5’ ACGTTGGATGGATGAAAAAAGGATGCCATAC 3’
R: 5’ ACGTTGGATGCCTGTGATGCAGCTTCTCCT 3’
E: 5’ accttTGATGCAGCTTCTCCTGGCACA 3’
1414938
Intron
G:A
A 0.37
rs10903479
F: 5’ ACGTTGGATGAGAACGCAATGCACTCTTCC 3’
1581881
Intron
A:T
T 0.40 Table 4
SNPs genotyped in ADARB1 and ADARB2 by Sequenom. REFERENCES R: 5’ ACGTTGGATGTCGGTTTTGGAGTCTAGAGG 3’
E: 5’ CCTCATTGTCACAGAGT 3’
rs7094094
F: 5’ ACGTTGGATGTCTAGAAAATGCAGAAGGG 3’
R: 5’ ACGTTGGATGCTTGGGCTATACTTTTTGTG 3’
E: 5’CATTCAGTTGTTCTAATATTATATTGA 3’
1652840
Intron
T:G
G 0.38
rs10903520
F: 5’ ACGTTGGATGTTACTCCTTAAGTGGAAGGG 3’
R: 5’ ACGTTGGATGACCATGATATCTACCCCTCC 3’
E: 5’ AAAGAAGCAGGCGTT 3’
1672481
Intron
G:A
A 0.39
rs884861
F: 5’ ACGTTGGATGGAAAAACAGATAGACAAAGC 3’
R: 5’ ACGTTGGATGCCCTGGAATAACTTCAGGGT 3’
E: 5’ ACAGATAGACAAAGCAGAATATAT 3’
1765662
Intron
C:G
C 0.43 aCEU MAF = HapMap Caucasian Minor Allele Frequency SNP
Group
Genotypes
Total
Genoty
aMAF
Allelic
Hap-Map
a
(n=)
pic p-
value
p-value
CEU MAF
ADARB1
CC
GC
GG
rs2838771
Cases
0.429
0.441
0.130
261
0.86
0.35
0.65
Controls
0.405
0.461
0.134
247
0.36
G allele, 0.36
GG
GA
AA
rs407133
Cases
0.178
0.519
0.303
264
0.95
0.44
0.35
Controls
0.179
0.529
0.292
291
0.44
G allele, 0.44
CC
CA
AA
rs422720
Cases
0.157
0.498
0.345
255
0.64
0.42
0.35
Controls
0.138
0.480
0.382
275
0.38
C allele, 0.38
AA
AG
GG
rs1051367
Cases
0.277
0.530
0.193
274
0.53
0.46
0.40
Controls
0.320
0.493
0.187
300
0.43
G allele, 0.43
ADARB2
AA
GA
GG
rs3793733
Cases
0.0
0.017
0.983
239
b0.32
0.008
0.32
Controls
0.0
0.031
0.969
228
0.015
A allele, 0.00
GG
AA
GA
rs7070629
Cases
0.138
0.466
0.395
253
0.70
0.37
0.41
Controls
0.125
0.444
0.430
279
0.35
A allele, 0.37 TT
CT
CC
rs10903467
Cases
0.183
0.470
0.347
251
0.16
0.42
0.11
Controls
0.205
0.524
0.271
288
0.47
T allele, 0.48
TT
TA
AA
rs10903479
Cases
0.195
0.489
0.316
266
0.33
0.44
0.15
Controls
0.153
0.490
0.357
294
0.40
T allele, 0.40
TT
CT
CC
rs11250642
Cases
0.200
0.459
0.341
255
0.34
0.42
0.31
Controls
0.200
0.500
0.300
270
0.45
T allele, 0.45
GG
GT
TT
rs7094094
Cases
0.100
0.420
0.480
246
0.83
0.37
0.62
Controls
0.104
0.442
0.454
269
0.33
G allele, 0.37
AA
GA
GG
rs10903520
Cases
0.114
0.493
0.393
280
0.55
0.36
0.62
Controls
0.144
0.476
0.380
290
0.38
A allele, 0.38
GG
GC
CC
rs884861
Cases
0.161
0.435
0.404
248
0.16
0.38
0.05
Controls
0.220
0.440
0.340
273
0.44
G allele, 0.43 bHeterozygous GA genotype versus homozygous AA genotype only as no homozygous GG
individuals were detected. ozygous GA genotype versus homozygous AA genotype only as no homozygous GG
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https://zenodo.org/records/4065842/files/52%2021Sep18%2010Oct17%209283-11293-2-%20ED%20iqbal.pdf
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English
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Elastic neural network method for load prediction in cloud computing grid
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International Journal of Electrical and Computer Engineering
| 2,019
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cc-by
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International Journal of Electrical and Computer Engineering (IJECE)
Vol. 9, No. 2, April 2019, pp. 1201~1208
ISSN: 2088-8708, DOI: 10.11591/ijece.v9i2.pp1201-1208 International Journal of Electrical and Computer Engineering (IJECE)
Vol. 9, No. 2, April 2019, pp. 1201~1208
ISSN: 2088-8708, DOI: 10.11591/ijece.v9i2.pp1201-1208 1201 1201 Corresponding Author: Corresponding Author:
Kefaya Qaddoum,
Department of Computer Information Systems,
Higher Colleges of Technology,
Traffic Department Road, Al Ain, United Arab Emirates. Email: K.s.Qaddoum@gmail.com Kefaya Qaddoum,
Department of Computer Information Systems,
Higher Colleges of Technology, Traffic Department Road, Al Ain, United Arab Emirates. Email: K.s.Qaddoum@gmail.com Traffic Department Road, Al Ain, United Arab Emirates. Email: K.s.Qaddoum@gmail.com Elastic neural network method for load prediction
in cloud computing grid Kefaya S. Qaddoum1, Nameer N. El Emam2, Mosleh. A Abualhaj3
1Department of Computer Information Systems, Higher Colleges of Technology, United Arab Emirates
2Department of Computer Science, Philadelphia University, United State
3Department of Information Technology, Amman Al Ahliyya University, Yordania ABSTRACT Cloud computing still has no standard definition, yet it is concerned with
Internet or network on-demand delivery of resources and services. It has
gained much popularity in last few years due to rapid growth in technology
and the Internet. Many issues yet to be tackled within cloud computing
technical challenges, such as Virtual Machine migration, server association,
fault tolerance, scalability, and availability. The most we are concerned with
in this research is balancing servers load; the way of spreading the load
between various nodes exists in any distributed systems that help to utilize
resource and job response time, enhance scalability, and user satisfaction. Load rebalancing algorithm with dynamic resource allocation is presented to
adapt with changing needs of a cloud environment. This research presents a
modified elastic adaptive neural network (EANN) with modified adaptive
smoothing errors, to build an evolving system to predict Virtual Machine
load. To evaluate the proposed balancing method, we conducted a series of
simulation studies using cloud simulator and made comparisons with
previously suggested approaches in the previous work. The experimental
results show that suggested method betters present approaches significantly
and all these approaches. Cloud computing
Load balancing
Neural networks
Virtual machine migration Corresponding Author:
Kefaya Qaddoum,
Department of Computer Information Systems,
Higher Colleges of Technology,
Traffic Department Road, Al Ain, United Arab Emirates. Email: K.s.Qaddoum@gmail.com Copyright © 2019 Institute of Advanced Engineering and Science.
All rights reserved. Journal homepage: http://iaescore.com/journals/index.php/IJECE Corresponding Author:
Kefaya Qaddoum,
Department of Computer Information Systems,
Higher Colleges of Technology,
Traffic Department Road, Al Ain, United Arab Emirates.
Email: K.s.Qaddoum@gmail.com 1.
INTRODUCTION According to the National Institute of Standards and Technology (NIST): "Cloud computing is a
model for enabling convenient, on-demand network access to a shared pool of configurable computing
resources [1]. Cloud is a huge scale distributed computing, transferring computer operations and storage from
desktop and portable PCs to a huge hosting center [1], where resources could be easily used remotely, with
effective cost [2], [3]. Data centers use Virtual Machine (VM) for integration of different solutions [4]. Infrastructure-as-a-Service (IaaS), Platform-as-a-Service (PaaS), and Software-a-a-Service (SaaS) are the
offered types of market services among cloud. yp
g
Many millions of online devices are connected through the Internet, where using cloud computing
service industries is increasing every day [4]. Cloud computing involves sharing supplies, software, which
supposed to reduced functioning cost, regarding the time needed to process data, adjust the system
permanence according to demands changes in the system as it goes. Load balancers are part of cloud
structure; its goal is to forward incoming request to the customer who demands the service. Depending on
availability and existing load, the load balancer task is to apply scheduling algorithm to decide the best server
that could handle the request [5]. It gets an update on client’s server's status and current job. If one server is Journal homepage: http://iaescore.com/journals/index.php/IJECE
1202 ISSN: 2088-8708 overloaded with tasks, then it is redirected to another server to keep load balanced [6-7]. In this paper, the
objective of load scheduling is minimizing the average shift time of a set of online loads, where shifting time
represents the total elapsed time for a load application from load submission to load completion, including
waiting time and execution time. overloaded with tasks, then it is redirected to another server to keep load balanced [6-7]. In this paper, the
objective of load scheduling is minimizing the average shift time of a set of online loads, where shifting time
represents the total elapsed time for a load application from load submission to load completion, including
waiting time and execution time. g
As elaborated in [8], load computing is a good way to understand job parallelism where each job
requires a single processor for execution and different tasks might run concurrently or follow superiority
restrictions. On the other side, parallel programs are written with some message-passing library, e.g.,
MPI [9], are a classic way to accomplish data parallelism. 1.
INTRODUCTION A load model can be used to describe the scheduling job to keep balance among the cloud VMs. As the high-performance computing platform evolves into grid and cloud environments, the underlying
speed-heterogeneous multi-cluster design [10] makes mixed-parallel load scheduling more laborious and
difficult than traditional task- parallel loads scheduling because of the resource division issue [11]
experienced by parallel task distribution. In [12], Online Load Management was proposed as the first step to
tackling such problems. ANN is widely used in different aspects and applications [17], in this work, we
propose an Elastic Artificial Neural Network (EANN) framework, which formalizes the online-load
scheduling process into job allocation. The job-clustering phase manages the job load within a server. The
job rearrangement phase allows some jobs reallocations to different servers to handle the request to increase
resource utilization on other servers. The job allocation phase assigns a proper set of resources to a job. We live in the cloud era, where users of the cloud are increasing fast, that caused heavy job on cloud
servers which made the load balancing as an urgent issue of cloud computing. The core goal of load
balancing is to allocate the job consistently to the whole cloud to guarantee no overloaded or underloaded
servers exists in the entire network [13]. Load balancing algorithms' goal is to monitor the nodes for
underloading or overloading and take appropriate action to normalize the load among VMs. So that
utilization of resources is better. Many load controlling techniques, like ASKALON [14], DAGman [15],
have developed systems to manage load uses in both parallel and distributed systems. Some existing load
management systems, e.g., DAGman and [16], make sure the execution of a load does not violate the
precedence constraints among tasks but pay little attention to improving scheduling time. Other systems
focus on completing task scheduling within loads to enhance desired final time, e.g. [17]. The rest of the paper covers. Section 2 discusses related work of load balancing; Section 3 discusses
the proposed system, including Load balancing, suggested framework, algorithm to predict cloud load,
server’s classification used in Cloud computing. Section 4 shows simulation and results of the suggested
system. Section 5 has the conclusion with some ideas for future work. Int J Elec & Comp Eng, Vol. 9, No. 2, April 2019 : 1201 - 1208 2.1. EANN design for load prediction We give c -1
rate, so that value we get from equation (1) is converted as (2) here. D(p) = - |c|* D(p - 1) + (1 + |c|) * O(p);-1 < c < 0 = O(p) + |c| * (O(p) - D(p – 1*c))
(2) D(p) = - |c|* D(p - 1) + (1 + |c|) * O(p);-1 < c < 0 = O(p) + |c| * (O(p) - D(p – 1*c))
(2) (2) The predicted value is concluded based on historical behaviors on the cloud. 2.1. EANN design for load prediction g
p
To forecast expected resource requests, we need to look inside a VM and analyze logs of pending
requests. To do this, VMs modifications are required, which might be difficult. The prediction is built on the
historical log behaviors of VMs. The algorithm deploys an artificial neural network to predict load balancing
in the cloud; this is achieved by training neural network on a big dataset containing different load situations. Back Propagation learning algorithm was used during EANN training phase so that it can manage incoming
jobs to the cloud. The suggested EANN consist of three tiers; the first tier is input, presenting the current job
for N nodes. The second is the hidden tier, while the third is output tier, which signifies the balanced job for
N nodes. Each node in the input tier act for either the current server's job or the current average job of a
cluster of servers where its given a number. On the other hand, the equivalent node in the output tier
represents either server’s job or cluster’s regular job after balancing correspondingly. The system proposed is an intelligent technique, where the values of the neural network connection
weights are modified within training phase, using a reformed optimization methodology; it consists of
building an elastic artificial neural network (EANN) based on modified adaptive smoothing errors (MASE). Then to adjust the neural network connection weights during the training phase. The algorithm uses EANN
technique work on reducing errors. First, we need to determine average with weights by a predetermined scheme, where the expected
and detected load at a given period p as seen in (1). D(p) = c* D(p - 1) + (1 - c) * O(p); 0 < c < 1
(1) (1) D(p) = c* D(p - 1) + (1 - c) * O(p); 0 < c < 1 as c indicates, the balance among steadiness and approachability. as c indicates, the balance among steadiness and approachability. EANN is used to predict the load of CPU on a defined server. We group the time c =0.5 and use it to
predict the next minute load and compare it with the current minute. The next step is monitoring decreased usage; we need to work on the observed values which are
higher by 69% normally. If balance values groups within [0 to 1], then the balance is within the desired range. 2.
RESEARCH METHODOLOGY Explaining research chronological, including research design, research procedure (in the form of
algorithms, Pseudocode or other), how to test and data acquisition [18], [19]. The description of the course of
research should be supported references so that the explanation can be accepted scientifically [20], [21]. Whenever there is a certain job on the Cloud, the load balancer sends the job request through the
suggested algorithm to be clustered to which server to be sent [22]. K-means clustering is one of the simplest
clustering algorithms used for unsupervised learning problems, MacQueen, 1967 introduced it, it works
mainly on minimizing the distance between the cluster center and the data point. K-means is used in this
research appointing jobs as data points on the server and based on the processor type and speed to cluster
these jobs. After determining hard disk capacity, another clustering is done based on their cost per hour. An
operator node to manage node performance exists, its job is to supervise the node then report utilization and
send it to manager controller, the manager is a cluster management system aimed at decreasing the effort
done to manage cluster resources while refining the facility of customer's demand and manage resources. The manager waits until the whole cluster is empty before looking for work from other clusters. Figure 1 [7]
shows system model, where the manager controller sends a report as input which is used in prediction
algorithm. Int J Elec & Comp Eng, Vol. 9, No. 2, April 2019 : 1201 - 1208
1203 1203 ISSN: 2088-8708 Int J Elec & Comp Eng Figure 1. Load balancer Figure 1. Load balancer Xn∗=Xn∗+α(Xs−Xn) Else, go back to step (2). The mean squared error (MSE) between the predicted and the required result represents the fitness
of the input. ( )
( )
(6)
z = (tz −Oz)
( ( ))
(7)
( )
(8) ( )
( )
(6)
z = (tz −Oz)
( ( ))
(7)
( )
(8) ( )
( )
z = (tz −Oz)
( ( )) ( )
( )
z = (tz −Oz)
( ( )) (6) (7) ( ) (8) The factor δz and the activation ht of the hidden neuron Ht, determines adjustment made to the value
of Vst and error factors be outlined in (8). The factor δz and the activation ht of the hidden neuron Ht, determines adjustment made to the value
of Vst and error factors be outlined in (8). 2.2. EANN architecture The suggested system includes three tiers, neurons in the first, second, and third tiers. EANN as
shown in Figure 2 goes as follows: Elastic neural network method for load prediction in cloud computing grid (Kefaya S. Qaddoum)
1204 ISSN: 2088-8708 Figure 2. Elastic ANN architecture Figure 2. Elastic ANN architecture Figure 2. Elastic ANN architecture Phase 1: Regroup neuron and allocate neurons with diverse routes locations. Phase 2: group a location route Xs of server node n as an input. Since there are three neurons in the
input tier, Xs should be the input of different neuron of input tiers each time. p
p
Phase 3: Create a group B relates server node s corresponds to the neuron n. Phase 3: Create a group B relates server node s corresponds to the neuron S = {i|Xi ∈ Sq ∩ kXs − Xik ≤ ri} (3) where Xi is the location route of neuron n and the associations related to Sq can shield server node n. Phase 4: If group S is unfilled, iterate this procedure by picking another random server node. Xn∗ =min kXs −Xnk. (4) Xn∗ =min kXs −Xnk. (4)
n∈Sq Xn∗ =min kXs −Xnk. (4) n∈Sq Phase 5: alter the chosen sites of neuron and next ones according to: Xn∗=Xn∗+α(Xs−Xn)
(5) (5) Xn∗=Xn∗+α(Xs−Xn) Int J Elec & Comp Eng For each task of a workflow, the number of processors required to execute it is set by picking up a random
number within a pre-configured range according to a specified probability distribution. For each task of a workflow, the number of processors required to execute it is set by picking up a random
number within a pre-configured range according to a specified probability distribution. Each experiment invokes 20 runs, of which each simulates 100 online workflows in a multi-cluster
environment composed of 5 clusters each containing 50-70 processors. EANN algorithm is used to give best
possible allocation results. Set p=16 s for example and a set of demands on service is D, where clustered sets
represented by C1, C2, C3. The outcome of 92 iterations was balanced and reasonable node allocation. Task
allocation using EANN goes as Association C1={S7, S15, S20}, that is, servers S7, S15, and S15 are elected
to track demand D1. Coalition C2={S2, S6, S22}, that is, servers S2, S6, and S22 are elected to track demand
D2. Coalition C3={S8, S17, S18}, that is, servers S8, S17, and S18 are elected to track demand D3. Results
are shown in Table 1. Table 1. Time of Execution of the Selected Customer's Cloud Tasks
Set-up
Retort
Processing time
First
Data center with 30 VMs
244.68
Second
Two data centers with 15 VMs each
260.77
Third
Two data centers with 30 VMs each
174.92 The predicted value, which is done with the help of past input value is clearly shown in Figure 3. The predicted value provides a higher efficiency of output. Since the predicted value provides greater
efficiency, the mean square error is slightly high. As Figure 3 illustrates output if the rate=0.7, and this is
selected on account of least error calculation for our jobs prediction on VM, where this result enhances the
prediction accuracy and work for the benefit of better scheduling in the next step. Figure 3. Error rate for best performance Figure 3. Error rate for best performance The load migration before and after is given in Figure 4, as we can see that over a period the number
of jobs executed comparing to available VMs is becoming reasonable after migrating jobs around. The result
of predicted jobs is keeping the balance of different servers as equal as possible. 3.
RESULTS AND ANALYSIS In this section, it is explained the results of research and at the same time is given the
comprehensive discussion. The experiments were conducted on a cloud simulator. In cloud simulator, the
cloudsim, that aids modeling, and simulation of Cloud computing systems and application settings. It provisions both system and behavior modeling of Cloud system components such as data centers, virtual
machines (VMs) and resource provisioning policies. Using the load balancing parameters of virtual machine
and data center, we have examined on cloudsim-3.0 a cloud-computing simulator and evaluate the
performance of the suggested method using job assigning to the cloud subset. Figure 4 represents the load
balancing in each cloud partition, and the proposed approaches such as EANN based load balancing
algorithm shows improved results than the existing techniques. In the beginning simulator first, generates the
input workload consisting of a set of workflows arriving at different time instants in an online manner. Int J Elec & Comp Eng, Vol. 9, No. 2, April 2019 : 1201 - 1208 Int J Elec & Comp Eng 1205 Int J Elec & Comp Eng ISSN: 2088-8708 Int J Elec & Comp Eng Figure 5 gives the graphical
demonstration of the load balancing in each cloud partition and the suggested approach EANN based load
balancing algorithm shows the better results than the other techniques like Ant colony and Honey behavior. Elastic neural network method for load prediction in cloud computing grid (Kefaya S. Qaddoum)
1206
206 ISSN: 2088-8708 Figure 4. Load migration on cloud
Figure 5. Load balancing comparison Figure 4. Load migration on cloud Figure 4. Load migration on cloud Figure 5. Load balancing comparison Int J Elec & Comp Eng, Vol. 9, No. 2, April 2019 : 1201 - 1208 4.
CONCLUSION Load prediction is needed for allocating data center resources depending on applications demands. The fluctuation of demand will be less detected using our approach. The system is managing resources and
avoids overload. The suggested technique uses some of the design processes of a load balancing. EANN
forecast the request then accordingly assign resources according to the request. That maintains the active
servers according to current demand. This paper, suggested a new effective load balancing technique-using
EANN based load-balancing technique to achieve detectable enhancements in resource deployment and
availability among cloud-computing atmosphere. For future, to further improve the balancing performance of load in cloud environments, for
instance, the proactive job allocation method could be extended to consider multiple running tasks for
simultaneous preemption and would increase the probability for high-priority tasks to start execution earlier
and thus improve the overall system performance. For the shortest- workflow-first policy, other metrics for
prioritizing workflows could be investigated in addition to the remaining execution time used in this paper. Int J Elec & Comp Eng, Vol. 9, No. 2, April 2019 : 1201 - 1208
1207 1207 ISSN: 2088-8708 Int J Elec & Comp Eng REFERENCES REFERENCES
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10th International Symposium on Pervasive Systems, Algorithms, and Networks, pp.701-705, 2009. 1] C. C. Hsu, K.C. Huang and F.J. Wang, “Online Scheduling of Load Applications in Grid Environments,” Futu
Generation Computer Systems 27, pp. 860–870, 2011. [12] Santanu Dam, Gopa Mandal, Kousik Dasgupta and Paramartha Dutta, “Genetic Algorithm and Gravitational
Emulation Based Hybrid Load Balancing Strategy in Cloud Computing”, in proc. third International Conference on
Computer, Communication, Control and Information Technology (C3IT), IEEE, pp. 1-7, February 2015. [13] ASKALON, http://www.dps.uibk.ac.at/projects/teuta/ (2015.3) [14] DAGman, http://research.cs.wisc.edu/htcondor/dagman/dagman.html (2015.3) [15] M. Wieczorek, M. Siddiqui, A. Villazón, R. Prodan, T. REFERENCES Fahringer, “Applying Advance Reservation to Increase
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and Grid Computing, pp. 82, December 4-6, Amsterdam, Netherlands, 2006. [16] Zeba Khan, Mahfooz Alam, Raza Abbas Haidri “Effective Load Balance Scheduling Schemes for Heterogeneous
Distributed System,” International Journal of Electrical and Computer Engineering (IJECE), Vol.7, No.5, October
2017, pp. 2757~2765, ISSN: 2088-8708, 2017. pp
[17] R. Prodan, T. Fahringer, “Dynamic Scheduling of Scientific Workflow Applications on the Grid: A Case S
Proceedings of the20th Symposium on Applied Computing (SAC 2005), pp. 687-694, 2005. [18] Kefaya Qaddoum, Evor Hines, and Daciana Iliescu, “Yield Prediction Technique Using Hybrid Adaptive Neural
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2012, DOI: http://dx.doi.org/10.1142/S1469026812500216. p
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[19] T. Sasidhar, V. Havisha, S. Koushik, M. Deep, VK. Reddy, "Load Balancing Techniques for Efficient Traffic
Management in Cloud Environment,” International Journal of Electrical and Computer Engineering (IJECE),
vol. 6, no. 3, pp. 963-973, 2016. [20] P. Qiao, “On the Security of a Dynamic and Secure Key Management Model Forhierarchical Heterogeneous Sensor
Networks,” TELKOMNIKA Indonesian Journal of Elec-trical Engineering, vol. 12, no.10, pp. 7459–7462, 2014. http://iaesjournal.com/online/index.php/TELKOMNIKA/article/view/5579 p
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1] Cao, X., Zhong, Y., Zhou, Y., Wang, J., Zhu, C., & Zhang, W. “Interactive Temporal Recurrent Convoluti
Network for Traffic Prediction in Data Centers,” IEEE Access, 6, 5276-5289, 2018. [22] Peng, G., Wang, H., Zhang, H., & Dong, J. “Knowledge-Based Resource Allocation for Collaborative Simulation
Development in a Multi-Tenant Cloud Computing Environment,” IEEE Transactions on Services Computing, 2016. DOI: 10.1109/TSC.2016.2518161 Int J Elec & Comp Eng, Vol. 9, No. 2, April 2019 : 1201 - 1208 BIOGRAPHIES OF AUTHORS Dr. Kefaya Qaddoum is an Assistant professor at Higher Colleges of Technology UAE, was an
Assistant professor in Al-Ahliyya Amman University. She received her first degree in Computer
Science from Philadelphia University, Jordan, in July 2003, master degree in Computer Information
System from Philadelphia University July 2008, and a doctorate degree in A.I from Warwick
University, the UK in 2013. Her research area of interest includes A.I, Machine Learning, Data
Mining, and Networking. Dr. Kefaya Qaddoum is an Assistant professor at Higher Colleges of Technology UAE, was an
Assistant professor in Al-Ahliyya Amman University. She received her first degree in Computer
Science from Philadelphia University, Jordan, in July 2003, master degree in Computer Information
System from Philadelphia University July 2008, and a doctorate degree in A.I from Warwick
University, the UK in 2013. Her research area of interest includes A.I, Machine Learning, Data
Mining, and Networking. Elastic neural network method for load prediction in cloud computing grid (Kefaya S. Qaddoum)
1208
1208 ISSN: 2088-8708 Dr. Mosleh M. Abu-Alhaj is a senior lecturer in Al-Ahliyya Amman University. He received his
first degree in Computer Science from Philadelphia University, Jordan, in July 2004, master degree
in Computer Information System from the Arab Academy for Banking and Financial Sciences,
Jordan in July 2007, and a doctorate in Multimedia Networks Protocols from Universiti Sains
Malaysia in 2011. His research area of interest includes VoIP, Multimedia Networking, and
Congestion Control. Apart from research, Dr. Mosleh M. Abu-Alhaj also does consultancy services
in the above research areas and directs the Cisco academy team at Al-Ahliyya Amman University. Nameer N. EL-Emam: He completed his Ph.D. in Computer Science with honor in 1997. He works
as an assistant professor in the Computer Science Department, Basra University. In 1998, he joined
the Department of Computer Science, Philadelphia University, as an Assistance Professor, and then
he got the rank "Associate Professor" in 2010. Now he is a Full Professor at the same university. He
works as a chair of Computer Science Department and the deputy dean of the faculty of Information
Technology, Philadelphia University. His research interest includes Computer Simulation with the
intelligent system, Parallel Algorithms, and Soft computing using Convolution Neural Network, GA,
ACO, and PSO for many kinds of applications like Image Processing, Sound Processing, Fluid Flow,
and Computer Security (Steganography). Dr. Mosleh M. Abu-Alhaj is a senior lecturer in Al-Ahliyya Amman University. BIOGRAPHIES OF AUTHORS He received his
first degree in Computer Science from Philadelphia University, Jordan, in July 2004, master degree
in Computer Information System from the Arab Academy for Banking and Financial Sciences,
Jordan in July 2007, and a doctorate in Multimedia Networks Protocols from Universiti Sains
Malaysia in 2011. His research area of interest includes VoIP, Multimedia Networking, and
Congestion Control. Apart from research, Dr. Mosleh M. Abu-Alhaj also does consultancy services
in the above research areas and directs the Cisco academy team at Al-Ahliyya Amman University. Dr. Mosleh M. Abu-Alhaj is a senior lecturer in Al-Ahliyya Amman University. He received his
first degree in Computer Science from Philadelphia University, Jordan, in July 2004, master degree
in Computer Information System from the Arab Academy for Banking and Financial Sciences,
Jordan in July 2007, and a doctorate in Multimedia Networks Protocols from Universiti Sains
Malaysia in 2011. His research area of interest includes VoIP, Multimedia Networking, and
Congestion Control. Apart from research, Dr. Mosleh M. Abu-Alhaj also does consultancy services
in the above research areas and directs the Cisco academy team at Al-Ahliyya Amman University. Nameer N. EL-Emam: He completed his Ph.D. in Computer Science with honor in 1997. He works
as an assistant professor in the Computer Science Department, Basra University. In 1998, he joined
the Department of Computer Science, Philadelphia University, as an Assistance Professor, and then
he got the rank "Associate Professor" in 2010. Now he is a Full Professor at the same university. He
works as a chair of Computer Science Department and the deputy dean of the faculty of Information
Technology, Philadelphia University. His research interest includes Computer Simulation with the
intelligent system, Parallel Algorithms, and Soft computing using Convolution Neural Network, GA,
ACO, and PSO for many kinds of applications like Image Processing, Sound Processing, Fluid Flow,
and Computer Security (Steganography). Int J Elec & Comp Eng, Vol. 9, No. 2, April 2019 : 1201 - 1208
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Linear regression with partially mismatched data: local search with theoretical guarantees
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MIT Open Access Articles The MIT Faculty has made this article openly available. Please share
how this access benefits you. Your story matters. Terms of use: Creative Commons Attribution Mathematical Programming
https://doi.org/10.1007/s10107-022-01863-y Mathematical Programming
https://doi.org/10.1007/s10107-022-01863-y FULL LENGTH PAPER
Series B FULL LENGTH PAPER
Series B Linear regression with partially mismatched data: local
search with theoretical guarantees Rahul Mazumder1
· Haoyue Wang1 Received: 31 May 2021 / Accepted: 14 June 2022
© The Author(s) 2022 A subset of this paper appeared in IPCO 2021 [12]. This research was supported in part, by grants from
the Office of Naval Research: ONR-N000141812298 (YIP), the National Science Foundation:
NSF-IIS-1718258, IBM and Liberty Mutual Insurance, awarded to Rahul Mazumder. B Rahul Mazumder
rahulmaz@mit.edu B Rahul Mazumder
rahulmaz@mit.edu
Haoyue Wang
haoyuew@mit.edu 1
Massachusetts Institute of Technology, Cambridge, MA 02139, USA 1 Introduction Linear regression and its extensions are among the most fundamental models in statis-
tics and related fields. In the classical and most common setting, we are given n samples
with features xi ∈Rd and response yi ∈R, where i denotes the sample indices. We
assume that the features and responses are perfectly matched i.e., xi and yi corre-
spond to the same record or sample. However, in important applications (for example,
due to errors in the data merging process), the correspondence between the response
and features may be broken [13, 14, 19]. This erroneous correspondence needs to
be adjusted before performing downstream statistical analysis. Thus motivated, we
consider a mismatched linear model with responses y = [y1, . . . , yn]⊤∈Rn and
covariates X = [x1, . . . , xn]⊤∈Rn×d satisfying P∗y = Xβ∗+ ϵ
(1.1) (1.1) where β∗∈Rd are the true regression coefficients, ϵ = [ϵ1, . . . , ϵn]⊤∈Rn is the
noise term, and P∗∈Rn×n is an unknown permutation matrix. We consider the
classical setting where n > d and X has full rank; and seek to estimate both β∗and
P∗based on the n observations {(yi, xi)}n
1. Note that the main computational difficulty
in this task arises from the unknown permutation. Linear regression with mismatched/permuted data—for example, model (1.1)—
has a long history in statistics dating back to 1960s [5, 6, 13]. In addition to the
aforementioned application in record linkage, similar problems also appear in robotics
[22], multi-target tracking [4] and signal processing [3], among others. Recently, this
problem has garnered significant attention from the statistics and machine learning
communities. A series of recent works [1, 2, 7–9, 11, 14, 15, 17, 19–21, 23, 24,
26] have studied the statistical and computational aspects of this model. To learn the
coefficients β∗and the matrix P∗, one can consider the following natural optimization
problem: min
β,P ∥P y −Xβ∥2
s.t. P ∈Πn
(1.2) (1.2) where Πn is the set of n × n permutation matrices. Solving problem (1.2) is difficult
as there are exponentially many choices for P ∈Πn. Given P however, it is easy
to estimate β via least squares. Abstract Linear regression is a fundamental modeling tool in statistics and related fields. In
this paper, we study an important variant of linear regression in which the predictor-
response pairs are partially mismatched. We use an optimization formulation to
simultaneously learn the underlying regression coefficients and the permutation cor-
responding to the mismatches. The combinatorial structure of the problem leads to
computational challenges. We propose and study a simple greedy local search algo-
rithm for this optimization problem that enjoys strong theoretical guarantees and
appealing computational performance. We prove that under a suitable scaling of the
number of mismatched pairs compared to the number of samples and features, and
certain assumptions on problem data; our local search algorithm converges to a nearly-
optimal solution at a linear rate. In particular, in the noiseless case, our algorithm
converges to the global optimal solution with a linear convergence rate. Based on this
result, we prove an upper bound for the estimation error of the parameter. We also
propose an approximate local search step that allows us to scale our approach to much
larger instances. We conduct numerical experiments to gather further insights into
our theoretical results, and show promising performance gains compared to existing
approaches. Keywords Linear regression · Mismatched data · Local search method · Permutation
learning · Mixed integer programming · Polynomial time algorithm Mathematics Subject Classification 90C06 · 90C27 · 62J99 Mathematics Subject Classification 90C06 · 90C27 · 62J99 A subset of this paper appeared in IPCO 2021 [12]. This research was supported in part, by grants from
the Office of Naval Research: ONR-N000141812298 (YIP), the National Science Foundation: NSF-IIS-1718258, IBM and Liberty Mutual Insurance, awarded to Rahul Mazumder. 1
Massachusetts Institute of Technology, Cambridge, MA 02139, USA 123 R. Mazumder, H. Wang 1 Introduction [24] shows that in the noiseless setting (ϵ = 0), a
solution ( ˆP, ˆβ) of problem (1.2) equals (P∗, β∗) with probability one if n ≥2d and
the entries of X are independent and identically distributed (iid) as per a distribution
that is absolutely continuous with respect to the Lebesgue measure. [11, 15] studies the
estimation of (P∗, β∗) under the noisy setting. It is shown in [15] that Problem (1.2)
is NP-hard if d ≥κn for some constant κ > 0. A polynomial-time approximation
algorithm appears in [11] for a fixed d. However, as noted in [11], this algorithm does not appear to be efficient in practice. [8] propose a branch-and-bound method, that can solve small problems with n ≤20
(within a reasonable time). [16] propose a branch-and-bound method for a concave
minimization formulation, which can solve problem (1.2) with d ≤8 and n ≈100 (the 123 Linear regression with partially mismatched data… authors report a runtime of 40 minutes to solve instances with d = 8 and n = 100). [23]
propose an approach using tools from algebraic geometry, which can handle problems
with d ≤6 and n = 103 ∼105—the cost of this method increases exponentially
with d. This approach is exact for the noiseless case but approximate for the noisy
case (ϵ ̸= 0). Several heuristics have been proposed for (1.2): Examples include,
alternating minimization [9, 26], Expectation Maximization [2]—as far as we can tell,
these methods are sensitive to initialization, and have limited theoretical guarantees. As discussed in [18, 19], in several applications, a small fraction of the samples
are mismatched — that is, the permutation P∗is sparse. In other words, if we let
r := |{i ∈[n] | P∗ei ̸= ei}| where e1, . . . , en are the standard basis elements of Rn,
then r is much smaller than n. In this paper, we focus on such sparse permutation
matrices, and assume the value of r is known or a good estimate is available to the
practitioner. This motivates a constrained version of (1.2), given by min
β,P ∥P y −Xβ∥2
s.t. P ∈Πn, dist(P, In) ≤R
(1.3) (1.3) where, the constraint dist(P, In) ≤R restricts the number of mismatches between
P and the identity permutation to be below R—See (1.4) for a formal definition of
dist(·, ·). 1 We draw inspiration from the local search method presented in [10] in the context of a different problem:
high dimensional sparse regression. 1 Introduction We also prove an upper
bound of the estimation error of β∗(in ℓ2 norm) and derive a bound on the number
of iterations taken by our proposed local search method to find a solution with this
estimation error. Notation and preliminaries: For a vector a, we let ∥a∥denote the Euclidean norm,
∥a∥∞the ℓ∞-norm and ∥a∥0 the ℓ0-pseudo-norm (i.e., number of nonzeros) of a. We let · 2 denote the operator norm for matrices. Let {e1, . . . , en} be the natural
orthogonal basis of Rn. For a finite set S, we let #S denote its cardinality. For any
permutation matrix P, let πP be the corresponding permutation of {1, 2, . . . ., n}, that
is, πP(i) = j if and only if e⊤
i P = e⊤
j if and only if Pi j = 1. We define the distance
between two permutation matrices P and Q as dist(P, Q) = #
i ∈[n] : πP(i) ̸= π Q(i)
. (1.4) (1.4) Recall that we assume r = dist(P∗, In). For a given permutation matrix P, define
the m-neighbourhood of P as Nm(P) := { Q ∈Πn : dist(P, Q) ≤m}. (1.5) (1.5) It is easy to check that N1(P) = {P}, and for any R ≥2, NR(P) contains more
than one element. For any permutation matrix P ∈Πn, we define its support as:
supp(P) := {i ∈[n] : πP(i) ̸= i} . For a real symmetric matrix A, let λmax(A) and
λmin(A) denote the largest and smallest eigenvalues of A, respectively. For two positive scalar sequences {an}, {bn}, we write an = O(bn) or equivalently,
an/bn = O(1), if there exists a universal constant C such that an ≤Cbn. We write
an = Ω(bn) or equivalently, an/bn = Ω(1), if there exists a universal constant c such
that an ≥cbn. We write an = Θ(bn) if both an = O(bn) and an = Ω(bn) hold. 1 Introduction Above, R is taken such that r ≤R ≤n (Further details on the choice of R
can be found in Sects. 3 and 5). Note that as long as r ≤R ≤n, the true parameters
(P∗, β∗) lead to a feasible solution to (1.3). In the special case when R = n, the
constraint dist(P, In) ≤R is redundant, and Problem (1.3) is equivalent to problem
(1.2). Interesting convex optimization approaches based on robust regression have
been proposed in [18] to approximately solve (1.3) when r ≪n. The authors focus on
obtaining an estimate of β∗. Similar ideas have been extended to consider problems
with multiple responses in [19]. Problem (1.3) can be formulated as a mixed-integer program (MIP) with O(n2)
binary variables (to model the unknown permutation matrix). Solving this MIP with
off-the-shelf MIP solvers (e.g., Gurobi) becomes computationally expensive for even
a small value of n (e.g. n ≈50). To the best of our knowledge, we are not aware of
computationally practical algorithms with theoretical guarantees that can optimally
solve the original problem (1.3), under suitable assumptions on the problem data. Addressing this gap is the main focus of this paper: We propose and study a novel
greedy local search method1 for Problem (1.3). Loosely speaking, our algorithm at
every step performs a greedy swap or transposition, in an attempt to improve the
cost function. This algorithm is typically efficient in practice based on our numerical
experiments. We also propose an approximate version of the greedy swap procedure
that scales to much larger problem instances. We establish theoretical guarantees on the
convergence of the proposed method under suitable assumptions on the problem data. Under a suitable scaling of the number of mismatched pairs compared to the number
of samples and features, and certain assumptions on the covariates and noise; our local
search method converges to an objective value that is at most a constant multiple of
the squared norm of the underlying noise term. From a statistical viewpoint, this is 12 3 3 R. Mazumder, H. Wang the best objective value that one can hope to obtain (due to the noise in the problem). Interestingly, in the special case of ϵ = 0 (i.e., the noiseless setting), our algorithm
converges to an optimal solution of (1.3) with a linear rate2. 2 The extended abstract [12] which is a shorter version of this manuscript, studies the noiseless setting. If ∥H P(k) y∥2 −∥H P(k+1) y∥2 ≤e or k = K, output P(k). At iteration k, Algorithm 1 finds a swap (within a distance of R from In) that leads
to the smallest objective value. To see the computational cost of (2.3), note that: ∥H P y∥2 = ∥H(P −P(k))y + H P(k) y∥2
= ∥H(P −P(k))y∥2 + 2⟨(P −P(k))y, H P(k) y⟩
+∥H P(k) y∥2 . (2.4) (2.4) For each P, with dist(P, P(k)) ≤2, the vector (P −P(k))y has at most two nonzero
entries. Since we pre-compute H, computing the first term in (2.4) costs O(1) opera-
tions. As we retain a copy of H P(k) y in memory, computing the second term in (2.4)
also costs O(1) operations. Therefore, computing (2.3) requires O(n2) operations, as
there are at most n2-many possible swaps to search over. The O(n2) per-iteration cost
is quite reasonable for medium-sized examples with n being a few hundred to a few
thousand, but might be expensive for larger examples. In Sect. 4, we propose a fast
method to find an approximate solution of (2.3) that scales to instances with n ≈107
in a few minutes (see Sect. 5 for numerical findings). 3 Theoretical guarantees for algorithm 1 Here we present theoretical guarantees for Algorithm 1. The main assumptions and
conclusions appear in Sect. 3.1. Section 3.2 presents the proofs of the main theorems. The development in Sects. 3.1 and 3.2 assumes that the problem data (i.e., y, X, ϵ) is
deterministic. Section 3.3 discusses conditions on the distribution of the features and
the noise term, under which the main assumptions hold true with high probability. 2 A local search method Here we present our local search method for (1.3). For any fixed P ∈Πn, by minimiz-
ing the objective function in (1.3) with respect to β, we have an equivalent formulation min
P
∥P y −H P y∥2 s.t. P ∈Πn, dist(P, In) ≤R ,
(2.1) (2.1) where H = X(X⊤X)−1X⊤is the projection matrix onto the columns of X. To
simplify notation, denote H := In −H, then (2.1) is equivalent to min
P
∥H P y∥2 s.t. P ∈Πn, dist(P, In) ≤R . (2.2) (2.2) 2 The extended abstract [12] which is a shorter version of this manuscript, studies the noiseless setting. 12 123 Linear regression with partially mismatched data… Our local search approach for the optimization of Problem (2.2) is summarized in
Algorithm 1. Our local search approach for the optimization of Problem (2.2) is summarized in
Algorithm 1. Algorithm 1 A local search method for Problem (2.2). Input: Initial permutation P(0) = In. Tolerance e ≥0 and maximum number of iterations, K. For k = 0, 1, 2, .... Input: Initial permutation P(0) = In. Tolerance e ≥0 and maximum number of iterations, K. For k = 0, 1, 2, .... P(k+1) ∈argmin
P
∥H P y∥2 : dist(P, P(k)) ≤2, dist(P, In) ≤R
. (2.3) (2.3) 3.1 Main results We state and prove the main theorems on the convergence of Algorithm 1. For any
m ≤n, define Bm :=
w ∈Rn : ∥w∥0 ≤m
. (3.1) (3.1) We first state the assumptions useful for our technical analysis. R. Mazumder, H. Wang Assumption 1 Suppose X, y, ϵ, β∗and P∗satisfy the model (1.1) with dist(P∗, In) ≤
r. Suppose the following conditions hold: Assumption 1 Suppose X, y, ϵ, β∗and P∗satisfy the model (1.1) with dist(P∗, In) ≤
r. Suppose the following conditions hold: (1) There exist constants U > L > 0 such that (1) There exist constants U > L > 0 such that (1) There exist constants U > L > 0 such that max
i, j∈[n] |yi −y j| ≤U,
and |(P∗y)i −yi| ≥L
∀i ∈supp(P∗) . max
i, j∈[n] |yi −y j| ≤U,
and |(P∗y)i −yi| ≥L
∀i ∈supp(P∗) . (2) Set R = 10C1rU 2/L2 + 4 for some constant C1 > 1. (2) Set R = 10C1rU 2/L2 + 4 for some constant C1 > 1. (3) There is a constant ρn = O(d log(n)/n) such that Rρn ≤L2/(90U 2), and ∥Hu∥2 ≤ρn∥u∥2 ∀u ∈B4, and ∥Hu∥2 ≤Rρn∥u∥2 ∀u ∈B2R . (3.2) ∥Hu∥2 ≤ρn∥u∥2 ∀u ∈B4, and ∥Hu∥2 ≤Rρn∥u∥2 ∀u ∈B2R . (3.2) (3.2) (4) There is a constant ¯σ ≥0 satisfying ¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U) such
that
∥Hϵ∥∞≤¯σ, ∥ϵ∥∞≤¯σ, and ∥Hϵ∥≤
√
d ¯σ . (3.3) (4) There is a constant ¯σ ≥0 satisfying ¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U) such
that
∥Hϵ∥∞≤¯σ, ∥ϵ∥∞≤¯σ, and ∥Hϵ∥≤
√
d ¯σ . (3.3) (3.3) Note that the lower bound in Assumption 1 (1) states that the y-value for a record
that has been mismatched is not too close to its original value (before mismatch). Assumption 1 (2) states that R is set to a constant multiple of r. This constant can
be large (≥10U 2/L2), and appears to be an artifact of our proof techniques. Our
numerical experience appears to suggest that this constant can be much smaller in
practice. Assumption 1 (3) is a restricted eigenvalue (RE)-type condition [25] stating
that: a multiplication of any (2R)-sparse vector by H will result in a vector with small
norm (in the case Rρn < 1). Section 3.3 discusses conditions on the distribution of
the rows of X under which Assumption 1 (3) holds true with high probability. 3.1 Main results Note
that if ρn = Θ(d log(n)/n), then for the assumption Rρn ≤L2/(90U 2) to hold true,
we require n/ log(n) = Ω(dr). Assumption 1 (4) limits the amount of noise ϵ in the
problem. Section 3.3 presents conditions on the distributions of ϵ and X (in a random
design setting) which ensures Assumption 1 (4) holds true with high probability. Assumption 1 (3) plays an important role in our technical analysis. In particular,
this allows us to approximate the objective function in (2.2) with one that is easier to
analyze. To provide some intuition, we write H P(k) y = H(P(k) y −P∗y) + Hϵ—
noting that H P∗y = H(Xβ∗+ ϵ) = Hϵ, and assuming that the noise ϵ is small, we
have: ∥H P(k) y∥2 ≈∥H(P(k) y −P∗y)∥≈∥P(k) y −P∗y∥2. (3.4) (3.4) Intuitively, the term on the right-hand side is the approximate objective that we analyze
in our theory. Lemma 1 presents a one-step decrease property on the approximate
objective function. Lemma 1 (One-step decrease) Given any y ∈Rn and P, P∗∈Πn, there exists
a permutation matrix P ∈Πn such that dist(P, P) = 2, supp(P(P∗)−1) ⊆
supp(P(P∗)−1) and ∥P y −P∗y∥2 −∥P y −P∗y∥2 ≥(1/2)∥P y −P∗y∥2
∞. (3.5) (3.5) 123 Linear regression with partially mismatched data… If in addition ∥P y −P∗y∥0 ≤m for some m ≤n, then
∥P y −P∗y∥2 ≤(1 −1/(2m)) ∥P y −P∗y∥2 . (3.6) ∥P y −P∗y∥2 ≤(1 −1/(2m)) ∥P y −P∗y∥2 . (3.6) (3.6) ThemainresultsmakeuseofLemma1andformalizetheintuitionconveyedin (3.4). We first present a result regarding the support of the permutation matrix P(k) delivered
by Algorithm 1. Proposition 1 (Support detection) Suppose Assumption 1 holds. Let {P(k)}k≥0 be the
permutation matrices generated by Algorithm 1. Then for all k ≥R/2, it holds
supp(P∗) ⊆supp(P(k)). Proposition 1 states that the support of P∗will be contained within the support
of P(k) after at most R/2 iterations. Intuitively, this result is because of Assumption
1 (1), which assumes that the mismatches represented by P∗have “strong signal”. Proposition 1 is also useful for the proofs of the main theorems below (e.g., see Claim
3.25 in the proof of Theorem 3 for details). We now present some additional assumptions required for the results that follow. Assumption 2 Let ρn and ¯σ be parameters appearing in Assumption 1. Assumption 2 Let ρn and ¯σ be parameters appearing in Assumption 1. (1) Suppose R2ρn ≤1/10. (1) Suppose R2ρn ≤1/10. 3.1 Main results pp
/
) There is a constant σ ≥0 such that ¯σ 2 ≤σ 2 min{n/(660R2), n/(5d R)}, and (2) There is a constant σ ≥0 such that ¯σ 2 ≤σ 2 min{n/(660R2), n/(5d R)}, and (2) There is a constant σ ≥0 such that ¯σ 2 ≤σ 2 min{n/(660R2), n/(5d R)}, and ∥Hϵ∥2 ≥(1/2)nσ 2. (3.7) (3.7) In light of the discussion following Assumption 1, Assumption 2 (1) places a stricter
condition on the size of n via the requirement R2ρn ≤1/10. If ρn = Θ(d log(n)/n),
then we would need n/ log(n) = Ω(dr2), which is stronger than the condition
n/ log(n) = Ω(dr) needed in Assumption 1. Assumption 2 (2) imposes a lower bound on ∥Hϵ∥– this can be equivalently
viewed as an upper bound on ∥Hϵ∥, in addition to the upper bound appearing in
Assumption 1 (4). Section 3.3 provides a sufficient condition for Assumption 2 (2) to
hold with high probability. In particular, in the noiseless case (ϵ = 0), Assumption 1 (4)
and Assumption 2 (2) hold with ¯σ = σ = 0. We now state the first convergence result. Theorem 3 (Linear convergence of objective up to noise level) Suppose Assumptions
1 and 2 hold with R being an even number. Let {P(k)}k≥0 be the permutation matrices
generated by Algorithm 1. Then for any k ≥0, we have ∥H P(k) y∥2 ≤
1 −
1
18R
k
∥H P(0) y∥2 + 36∥Hϵ∥2 . (3.8) (3.8) In the special (noiseless) setting when ϵ = 0, Theorem 3 establishes that the
sequence of objective values generated by Algorithm 1 converges to zero i.e., the
optimalobjectivevalue,atalinearrate.Theparameterforthelinearrateofconvergence
depends upon the search width R. Following the discussion after Assumption 2, the 123 R. Mazumder, H. Wang sample-size requirement n/ log(n) = Ω(dr2) is more stringent than that needed in
orderforthemodeltobeidentifiable(n ≥2d)[24]inthenoiselesssetting.Inparticular,
when n/(d log n) = O(1), the number of mismatched pairs r needs to be bounded by
a constant. Numerical evidence presented in Sect. 5 (for the noiseless case) appears to
suggest that the sample size n needed to recover P∗is smaller than what is suggested
by our theory. In the noisy case (i.e. ϵ ̸= 0), the bound (3.8) provides an upper bound on the
objective value consisting of two terms. The first term converges to 0 with a linear rate
similar to the noiseless case. 3.1 Main results The second term is a constant multiple of the squared
norm of the unavoidable noise term3: ∥Hϵ∥2. In other words, Algorithm 1 finds a
solution whose objective value is at most a constant multiple of the objective value at
the true permutation P∗. Theorem 3 proves a convergence guarantee on the objective value. The next result
provides upper bounds on the ℓ∞-norm of the mismatched entries i.e., ∥P(k) y −
P∗y∥∞. For any Q ∈Πn, define G( Q) := ∥H Q y∥2 −
min
P∈N2( Q)∩NR(In) ∥H P y∥2
(3.9) (3.9) that is, G( Q) is the decrease in the objective value after one step of local search
starting at Q. For the permutation matrices {P(k)}k≥0 generated by Algorithm 1, we
know G(P(k)) = ∥H P(k) y∥2 −∥H P(k+1) y∥2. Theorem 4 (ℓ∞-bound on mismatched pairs) Suppose Assumptions 1 and 2 hold, and
let {P(k)}k≥0 be the permutation matrices generated by Algorithm 1. Then for all
k
0 i h ld k ≥0 it holds ∥P(k) y −P∗y∥2
∞≤800 ¯σ 2 + 10G(P(k)) . Theorem 4 states that the largest squared error of the mismatched pairs (i.e., ∥P(k) y−
P∗y∥2
∞) is bounded above by a constant multiple of the one-step decrease in objective
value (i.e. G(P(k))) plus a term comparable to the noise level O(¯σ 2). In particular, if
Algorithm 1 is terminated at an iteration P(k) with G(P(k)) of the order of ¯σ 2, then
∥P(k) y −P∗y∥2
∞is bounded by a constant multiple of ¯σ 2. Note that the constant 800 in (3.10) is conservative and may be improved with a
careful adjustment of the constants appearing in the proof and in the assumptions. In light of Theorem 4, we can prove an upper bound on the estimation error of β∗,
using an additional assumption stated below. Assumption 5 There exists a constant ¯γ > 0 such that Assumption 5 There exists a constant ¯γ > 0 such that Assumption 5 There exists a constant ¯γ > 0 such that (1) λmin
1
n X⊤X
≥¯γ
(2) ∥(X⊤X)−1X⊤ϵ∥≤¯σ
d
n ¯γ . (3.10) (3.10) 3 Recall that the objective value at P = P∗is ∥H P∗y∥2 = ∥H(Xβ∗+ ϵ)∥2 = ∥Hϵ∥2. where ¯σ is as defined in Assumption 1. where ¯σ is as defined in Assumption 1. where ¯σ is as defined in Assumption 1. 3 Recall that the objective value at P = P∗is ∥H P∗y∥2 = ∥H(Xβ∗+ ϵ)∥2 = ∥Hϵ∥2. Linear regression with partially mismatched data… Section 3.3 presents conditions on X, ϵ under which Assumption 5 is satisfied with
high probability. Section 3.3 presents conditions on X, ϵ under which Assumption 5 is satisfied with
high probability. Theorem 6 (Estimation error) Suppose Assumptions 1, 2 and 5 hold. Suppose iter-
ation k of Algorithm 1 satisfies G(P(k)) ≤c ¯σ 2 for a constant c > 0. Let β(k) :=
(X⊤X)−1X⊤P(k) y and denote ¯c := 800 + 10c. Then we have ∥β(k) −β∗∥2 ≤4 ¯γ −1 ¯c R ¯σ 2
n
+ 2 ¯γ −1 d ¯σ 2
n
(3.11) (3.11) and and 1
n
(P(k))−1Xβ(k) −(P∗)−1Xβ∗
2
≤2
√
2¯c + 3
2 R ¯σ 2
n
+ 2d ¯σ 2
n
. (3.12) (3.12) Theorem 6 (cf bound (3.11)) states that as long as k is sufficiently large4, the
estimationerror ∥β(k)−β∗∥2 is of theorder O((r + d)¯σ 2/n), assuming ¯γ is aconstant. Therefore, as n →∞(with r, d fixed), the estimator delivered by our algorithm (after
sufficiently many iterations) will converge to the true regression coefficient vector, β∗. In addition, (3.12) provides an upper bound on the entrywise “denoising error" (left
hand side of (3.12))—this is of the order O((r + d)¯σ 2/n). See [14] for past works
and discussions on this error metric. The following theorem provides an upper bound on the total number of local search
steps needed to find a P(k) with G(P(k)) ≤c ¯σ 2. Theorem 7 (Iteration complexity) Suppose Assumptions 1 and 2 hold. Let {P(k)}k≥0
be the permutation matrices generated by Algorithm 1. Given any c > 0, define K † :=
log
36∥Hϵ∥2
∥H P(0) y∥2
log
1 −
1
18R
+ 72n
c
+ 1 . (3.13) (3.13) Then there exists 0 ≤k ≤K † such that G(P(k)) ≤c ¯σ 2. Then there exists 0 ≤k ≤K † such that G(P(k)) ≤c ¯σ 2. Proof Denote Then there exists 0 ≤k ≤K † such that G(P(k)) ≤c ¯σ 2. K1 :=
log
36∥Hϵ∥2
∥H P(0) y∥2
log
1 −
1
18R
. 4 We note that in Algorithm 1, as k →∞, the quantity G(P(k)) →0, and the condition G(P(k)) ≤c ¯σ 2
will hold for k sufficiently large. 3.2 Proofs of main theorems In this section, we present the proofs of Proposition 1, Theorem 3, Theorem 4 and
Theorem 6. We first present a technical result used in our proofs. Lemma 2 Suppose Assumption 1 holds. Let {P(k)}k≥0 be the permutation matrices
generated by Algorithm 1. Suppose ∥P(k) y −P∗y∥∞≥L for some k ≥1. Suppose
at least one of the two conditions holds: (i) k ≤R/2; or (ii) k ≥R/2 + 1, and
supp(P∗) ⊆supp(P(k′)) for all R/2 ≤k′ ≤k −1. Then for all t ≤k −1, we have ∥P(t+1) y −P∗y∥2 −∥P(t) y −P∗y∥2 ≤−L2/5 . (3.16) (3.16) The proof of Lemma 2 is presented in Sect. A.3. As mentioned earlier, our analy-
sis makes use of the one-step decrease condition in Lemma 1. Note however, if the
permutation matrix at the current iteration, denoted by P(k), is on the boundary, i.e. dist(P(k), In) = R, it is not clear whether the permutation found by Lemma 1 is
within the search region NR(In). Lemma 2 helps address this issue (See the proof of
Theorem 3 below for details). where ¯σ is as defined in Assumption 1. (3.14) (3.14) Then by Theorem 3, after K1 iterations, it holds Then by Theorem 3, after K1 iterations, it holds ∥H P(K1) y∥2 ≤36∥Hϵ∥2 + 36∥Hϵ∥2 = 72∥Hϵ∥2 ≤72n ¯σ 2
(3.15) ∥H P(K1) y∥2 ≤36∥Hϵ∥2 + 36∥Hϵ∥2 = 72∥Hϵ∥2 ≤72n ¯σ 2
(3.15) (3.15) where the second inequality follows Assumption 1 (4). Suppose G(P(k)) > c ¯σ 2 for
all K1 ≤k ≤K † −1, then where the second inequality follows Assumption 1 (4). Suppose G(P(k)) > c ¯σ 2 for
all K1 ≤k ≤K † −1, then 4 We note that in Algorithm 1, as k →∞, the quantity G(P(k)) →0, and the condition G(P(k)) ≤c ¯σ 2
will hold for k sufficiently large. R. Mazumder, H. Wang ∥H P(K †) y∥2 = ∥H P(K1) y∥2 −
K †−1
k=K1
G(P(k)) < 72n ¯σ 2 −72n
c c ¯σ 2 = 0 , which is a contradiction. So there must exist some K1 ≤k ≤K † −1 such that
G(P(k)) ≤c ¯σ 2. ⊓⊔ which is a contradiction. So there must exist some K1 ≤k ≤K † −1 such that
G(P(k)) ≤c ¯σ 2. ⊓⊔ ⊓⊔ Note that if R and ∥Hϵ∥2/∥H P(0) y∥2 are bounded by a constant, then the number
of iterations K † = O(n). Therefore, in this situation, one can find an estimate β(k)
satisfying ∥β(k) −β∗∥2 ≤O((d + r)¯σ 2/n) within O(n) iterations of Algorithm 1. ( )
,
,
β
tisfying ∥β(k) −β∗∥2 ≤O((d + r)¯σ 2/n) within O(n) iterations of Algorithm 1. 3.2.1 Proof of proposition 1 We show this result by contradiction. Suppose that there exists a k ≥R/2 such that
supp(P∗) ⊈supp(P(k)). Let T ≥R/2 be the first iteration (≥R/2) such that
supp(P∗) ⊈supp(P(T )), i.e., supp(P∗) ⊈supp(P(T )) and supp(P∗) ⊆supp(P(k)) ∀R/2 ≤k ≤T −1 . supp(P∗) ⊈supp(P(T )) and supp(P∗) ⊆supp(P(k)) ∀R/2 ≤k ≤T −1 . Let i ∈supp(P∗) but i /∈supp(P(T )), then by Assumption 1 (1), we have ∥P(T ) y −P∗y∥∞≥|e⊤
i (P(T ) y −P∗y)| = |e⊤
i (y −P∗y)| ≥L. By Lemma 2, we have ∥P(k+1) y −P∗y∥2 −∥P(k) y −P∗y∥2 ≤−L2/5 for al
k
T
1 A
l ∥P(T ) y −P∗y∥∞≥|e⊤
i (P(T ) y −P∗y)| = |e⊤
i (y −P∗y)| ≥L. By Lemma 2, we have ∥P(k+1) y −P∗y∥2 −∥P(k) y −P∗y∥2 ≤−L2/5 for all
k ≤T −1. As a result, By Lemma 2, we have ∥P(k+1) y −P∗y∥2 −∥P(k) y −P∗y∥2 ≤−L2/5 for all
k ≤T −1. As a result, k ≤T −1. As a result, ∥P(T ) y −P∗y∥2 −∥P(0) y −P∗y∥2 ≤−T L2/5 ≤−RL2/10 . 123 123 Linear regression with partially mismatched data… Since by Assumption 1 (1), ∥P(0) y −P∗y∥2 = ∥y −P∗y∥2 ≤rU 2 , we have
∥P(T ) y −P∗y∥2 ≤rU 2 −RL2/10 ≤rU 2 −L2
10
10C1rU 2
L2
= (1 −C1)rU 2 < 0 . This is a contradiction, so such an iteration counter T does not exist; and for all
k ≥R/2, we have supp(P∗) ⊆supp(P(k)). 3.2.2 Proof of theorem 3 Let a = 2.2. Because R ≥10r, we have r + R ≤1.1R = aR/2; and for any k ≥0:
∥P(k) y −P∗y∥0 ≤dist(P(k), In) + dist(P∗, In) ≤R + r ≤aR/2. Hence, by Lemma 1, there exists a permutation matrix P(k) ∈Πn such that
dist(P(k), P(k)) ≤2, supp(P(k)(P∗)−1) ⊆supp(P(k)(P∗)−1) and Hence, by Lemma 1, there exists a permutation matrix P(k) ∈Πn such that
dist(P(k), P(k)) ≤2, supp(P(k)(P∗)−1) ⊆supp(P(k)(P∗)−1) and ∥P(k) y −P∗y∥2 ≤(1 −1/(aR))∥P(k) y −P∗y∥2 . As a result, ∥H(P(k) y −P∗y)∥2 ≤∥P(k) y −P∗y∥2 ≤(1 −1/(aR))∥P(k) y −P∗y∥2
≤1 −1/(aR)
1 −Rρn
∥H(P(k) y −P∗y)∥2 , where the last inequality is from Assumption 1 (3). Note that by Assumption 2 (1),
we have R2ρn ≤1/10 ≤1/(2a), so Rρn ≤1/(2aR). Because (1 −1/(aR)) ≤
(1 −1/(2aR))2, we have ∥H(P(k) y −P∗y)∥2 ≤1 −1/(aR)
1 −1/(2aR)∥H(P(k) y −P∗y)∥2
≤(1 −1/(2aR))∥H(P(k) y −P∗y)∥2 . Recall that (1.1) leads to H P∗y = Hϵ, so we have Recall that (1.1) leads to H P∗y = Hϵ, so we have ∥HP(k) y −Hϵ∥2 ≤(1 −1/(2aR))∥H P(k) y −Hϵ∥2 . (3.17) (3.17) Let η := 1/(2aR) and z := P(k) y −P(k) y, then (3.17) leads to: Let η := 1/(2aR) and z := P(k) y −P(k) y, then (3.17) leads to: ∥HP(k) y∥2 ≤(1 −η)∥H P(k) y∥2 −η∥Hϵ∥2 + 2⟨HP(k) y, Hϵ⟩−2(1 −η)⟨H P(k) y, Hϵ⟩
≤(1 −η)∥H P(k) y∥2 + 2(1 −η)⟨Hz, Hϵ⟩+ 2η⟨HP(k) y, Hϵ⟩
≤(1 −η)∥H P(k) y∥2 + 2|⟨Hz, Hϵ⟩| + 2η|⟨HP(k) y, Hϵ⟩|
(3.18) (3.18) (3.18) 123 123 R. Mazumder, H. Wang where, to arrive at the second inequality, we drop the term −η∥Hϵ∥2. We now make
use of the following claim whose proof is in Sect. A.7: where, to arrive at the second inequality, we drop the term −η∥Hϵ∥2. We now make
use of the following claim whose proof is in Sect. A.7: Claim. 2|⟨Hz, Hϵ⟩| ≤η
4∥HP(k) y∥2 + η
4∥H P(k) y∥2 + 4η∥Hϵ∥2 . (3.19) Claim. 2|⟨Hz, Hϵ⟩| ≤η
4∥HP(k) y∥2 + η
4∥H P(k) y∥2 + 4η∥Hϵ∥2 . (3.19) (3.19) On the other hand, by Cauchy-Schwarz inequality, On the other hand, by Cauchy-Schwarz inequality, 2η|⟨HP(k) y, Hϵ⟩| ≤(η/4)∥HP(k) y∥2 + 4η∥Hϵ∥2 . (3.20) (3.20) Combining (3.18), (3.19) and (3.20), we have ∥HP(k) y∥2 ≤(1 −η)∥H P(k) y∥2 + (η/2)∥HP(k) y∥2
+ (η/4)∥H P(k) y∥2 + 8η∥Hϵ∥2 . 3.2.2 Proof of theorem 3 After some rearrangement, the above leads to: After some rearrangement, the above leads to: ∥HP(k) y∥2 ≤1 −3η/4
1 −η/2 ∥H P(k) y∥2 +
8η
1 −η/2∥Hϵ∥2
≤(1 −η/4)∥H P(k) y∥2 + 9η∥Hϵ∥2
(3.21) (3.21) where the second inequality uses 1−3η/4 ≤(1−η/2)(1−η/4) and (1−η/2)−1 ≤9/8
(recall, η = 1/(2aR)). To complete the proof, we use another claim whose proof is in Sect. A.6: Claim. For any k ≥0 it holds that P(k) ∈NR(In) ∩N2(P(k)) . (3.22)
By the above claim, the update rule (2.3) and inequality (3.21), we have Claim. For any k ≥0 it holds that P(k) ∈NR(In) ∩N2(P(k)) . (3.22) Claim. For any k ≥0 it holds that P(k) ∈NR(In) ∩N2(P(k)) . (3.22)
By the above claim, the update rule (2.3) and inequality (3.21), we have (3.22) (3.22) By the above claim, the update rule (2.3) and inequality (3.21), we have ∥H P(k+1) y∥2 ≤∥HP(k) y∥2 ≤(1 −η/4)∥H P(k) y∥2 + 9η∥Hϵ∥2 . Using the notation ak := ∥H P(k) y∥2, λ = 1 −η/4 and ˜e = 9η∥Hϵ∥2, the above
inequality leads to: ak+1 ≤λak + ˜e for all k ≥0. Therefore, we have ak+1
λk+1 ≤ak
λk +
˜e
λk+1 ≤ak−1
λk−1 + ˜e
λk +
˜e
λk+1 ≤· · · ≤a0
λ0 + ˜e
k+1
i=1
1
λi , which implies ak ≤a0λk + ˜e k
i=1 λi−1 ≤a0λk + (˜e/(1 −λ)). This leads to which implies ak ≤a0λk + ˜e k
i=1 λi−1 ≤a0λk + (˜e/(1 −λ)). This leads to which implies ak ≤a0λk + ˜e k
i=1 λi−1 ≤a0λk + (˜e/(1 −λ)). This leads to ∥H P(k) y∥2 ≤(1 −η/4)k∥H P(0) y∥2 + 9η∥Hϵ∥2
η/4
≤(1 −1/(8aR))k∥H P(0) y∥2 + 36∥Hϵ∥2 . Recalling that 8a ≤18, we conclude the proof of the theorem. 123 12 Linear regression with partially mismatched data… 3.2.3 Proof of theorem 4 he definition of G(·), we have By the definition of G(·), we have ∥H P(k) y∥2 ≤∥H P y∥2 + G(P(k)) ∀P ∈N2(P(k)) ∩NR(In) . (3.23) (3.23) By Lemma 1, there exists a permutation matrix P(k) ∈Πn such that By Lemma 1, there exists a permutation matrix P(k) ∈Πn such that dist(P(k), P(k)) ≤2, supp(P(k)(P∗)−1) ⊆supp(P(k)(P∗)−1) and ∥P(k) y −P∗y∥2 −∥P(k) y −P∗y∥2 ≤−(1/2)∥P(k) y −P∗y∥2
∞. (3.24) By Claim (3.22) we have By Claim (3.22) we have By Claim (3.22) we have P(k) ∈NR(In) ∩N2(P(k)) . (3.25) (3.25) Therefore, by (3.23) and (3.25), we have Therefore, by (3.23) and (3.25), we have ∥H P(k) y∥2 ≤∥HP(k) y∥2 + G(P(k)) . Let z := P(k) y −P(k) y. Recall that H P∗y = Hϵ, so by the inequality above we
have Let z := P(k) y −P(k) y. Recall that H P∗y = Hϵ, so by the inequality above we
have ∥H(P(k) y −P∗y) + Hϵ∥2 ≤∥H(P(k) y −P∗y) + Hϵ + Hz∥2 + G(P(k)) ∥H(P(k) y −P∗y) + Hϵ∥2 ≤∥H(P(k) y −P∗y) + Hϵ + Hz∥2 + G(P(k)) which is equivalent to which is equivalent to −2⟨H(P(k) y −P∗y), Hz⟩−∥Hz∥2 ≤2⟨Hz, Hϵ⟩+ G(P(k)) . (3.26) (3.26) On the other hand, from (3.24) we have On the other hand, from (3.24) we have ∥P(k) y −P∗y + z∥2 −∥P(k) y −P∗y∥2 ≤−(1/2)∥P(k) y −P∗y∥2
∞ or equivalently, 2⟨P(k) y −P∗y, z⟩+ ∥z∥2 ≤−(1/2)∥P(k) y −P∗y∥2
∞. (3.27) Summing up (3.26) and (3.27) we have Summing up (3.26) and (3.27) we have 2⟨H(P(k) y −P∗y), Hz⟩+ ∥Hz∥2
≤2⟨Hz, Hϵ⟩−(1/2)∥P(k) y −P∗y∥2
∞+ G(P(k)) . (3.28)
123 2⟨H(P(k) y −P∗y), Hz⟩+ ∥Hz∥2
≤2⟨Hz, Hϵ⟩−(1/2)∥P(k) y −P∗y∥2
∞+ G(P(k)) . (3.28) (3.28) R. Mazumder, H. Wang ote that Note that Note that 2|⟨H(P(k) y −P∗y), Hz⟩| ≤2∥H(P(k) y −P∗y)∥· ∥Hz∥
≤2
Rρn∥P(k) y −P∗y∥∥Hz∥≤2
√
2R√ρn∥P(k) y −P∗y∥∞∥Hz∥
(3.29) (3.29) where the second inequality is by Assumption 1 (3) and the third inequality uses
∥P(k) y −P∗y∥0 ≤2R. From Assumption 2 (1) we have R√ρn ≤1/
√
10, hence 2|⟨H(P(k) y −P∗y), Hz⟩| ≤
2
√
5
∥P(k) y −P∗y∥∞∥Hz∥
≤1
5∥P(k) y −P∗y∥2
∞+ ∥Hz∥2
(3.30) (3.30) where the last inequality is by Cauchy-Schwarz inequality. Rearranging terms
in (3.28), and making use of (3.30), we have where the last inequality is by Cauchy-Schwarz inequality. 3.2.3 Proof of theorem 4 Rearranging terms
in (3.28), and making use of (3.30), we have ∥Hz∥2 + (1/2)∥P(k) y −P∗y∥2
∞
≤2⟨Hz, Hϵ⟩−2⟨H(P(k) y −P∗y), Hz⟩+ G(P(k))
≤2⟨Hz, Hϵ⟩+ (1/5)∥P(k) y −P∗y∥2
∞+ ∥Hz∥2 + G(P(k)) . As a result, As a result, (3/10)∥P(k) y −P∗y∥2
∞≤2⟨Hz, Hϵ⟩+ G(P(k))
= 2⟨z, Hϵ⟩+ G(P(k)). (3.31) (3.31) By the definition of z, we know there exist i, j ∈[n] such that By the definition of z, we know there exist i, j ∈[n] such that z = ∥z∥∞(ei −e j) = (∥z∥/
√
2)(ei −e j). Therefore Therefore 2⟨z, Hϵ⟩=
√
2∥z∥⟨ei −e j, Hϵ⟩≤2
√
2∥z∥∥Hϵ∥∞≤2
√
2 ¯σ∥z∥
(3.32) (3.32) where the last inequality makes use of Assumption 1 (4). On the other hand, by (3.27)
we have where the last inequality makes use of Assumption 1 (4). On the other hand, by (3.27)
we have ∥z∥2 ≤2|⟨P(k) y −P∗y, z⟩| =
√
2∥z∥|⟨P(k) y −P∗y, ei −e j⟩| , and hence ∥z∥≤
√
2|⟨P(k) y −P∗y, ei −e j⟩| ≤2
√
2∥P(k) y −P∗y∥∞. (3.33) (3.33) 123 Linear regression with partially mismatched data… Linear regression with partially mismatched data… ombining (3.31), (3.32) and (3.33), we have Combining (3.31), (3.32) and (3.33), we have (3/10)∥P(k) y −P∗y∥2
∞≤8¯σ∥P(k) y −P∗y∥∞+ G(P(k))
≤80 ¯σ 2 + (1/5)∥P(k) y −P∗y∥2
∞+ G(P(k))(3.34) where the second inequality is by Cauchy-Schwarz inequality. Inequality in dis-
play (3.34) leads to where the second inequality is by Cauchy-Schwarz inequality. Inequality in dis-
play (3.34) leads to ∥P(k) y −P∗y∥2
∞≤800 ¯σ 2 + 10G(P(k)) which completes the proof of this theorem. which completes the proof of this theorem. which completes the proof of this theorem. 3.2.4 Proof of theorem 6 Recall that P∗y = Xβ∗+ ϵ, so it holds β∗= (X⊤X)−1X⊤(P∗y −ϵ). Therefore
β(k) −β∗= (X⊤X)−1X⊤(P(k) y −P∗y + ϵ) . (3.35) (3.35) Hence we have Hence we have ∥β(k) −β∗∥≤∥(X⊤X)−1X⊤(P(k) y −P∗y)∥+ ∥(X⊤X)−1X⊤ϵ∥
≤∥(X⊤X)−1X⊤(P(k) y −P∗y)∥+ ¯σ
d/(n ¯γ )
(3.36) (3.36) where the second inequality is by Assumption 5 (2). Note that ∥P(k) y −P∗y∥≤
√
2R∥P(k) y −P∗y∥∞≤¯σ
√
2R ¯c
(3.37) (3.37) where the first inequality is because P(k) y−P∗y has at most 2R non-zero coordinates;
the second inequality makes use of Theorem 4 and the definition of ¯c in Theorem 6. On the other hand, by Assumption 5 (1) we have where the first inequality is because P(k) y−P∗y has at most 2R non-zero coordinates;
the second inequality makes use of Theorem 4 and the definition of ¯c in Theorem 6. On the other hand, by Assumption 5 (1) we have (X⊤X)−1X⊤2 =
(X⊤X)−12 ≤1/
n ¯γ . (3.38) (3.38) Combining (3.36), (3.37) and (3.38) we have Combining (3.36), (3.37) and (3.38) we have Combining (3.36), (3.37) and (3.38) we have ∥β(k) −β∗∥≤(X⊤X)−1X⊤2 ∥P(k) y −P∗y∥+ ¯σ
d/(n ¯γ )
≤¯σ
2 ¯γ −1R ¯c/n + ¯σ
d ¯γ −1/n . Squaring both sides of the above, we get Squaring both sides of the above, we get ∥β(k) −β∗∥2 ≤4 ¯γ −1 ¯c R ¯σ 2
n
+ 2 ¯γ −1 d ¯σ 2
n which completes the proof of (3.11). which completes the proof of (3.11). 123 123 R. Mazumder, H. Wang We will now prove (3.12). Let us denote J := (P(k))−1Xβ(k) −(P∗)−1Xβ∗. Note
that we can write J = (P(k))−1X(β(k) −β∗) + ((P(k))−1 −(P∗)−1)Xβ∗. (3.39)
ng both sides of (3.35) by (P(k))−1X, we have J = (P(k))−1X(β(k) −β∗) + ((P(k))−1 −(P∗)−1)Xβ∗. (3.39) (3.39) Multiplying both sides of (3.35) by (P(k))−1X, we have Multiplying both sides of (3.35) by (P(k))−1X, we have (P(k))−1X(β(k) −β∗) = (P(k))−1H(P(k) y −P∗y + ϵ) . (3.40) n the other hand, On the other hand, ((P(k))−1 −(P∗)−1)Xβ∗
= ((P(k))−1 −(P∗)−1)(P∗y −ϵ)
= (P(k))−1(P∗y −P(k) y) −((P(k))−1 −(P∗)−1)ϵ . (3.41) (3.41) Combining (3.39), (3.40) and (3.41) we have Combining (3.39), (3.40) and (3.41) we have J = (P(k))−1 H(P∗y −P(k) y)
:=J1
+ (P(k))−1Hϵ
:=J2
+ ((P∗)−1 −(P(k))−1)ϵ
:=J3
= J1 + J2 + J3 . 3.2.4 Proof of theorem 6 (3.42)
By (3 37) we know = J1 + J2 + J3 . = J1 + J2 + J3 . (3.42) By (3.37), we know ∥J1∥≤∥P∗y −P(k) y∥≤¯σ
√
2R ¯c . (3.43) By Assumption 1 (4) we have By Assumption 1 (4) we have ∥J2∥= ∥Hϵ∥≤
√
d ¯σ . (3.44) Since dist((P∗)−1, (P(k))−1) ≤2R, it holds Since dist((P∗)−1, (P(k))−1) ≤2R, it holds
∥J3∥= ∥(P∗)−1ϵ −(P(k))−1ϵ∥≤2
√
2R∥ϵ∥∞≤3
√
R ¯σ
(3.45) ∥J3∥= ∥(P∗)−1ϵ −(P(k))−1ϵ∥≤2
√
2R∥ϵ∥∞≤3
√
R ¯σ
(3.45) (3.45) where the last inequality makes use of Assumption 1 (4). Using (3.43), (3.44) and (3.45), to bound the r.h.s of (3.42), we have where the last inequality makes use of Assumption 1 (4). Using (3.43), (3.44) and (3.45), to bound the r.h.s of (3.42), we have ∥J∥≤¯σ
√
2R ¯c +
√
d ¯σ + 3
√
R ¯σ . As a result, 1
n ∥J∥2 ≤2(
√
2¯c + 3)2 R ¯σ 2
n
+ 2d ¯σ 2
n
. This completes the proof of (3.12). This completes the proof of (3.12). 123 Linear regression with partially mismatched data… 3.3 Sufficient conditions for assumptions to hold Our analysis in Sects. 3.1 and 3.2 was completely deterministic in nature under
Assumptions 1, 2 and 5. To provide some intuition, in the following, we discuss some
probability models on X and ϵ under which Assumption 1 (3), (4), Assumption 2 (2)
and Assumption 5 hold true with high probability. 5 The probability statements here are conditional on X. Suppose n is large enough such that
δn,m ≥2/n and 3b2 Σ 2 log(2d/τ)/n ≤
(1/4)γ 2. Then with probability at least 1 −2τ, it holds λmin( 1
n X⊤X) ≥γ/2, and Suppose n is large enough such that
δn,m ≥2/n and 3b2 Σ 2 log(2d/τ)/n ≤
(1/4)γ 2. Then with probability at least 1 −2τ, it holds λmin( 1
n X⊤X) ≥γ/2, and ∥Hu∥2 ≤δn,m∥u∥2
∀u ∈Bm . (3.46) (3.46) The proof of Lemma 3 is presented in Sect. A.4. Suppose there are universal con-
stants ¯c > 0 and ¯C > 0 such that the parameters (γ, V , b, Σ2, τ) in Lemma 3
satisfy ¯c ≤γ, V , b, Σ2, τ ≤¯C. Given a pre-specified probability level (e.g.,
1 −2τ = 0.99), under the setting of Lemma 3, if we set ρn = δn,4 and ¯σ = γ/2, then
Assumption 1 (3) and Assumption 5 (1) are true with high probability (≥1 −2τ). Note that the almost sure boundedness assumption on ∥xi∥can be relaxed to cases
when ∥xi∥is bounded with high probability (e.g. xi
iid∼N(0, Σ)). 3.3.1 A random model matrix X When the rows of X are iid draws from a well behaved probability distribution,
Assumption 1 (3) and Assumption 5 (1) hold true with high probability. This is for-
malized via the following lemma. Lemma 3 Suppose the rows of the matrix X: x1, . . . , xn are iid zero-mean random
vectors in Rd with covariance matrix Σ ∈Rd×d. Suppose there exist constants
γ, b, V > 0 such that λmin(Σ) ≥γ , ∥xi∥≤b and ∥xi∥∞≤V almost surely. Given any τ > 0, define δn,m := 16V 2
d
nγ log(2d/τ) + dm
nγ log(3n2)
. Suppose n is large enough such that
δn,m ≥2/n and 3b2 Σ 2 log(2d/τ)/n ≤
(1/4)γ 2. Then with probability at least 1 −2τ, it holds λmin( 1
n X⊤X) ≥γ/2, and ¯σ = σ
2 log(6n/τ) and ¯γ = γ/2. ¯σ = σ
2 log(6n/τ) and ¯γ = γ/2.
If in addition the conditions in Assumption 1 (1) and (2) hold true and the following
four inequalities Rρn ≤L2/(90U 2),
¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U) ,
R2ρn ≤1/10,
¯σ 2 ≤σ 2 min{n/(660R2), n/(5d R)}
(3.49)
are true, then all the statements in Assumptions 1, 2 and 5 are satisfied. Rρn ≤L2/(90U 2),
¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U) ,
R2ρn ≤1/10,
¯σ 2 ≤σ 2 min{n/(660R2), n/(5d R)}
(3.49) Rρn ≤L2/(90U 2),
¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U) , Rρn ≤L2/(90U 2),
¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U) ,
R2ρ ≤1/10
¯σ 2 ≤σ 2 min{n/(660R2) n/(5d R)} R2ρn ≤1/10,
¯σ 2 ≤σ 2 min{n/(660R2), n/(5d R)}
(3.49) (3.49) are true, then all the statements in Assumptions 1, 2 and 5 are satisfied. are true, then all the statements in Assumptions 1, 2 and 5 are satisfied. 3.3.2 The error distribution Inthefollowing, wediscuss acommonlyusedrandomsettingunder whichAssumption
1 (4) and Assumption 2 (2) hold with high probability. A random variable ξ is called
sub-Gaussian [25] with variance proxy ϑ2 (denoted by ξ ∈subG(ϑ2)) if E[ξ] = 0
and Eetξ ≤eϑ2t2/2 for all t ∈R. Lemma 4 Suppose ϵ = [ϵ1, . . . , ϵn]⊤with ϵ1, . . . , ϵn
iid∼subG(σ 2) for some σ > 0. Suppose ϵ is independent of X. Then with probability5 at least 1 −τ it holds Lemma 4 Suppose ϵ = [ϵ1, . . . , ϵn]⊤with ϵ1, . . . , ϵn
iid∼subG(σ 2) for some σ > 0. Suppose ϵ is independent of X. Then with probability5 at least 1 −τ it holds 123 123 R. Mazumder, H. Wang (a) max{∥ϵ∥∞, ∥Hϵ∥∞} ≤σ
2 log(6n/τ). (a) max{∥ϵ∥∞, ∥Hϵ∥∞} ≤σ
2 log(6n/τ).
(b) ∥Hϵ∥≤σ
2d log(6d/τ).
(c) ∥(X⊤X)−1X⊤ϵ∥≤λ−1/2
min ( 1
n X⊤X) · σ
2d log(6d/τ)/n.
(c) ∥(X⊤X)−1X⊤ϵ∥≤λ−1/2
min ( 1
n X⊤X) · σ
2d log(6d/τ)/n. In addition, if σ 2 := Var(ϵi) ≥(3/4)σ 2, then there exists a universal constant
C > 0 such that if
log(4/τ)/(Cn) + 2d log(4d/τ)/n ≤1/4, then with probability
at least 1 −τ, ∥Hϵ∥2 ≥(1/2)nσ 2 . (3.47) (3.47) The proof of Lemma 4 is presented in Sect. A.5. Note that in Lemma 4 the assump-
tion Var(ϵi) ≥(3/4)σ 2 can be replaced by Var(ϵi) ≥C0σ 2 for any constant C0,
with the conclusion changing accordingly (i.e., 1/2 in (3.47) will be replaced by
another constant). In particular, Lemma 4 holds true when ϵi
iid∼N(0, σ 2). Given a
pre-specified probability level (e.g., 1−τ = 0.99), under the setting of Lemma 4, if we
set ¯σ = σ
2 log(6n/τ), then Assumption 1 (4), Assumption 2 (2) and Assumption
5 (2) hold with probability at least 1 −2τ. 3.3.3 Summary We summarize parameter choices informed by the results above in the following
corollary. Corollary 1 Suppose the matrix X is drawn from a probability model as discussed in
Lemma 3 with m = R; and the noise term ϵ satisfies the assumptions in Lemma 4. Then with probability at least 1 −6τ, the inequalities in (3.2), (3.3), (3.7) and (3.10)
hold true with the following parameters ρn = 16V 2
d
nγ log(2d/τ) + 4d
nγ log(3n2)
,
(3.48) (3.48) 4 Approximate local search steps for computational scalability As discussed in Sect. 2, the local search step (2.3) in Algorithm 1 costs O(n2) for
each iteration k—this can limit the scalability of Algorithm 1 to problems with a
large n. Here we discuss an efficient method to find an approximate solution for 123 Linear regression with partially mismatched data… step (2.3). Suppose that in the k-th iteration of Algorithm 1 the permutation P(k)
satisfies dist(P(k), In) ≤R −2, then update (2.3) is step (2.3). Suppose that in the k-th iteration of Algorithm 1 the permutation P(k)
satisfies dist(P(k), In) ≤R −2, then update (2.3) is min
P
∥H P y∥2
s.t. dist(P, P(k)) ≤2. (4.1) (4.1) Problem (4.1) can be equivalently formulated as Problem (4.1) can be equivalently formulated as min
i, j∈[n] ∥H(P(k) y −(yi −y j)(ei −e j))∥2
= min
i, j∈[n]
∥H P(k) y∥2 −2(yi −y j)⟨ei −e j, H P(k) y⟩
+ (yi −y j)2∥H(ei −e j)∥2
. (4.2) (4.2) In view of Assumption 1 (3), for n ≫d, we have ∥H(ei −e j)∥2 ≈2. Note that
in general, ∥H(ei −e j)∥2 ≤2. Hence, one can approximately optimize (4.2) by
minimizing an upper bound of the last two terms in the second line of display (4.2). This is given by: min
i, j∈[n]
−2(yi −y j)⟨ei −e j, H P(k) y⟩+ 2(yi −y j)2
. (4.3) (4.3) Denoting w := H P(k) y and v := y −w, the objective in (4.3) is given by Denoting w := H P(k) y and v := y −w, the objective in (4.3) is given by −2(yi −y j)⟨ei −e j, H P(k) y⟩+ 2(yi −y j)2
= 2(yi −y j)(−wi + w j + yi −y j) = 2(yi −y j)(vi −v j) . So problem (4.3) is equivalent to So problem (4.3) is equivalent to min
i, j∈[n] (yi −y j)(vi −v j). (4.4) (4.4) As we discuss below, the computation cost of the above problem can be reduced by
making use of its structural properties. Let us denote zi = (yi, vi) ∈R2. Among the
set of points {z1, . . . , zn}, we say zi is a “left-top" point if for all j ∈[n], As we discuss below, the computation cost of the above problem can be reduced by
making use of its structural properties. Let us denote zi = (yi, vi) ∈R2. 4 Approximate local search steps for computational scalability Among the
set of points {z1, . . . , zn}, we say zi is a “left-top" point if for all j ∈[n], z j /∈{(u1, u2) ∈R2 | u1 ≤yi, u2 ≥vi} \ {zi} . We say zi is a “right-bottom" point if for all j ∈[n], z j /∈{(u1, u2) ∈R2 | u1 ≥yi, u2 ≤vi} \ {zi} . Figure 1 shows an example of left-top and right-bottom points for a collection of zi’s
with noisy y. It can be seen that the number of left-top and right-bottom points can be
much smaller than the total number of points. Figure 1 shows an example of left-top and right-bottom points for a collection of zi’s
with noisy y. It can be seen that the number of left-top and right-bottom points can be
much smaller than the total number of points. 123 R. Mazumder, H. Wang - 3
- 2
- 1
0
1
2
3
- 4
- 2
0
2
4
y
v
left- top points
right- bottom points
Fig. 1 Figure illustrating the set Left-top points and right-bottom points in a 2D collection of points
zi = (yi, vi) ∈R2 Fig. 1 Figure illustrating the set Left-top points and right-bottom points in a 2D collection of points
zi = (yi, vi) ∈R2 Let (i∗, j∗) be an optimal solution to (4.4), then it must hold that one of {zi∗, z j∗}
is a left-top point and the other is a right-bottom point. Let Zlt and Zrb be the set of
left-top and right-bottom points respectively, and define Slt := {i ∈[n] | zi ∈Zlt},
Srb := {i ∈[n] | zi ∈Zrb} . Then Problem (4.4) is equivalent to Then Problem (4.4) is equivalent to min
i∈Slt, j∈Srb
(yi −y j)(vi −v j)
(4.5) (4.5) implying that it suffices to compute values of (yi −y j)(vi −v j) for i ∈Slt and j ∈Srb. Algorithm 2 discusses how to compute Slt and Srb—this requires (a) performing a
sorting operation on y, which can be done once with a cost of O(n log(n)); and (b) two
additional passes over the data with cost O(n) (to be performed at every iteration of
Algorithm 1). The computation of (4.5) can be further simplified as discussed in the following
section. Algorithm 2 Fast algorithm for Problem (4.4) There exist ¯t, ¯b ∈[L′] with ¯b ≤¯t such that (ym −yit )(vm −vit ) ≤0 for all
¯b ≤t ≤¯t, and (ym −yit )(vm −vit ) > 0 for all t > ¯t or t < ¯b. 2. There exist ¯t, ¯b ∈[L′] with ¯b ≤¯t such that (ym −yit )(vm −vit ) ≤0 for a
¯b ≤t ≤¯t, and (ym −yit )(vm −vit ) > 0 for all t > ¯t or t < ¯b. 2. There exist ¯t, ¯b ∈[L′] with ¯b ≤¯t such that (ym −yit )(vm −vit ) ≤0 for all
¯b ≤t ≤¯t, and (ym −yit )(vm −vit ) > 0 for all t > ¯t or t < ¯b. Because Zrb is nicely ordered as in (4.6), Case 1 above can be identified by a bisection
method with cost (at most) O(log n). Similarly, for Case 2, the values of ¯t and ¯b can
be found using bisection. Since the optimal value of (4.4) must be non-positive, we
can compute (ym −yit )(vm −vit ) only for ¯b ≤t ≤¯t. The methods described for solving (4.4) are summarized in Algorithm 2. Finally,
note that when dist(P(k), In) ≥R −1, similar ideas are still applicable. When
dist(P(k), In) = R −1, we consider the following problem: min
i, j
(yi −y j)(vi −v j)
s.t. i ∈[n], j ∈supp(P(k)). (4.7) (4.7) Similarly, when dist(P(k), In) = R, we consider: Similarly, when dist(P(k), In) = R, we consider: Similarly, when dist(P(k), In) = R, we consider: Similarly, when dist(P(k), In) = R, we consider:
min
i, j
(yi −y j)(vi −v j)
s.t. i, j ∈supp(P(k)). (4.8) min
i, j
(yi −y j)(vi −v j)
s.t. i, j ∈supp(P(k)). (4.8) (4.8) Problems (4.7) and (4.8) can also be efficiently solved by finding the sets of left-top
and right-bottom points and using the partial order to simplify the computation. We
omit the details for brevity. Problems (4.7) and (4.8) can also be efficiently solved by finding the sets of left-top
and right-bottom points and using the partial order to simplify the computation. We
omit the details for brevity. 4.1 Faster computation of problem (4.5) To simplify the computation of (4.5), we introduce a partial order ‘⪯’ on the points
in R2: For p, q ∈R2, denote p ⪯q if p1 ≤q1 and p2 ≤q2. It is easy to check that
for any two points zi, z j ∈Zrb, either it holds zi ⪯z j, or it holds z j ⪯zi. So we can
write Zrb = {zi1, ...., ziL′ } with zi1 ⪯zi2 ⪯· · · ⪯ziL′ . (4.6) (4.6) For any zm ∈Zlt, two cases can happen: 123 Linear regression with partially mismatched data… Algorithm 2 Fast algorithm for Problem (4.4) Algorithm 2 Fast algorithm for Problem (4.4)
Input: Vectors v, y ∈Rn. Step 1: (Sorting) Find a permutation π such that yπ(1) ≤yπ(2) ≤· · · ≤yπ(n). Define ¯yi = yπ(i) and
¯vi = vπ(i) for all i ∈[n]. Step 2: (Construct Slt) Initialize Slt = {π(1)} and T = ¯v1. For i = 2, 3, . . . , n:
If ¯vi > T : Slt = Slt ∪{π(i)}; T = ¯vi
end if
end for
Step 3: (Construct Srb) Initialize Srb = {π(n)} and B = ¯vn. For i = n −1, n −2, ...., 1:
If ¯vi < B: Srb = Srb ∪{π(i)}; B = ¯vi
end if
end for
St
4 S l
(4 5)
i
th
th d di
d i S
ti
4 1 Input: Vectors v, y ∈R . Step 1: (Sorting) Find a permutation π such that yπ(1) ≤yπ(2) ≤· · · ≤yπ(n). Define ¯yi = yπ(i) and
¯vi = vπ(i) for all i ∈[n]. i
π(i)
Step 2: (Construct Slt) Initialize Slt = {π(1)} and T = ¯v1. For i = 2, 3, . . . , n:
If ¯vi > T : Slt = Slt ∪{π(i)}; T = ¯vi
end if
end for i
π(i)
Step 2: (Construct Slt) Initialize Slt = {π(1)} and T = ¯v1. For i = 2, 3, . . . , n:
If ¯vi > T : Slt = Slt ∪{π(i)}; T = ¯vi
end if
end for Step 3: (Construct Srb) Initialize Srb = {π(n)} and B = ¯vn. For i = n −1, n −2, ...., 1:
If ¯vi < B: Srb = Srb ∪{π(i)}; B = ¯vi
end if
end for Step 3: (Construct Srb) Initialize Srb = {π(n)} and B = ¯vn. For i = n −1, n −2, ...., 1:
If ¯vi < B: Srb = Srb ∪{π(i)}; B = ¯vi
end if
end for end for
Step 4: Solve (4.5) using the methods discussed in Section 4.1 1. There is no point zit ∈Zrb satisfying (yit −ym)(vit −vm) ≤0. 2. There exist ¯t, ¯b ∈[L′] with ¯b ≤¯t such that (ym −yit )(vm −vit ) ≤0 for all 1. There is no point zit ∈Zrb satisfying (yit −ym)(vit −vm) ≤0. ¯
¯ 1. There is no point zit ∈Zrb satisfying (yit −ym)(vit −vm) ≤0. ¯
¯ 1. There is no point zit ∈Zrb satisfying (yit
ym)(vit
vm) ≤0. 2. 5 Experiments We perform numerical experiments to study the performance of Algorithm 1. We perform numerical experiments to study the performance of Algorithm 1. Data generation. We consider the setup in our basic model (1.1), where entries of
X ∈Rn×d are iid N(0, 1); β∗is generated uniformly from the unit sphere in Rd (i.e.,
∥β∗∥= 1), and β∗is independent of X. We consider two schemes for generating the
permutation P∗: (a) Random scheme: select r coordinates uniformly from {1, . . . , n}. (b) Equi-spaced scheme: Assume y1 ≤· · · ≤yn (otherwise re-order the data). Let
a1 < · · · < ar bethesequenceofr equi-spacedrealnumberswitha1 = mini∈[n] yi and
an = maxi∈[n] yi.Selectr indicesi1 < · · · < ir suchthati1 = argmini∈[n] |yi−a1|and R. Mazumder, H. Wang is = argminis−1+1≤i≤n |yi −as| for all 2 ≤s ≤r. After the r coordinates are chosen,
we generate a uniformly distributed random permutation on these r coordinates.6 is = argminis−1+1≤i≤n |yi −as| for all 2 ≤s ≤r. After the r coordinates are chosen,
we generate a uniformly distributed random permutation on these r coordinates.6 We generate ϵ (independent of X and β∗) with ϵi
iid∼N(0, σ 2) for some σ ≥0
(σ = 0 corresponds to the noiseless setting). Unless otherwise specified, we set the
tolerance e = 0 and K = ∞in Algorithm 1. 6 Note that P∗may not satisfy dist(P∗, In) = r, but dist(P∗, In) will be close to r. 5.1 Experiments for the noiseless setting We first consider the noiseless setting (ϵ = 0) with different combinations of (d,r, n). We use the random scheme to generate the unknown permutation P∗. We set R = n in
Algorithm 1 and a maximum iteration limit of 1000. While our algorithm parameter
choices are not covered by our theory, in practice when r is small, our local search
algorithm converges to optimality; and the number of iterations is bounded by a small
constant multiple of r (e.g., for r = 50, the algorithm converges to optimality within
around 60 iterations). Figure 2 presents preliminary results on examples with n
=
500, d
∈
{20, 50, 100, 200}, and 40 roughly equi-spaced values of r ∈[10, 400]. In Fig. 2
[left panel], we plot the Hamming distance of the solution ˆP computed by Algorithm
1 and the underlying permutation P∗(i.e. dist( ˆP, P∗)) versus r. In Fig. 2 [right panel],
we present errors in estimating β∗versus r. More precisely, let ˆβ be the solution com-
puted by Algorithm 1 (i.e. ˆβ = (X⊤X)−1X⊤ˆP y), then the beta error is defined as
∥ˆβ −β∗∥/∥β∗∥. For each choice of (r, d), we consider the average over 50 indepen-
dent replications (the vertical bars show standard errors, which are hardly visible in the
figures). As shown in Fig. 2, when r is small, the underlying permutation P∗can be
exactly recovered, and thus the corresponding beta error is also 0. As r becomes larger,
Algorithm 1 fails to recover P∗exactly; and dist(P∗, ˆP) is close to the maximal pos-
sible value n = 500. In contrast, the estimation error appears to vary more smoothly:
As the value of r increases, beta error increases. We also observe that the recovery of
P∗depends upon the number of covariates d — permutation recovery performance
deteriorates with increasing d. This is consistent with our theory suggesting that the
performance of our algorithm depends upon both r and d. 5.2 Experiments for the noisy setting We explore the performance of Algorithm 1 under the noisy setting (ϵ ̸= 0). Performance for different values of R: We denote Relative Obj as the objective value
computed by Algorithm 1 divided by ∥Hϵ∥2. Figure 3 presents the Relative Obj, beta
error and Hamming distance of the local search algorithm with different values of R
(x-axis corresponds to the values of R). Here we consider n = 1000, d = 10, r = 10
and σ = 0.1; and use the equi-spaced scheme to choose the mismatched coordinates
in P∗. We highlight the value at R = r = 10 by a red point. As shown in Fig. 3, as
R increases, the Relative Obj decreases below 1 – this is consistent with our theory 123 12 Linear regression with partially mismatched data… Fig. 2 Left: Values of Hamming distance dist( ˆP, P∗) versus r. Right: Values of beta error ∥ˆβ −β∗∥/∥β∗∥
versus r Fig. 2 Left: Values of Hamming distance dist( ˆP, P∗) versus r. Right: Values of beta error ∥ˆβ −β∗∥/∥β∗∥
versus r Fig. 3 Experiment on an instance with n = 1000, d = 10, r = 10 and σ = 0.1. Left: Relative Obj vs R. Middle: beta error vs R. Right: Hamming distance vs R. The circled red point corresponds to R = r Fig. 3 Experiment on an instance with n = 1000, d = 10, r = 10 and σ = 0.1. Left: Relative Obj vs R
Middle: beta error vs R. Right: Hamming distance vs R. The circled red point corresponds to R = r stating that with a proper choice of R, the final objective value will be below a constant
multiple of ∥Hϵ∥2. As R increases, different from the Relative Obj profile, the beta error and
Hamming distance first decrease then increase. This appears to suggest that when
R is too large, Algorithm 1 can overfit and further regularization may be necessary to
mitigate overfitting. A detailed investigation of this matter is left as future work. In
this example, the best beta error and Hamming distance are achieved when R equals
r. Note that in Fig. 3 [left panel], the Relative Obj is close to 1 when we choose R
close to r. 7 https://github.com/abidlabs/stochastic-em-shuffled-regression. 5.2 Experiments for the noisy setting Therefore, if we have a good estimate of the noise level σ (but the exact
value of r is not available), we can choose a value of R at which the Relative Obj is
approximately 1. Finally, we note that in the noisy case, the local search method cannot exactly
recover P∗. Indeed, in the noisy case, if a solution to (1.2) has to exactly recover P∗,
we need to take a smaller value of σ (see discussions in [15]). Even though in our
example, we cannot exactly recover P∗, we may still be able to obtain a good estimate
for β∗—see Fig. 3 [middle panel]. Estimating P∗, β∗under different noise levels: For a given σ (standard deviation
of the noise), let relative beta error be the value ∥ˆβ −β∗∥/(σ∥β∗∥), where ˆP and ˆβ
are the estimates available from Algorithm 1 upon termination. Consider an example with n = 500 and d = r = 10 and different values of
σ ∈{0.01, 0.03, 0.1, 0.3, 1.0}, and use the random scheme to generate the unknown
permutation P∗. We run Algorithm 1 with the setting R = r. Figure 4 presents the 123 R. Mazumder, H. Wang Fig. 4 Experiment on an instance with n = 500, d
= 10, r
= 10, and different noise levels
σ ∈{0.01, 0.03, 0.1, 0.3, 1.0}. Left: Hamming distance vs σ. Right: relative beta error vs σ Fig. 4 Experiment on an instance with n = 500, d
= 10, r
= 10, and different noise leve
σ ∈{0.01, 0.03, 0.1, 0.3, 1.0}. Left: Hamming distance vs σ. Right: relative beta error vs σ values of Hamming distance and relative beta error under different noise levels. In Fig. 4 [left panel], it can be seen that as σ increases, the Hamming distance also increases,
and recovering P∗becomes harder as the noise level becomes larger. In Fig. 4 [right
panel], we can see that the relative beta error almost does not change under different
values of σ. This appears to be consistent with our conclusion in Theorem 6 that
∥ˆβ −β∗∥will be bounded by a value proportional to σ. 5.3 Comparisons with existing methods We compare across the following methods for (1.3): We compare across the following methods for (1.3): – AltMin: The alternating minimization method of [9]. We initialize with P = In
and β = 0, and alternately minimize over P and β until no improvement can be
made. – StoEM: The stochastic expectation maximization method [2]. We run the algo-
rithm for 30 steps under the default setting. – S-BD: The robust regression relaxation method of [18]. We set the regularization
parameter λ = 4(1 + M)σ
2 log(n)/n with M = 1 (this value of λ is given by
Theorem 2 of [18]). or both AltMin and StoEM, we use the implementation provided in the repository7 For both AltMin and StoEM, we use the implementation provided in the repository For both AltMin and StoEM, we use the implementation provided in the repository7
accompanying paper [2]. We compare these methods with Algorithm 1 (denoted by
Alg-1) and the variant of Algorithm 1 with the fast approximate local search steps
introduced in Sect. 4 (denoted by Fast-Alg-1). We run Alg-1 and Fast-Alg-1
by setting R = r. For both AltMin and StoEM, we use the implementation provided in the repository7
accompanying paper [2]. We compare these methods with Algorithm 1 (denoted by
Alg-1) and the variant of Algorithm 1 with the fast approximate local search steps
introduced in Sect. 4 (denoted by Fast-Alg-1). We run Alg-1 and Fast-Alg-1
by setting R = r. For both AltMin and StoEM, we use the implementation provided in the repository
accompanying paper [2]. We compare these methods with Algorithm 1 (denoted b Table 1 presents the beta errors of different methods on an example with n = 500,
d = 10; and P∗chosen by the random scheme. The presented values are the average
of 10 independent replications. As shown in Table 1, in the noiseless setting, Alg-1 can recover the true value of
β∗for r up to 300. Fast-Alg-1 is quite similar to Alg-1, though the performance
is marginally worse for larger values of r. 5.3 Comparisons with existing methods In contrast, AltMin and StoEM are not
able to exactly estimate β∗even for small values of r, and for all values of r they 123 Linear regression with partially mismatched data… Table 1 Comparison with existing methods on an example with n = 500, d = 10
σ
r
beta error
Alg-1
Fast-Alg-1
AltMin
StoEM
S-BD
0
50
0.000
0.000
0.001
0.154
0.000
100
0.000
0.000
0.027
0.483
0.000
200
0.000
0.001
0.121
0.884
0.000
300
0.000
0.001
0.190
0.944
0.123
0.1
50
0.018
0.018
0.061
0.207
0.021
100
0.021
0.021
0.080
0.421
0.029
200
0.019
0.022
0.116
0.881
0.063
300
0.047
0.052
0.213
0.942
0.282 Table 1 Comparison with existing methods on an example with n = 500, d = 10 have large values of beta error. S-BD which is based on convex optimization, can also
recover β∗for r ≤200, but for r = 300 its performance degrades and has a large beta
error. In the noisy setting, with σ = 0.1, Alg-1 and Fast-Alg-1 have similar perfor-
mance, and compute a value of β with much smaller beta error compared to AltMin
and StoEM. For small values of r (≤100), S-BD has a similar performance to Alg-1
and Fast-Alg-1, while for r = 200 and 300, its performance degrades and has a
much larger beta error than Alg-1 and Fast-Alg-1. 5.4 Scalability to large instances Interestingly, it can be seen that the runtimes for the noisy case (σ = 0.1)
are smaller than the noiseless case (σ = 0). We believe this is because Algorithm 2 is
faster for the noisy case. In particular, for the noisy case, we empirically observe the
number of “left-top" points and “right-bottom" points to be fewer than those in the
noiseless case. and/or R). Interestingly, it can be seen that the runtimes for the noisy case (σ = 0.1)
are smaller than the noiseless case (σ = 0). We believe this is because Algorithm 2 is
faster for the noisy case. In particular, for the noisy case, we empirically observe the
number of “left-top" points and “right-bottom" points to be fewer than those in the
noiseless case. and/or R). Interestingly, it can be seen that the runtimes for the noisy case (σ = 0.1)
are smaller than the noiseless case (σ = 0). We believe this is because Algorithm 2 is
faster for the noisy case. In particular, for the noisy case, we empirically observe the
number of “left-top" points and “right-bottom" points to be fewer than those in the
noiseless case. Acknowledgements The authors would like to thank the anonymous referees for their constructive com-
ments that led to an improvement of the paper. Funding Open Access funding provided by the MIT Libraries Funding Open Access funding provided by the MIT Libraries OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,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/. 5.4 Scalability to large instances We explore the scalability of our proposed approach to large n problems (from n ≈104
up to n ≈107)—for these instances, Fast-Alg-1 appears to be computationally
attractive. All these experiments are run on the MIT engaging cluster with 1 CPU and
16GB memory. The codes are written in Julia 1.2.0. We consider examples with d = 100, r = 50 and n ∈{104, 105, 106, 107}. Here,
the mismatched coordinates of P∗are chosen based on the random scheme. We set
R = r for all instances. For these examples, we do not form the n × n matrices
H or H explicitly, but compute a thin QR decomposition of X ( Q ∈Rn×d) and
maintain Q in memory. The results are presented in Table 2, where “total time" is the
total runtime of Fast-Alg-1 upon termination, “QR time" is the time used for the
QR-decomposition, and “iterations" are the number of iterations taken by the local
search method till convergence. All numbers reported in the table are averaged across
10 independent replications. As shown in Table 2, Fast-Alg-1 can solve examples
with n up to 107 (and d = 100) within around 200 seconds — this runtime (s) includes
the time to complete around 60 iterations of local search steps and the time to do the
QR decomposition. The total runtime is empirically seen to be of the order O(n)
as n increases. Note that the QR time (i.e., time to perform the QR decomposition)
can be viewed as a benchmark runtime for ordinary least squares. Hence, for the
examples considered, the runtime of Fast-Alg-1 appears to be a constant multiple
of the runtime of performing ordinary least squares (the total time will increase with r 123 R. Mazumder, H. Wang Table 2 Runtimes of Fast-Alg-1 on instances with d = 100, r = 50 and different n. For reference, the
time taken by Alg-1 in the case n = 104 is 100 (s), which is 500X slower than Fast-Alg-1 y
g
0
00 ( ), w
500
w
ast
g
n
σ = 0
σ = 0.1
Total time(s)
QR time(s)
Iterations
total time(s)
QR time(s)
Iterations
104
0.2
0.1
57.8
0.2
0.1
58.3
105
2.2
0.7
58.9
1.5
0.7
58.8
106
21.3
6.3
58.7
13.9
6.4
56.4
107
212.6
64.5
58.5
152.9
64.8
60.7 and/or R). A.1 Technical lemmas Lemma 5 (Covariance estimation) Let X = [x1, . . . , xn]⊤be a random matrix in
Rn×d. Suppose rows x1, . . . , xn are iid zero-mean random vectors in Rd with covari-
ance matrix Σ ∈Rd×d. Suppose ∥xi∥≤b almost surely. Then for any t > 0, it
holds P
1
n X⊤X −Σ2 ≥t Σ2
≤2d exp
−nt2 Σ2
2b2(1 + t)
. See e.g. Corollary 6.20 of [25] for a proof. See e.g. Corollary 6.20 of [25] for a proof. Lemma 6 Suppose three permutation matrices P, P, Q ∈Πn satisfy Lemma 6 Suppose three permutation matrices P, P, Q ∈Πn satisfy supp(P Q−1) ⊆supp(P Q−1) and supp( Q) ⊆supp(P), 123 123 12 Linear regression with partially mismatched data… then it holds supp(P) ⊆supp(P). then it holds supp(P) ⊆supp(P). then it holds supp(P) ⊆supp(P). Proof We just need to show that for any i /∈supp(P), it holds i /∈supp(P). Let i /∈
supp(P), then e⊤
i P = e⊤
i . Since supp( Q) ⊆supp(P), we also have e⊤
i Q = e⊤
i . So
it holds e⊤
i P Q−1 = e⊤
i or equivalently i /∈supp(P Q−1). Because supp(P Q−1) ⊆
supp(P Q−1), we have i /∈supp(P Q−1), or equivalently e⊤
i P Q−1 = e⊤
i . This
implies e⊤
i P = e⊤
i Q = e⊤
i , or equivalently, i /∈supp(P). ⊓⊔ Lemma 7 Suppose Assumption 1 holds. Let P, P ∈NR(In) ⊆Πn with dist(P, P) ≤
4. Let Δ(P, P):=∥P y−P∗y∥2−∥P y −P∗y∥2,
ΔH(P, P):=∥HP y∥2 −∥H P y∥2,
then it holds |Δ(P, P) −ΔH(P, P)| < L2/5. Proof Let z := P y −P y. Note that Proof Let z := P y −P y. Note that et z := P y −P y. Note that Δ(P, P) = ∥z + P y −P∗y∥2 −∥P y −P∗y∥2
= ∥z∥2 + 2⟨z, P y −P∗y⟩
(A.1) (A.1) and ΔH(P, P) = ∥Hz + H P y∥2 −∥H P y∥2
= ∥Hz∥2 + 2⟨Hz, H P y⟩
= ∥Hz∥2 + 2⟨Hz, H(P y −P∗y)⟩+ 2⟨Hz, H P∗y⟩
= ∥Hz∥2 + 2⟨Hz, H(P y −P∗y)⟩+ 2⟨Hz, Hϵ⟩,
(A.2) ΔH(P, P) = ∥Hz + H P y∥2 −∥H P y∥2
= ∥Hz∥2 + 2⟨Hz, H P y⟩
= ∥Hz∥2 + 2⟨Hz, H(P y −P∗y)⟩+ 2⟨Hz, H P∗y⟩
= ∥Hz∥2 + 2⟨Hz, H(P y −P∗y)⟩+ 2⟨Hz, Hϵ⟩,
(A.2) (A.2) where the last equality uses the fact H P∗y = H(Xβ∗+ ϵ) = Hϵ. From (A.1) and
(A.2) we have where the last equality uses the fact H P∗y = H(Xβ∗+ ϵ) = Hϵ. From (A.1) and
(A.2) we have Δ(P, P) −ΔH(P, P) = ∥Hz∥2 + 2⟨Hz, H(P y −P∗y)⟩−2⟨Hz, Hϵ⟩
= ∥Hz∥2 + 2⟨Hz, H(P y −P∗y)⟩+ 2⟨Hz, Hϵ⟩−2⟨z, ϵ⟩, where the second equality is because H = In −H. As a result, |Δ(P, P) −ΔH(P, P)|
≤∥Hz∥2 + 2∥Hz∥∥H(P y −P∗y)∥+ 2∥Hz∥∥Hϵ∥+ 2|⟨z, ϵ⟩|. (A.3) Δ(P, P) −ΔH(P, P)|
≤∥Hz∥2 + 2∥Hz∥∥H(P y −P∗y)∥+ 2∥Hz∥∥Hϵ∥+ 2|⟨z, ϵ⟩|. then it holds supp(P) ⊆supp(P). (A.3 (A.3) Since dist(P, P) ≤4, we have z = P y −P y ∈B4, hence by Assumption 1 (3) and
(1) we have Since dist(P, P) ≤4, we have z = P y −P y ∈B4, hence by Assumption 1 (3) and
(1) we have ∥Hz∥2 ≤ρn∥z∥2 ≤4ρnU 2 . (A.4) (A.4) Since dist(P, P∗) ≤dist(P, In) + dist(P∗, In) ≤R + r, we have P y −P∗y ∈
BR+r ⊆B2R, hence by Assumption 1 (3) and (1), Since dist(P, P∗) ≤dist(P, In) + dist(P∗, In) ≤R + r, we have P y −P∗y ∈
BR+r ⊆B2R, hence by Assumption 1 (3) and (1), ∥H(P y −P∗y)∥≤
Rρn∥P y −P∗y∥≤√ρn(R + r)U ≤2√ρn RU . (A.5) 123 R. Mazumder, H. Wang gain because z ∈B4 we have |⟨z, ϵ⟩| ≤4U∥ϵ∥∞. (A.6) (A.6) Combining (A.3) – (A.6) we have ombining (A.3) – (A.6) we have Combining (A.3) – (A.6) we have |Δ(P, P) −ΔH(P, P)| ≤4ρnU2 + 4√ρnU · 2√ρn RU + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞
= (4 + 8R)ρnU2 + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞
< 9RρnU2 + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞, |Δ(P, P) −ΔH(P, P)| ≤4ρnU2 + 4√ρnU · 2√ρn RU + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞
= (4 + 8R)ρnU2 + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞
< 9RρnU2 + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞, |Δ(P, P) −ΔH(P, P)| ≤4ρnU2 + 4√ρnU · 2√ρn RU + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞
= (4 + 8R)ρnU2 + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞
< 9RρnU2 + 4√ρnU∥Hϵ∥+ 8U∥ϵ∥∞, where in the last inequality we use R > 4. By Assumption 1 (4) we know ∥ϵ∥∞≤¯σ
and ∥Hϵ∥≤
√
d ¯σ, so we have |Δ(P, P) −ΔH(P, P)| < 9ρn RU 2 + 4
dρnU ¯σ + 8U ¯σ . (A.7) (A.7) |Δ(P, P) −ΔH(P, P)| < 9ρn RU 2 + 4
dρnU ¯σ + 8U ¯σ . (A.7)
From Assumption 1 (4) we have ¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U). This implies From Assumption 1 (4) we have ¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U). This implies From Assumption 1 (4) we have ¯σ ≤min{0.5, (ρnd)−1/2}L2/(80U). This implies 4
dρnU ¯σ + 8U ¯σ ≤L2/20 + L2/20 ≤L2/10 . (A.8) (A.8) Note that by Assumption 1 (3) we have Rρn
≤L2/(90U 2), or equivalently,
9RρnU 2 ≤L2/10. Combining this with (A.7) and (A.8), we have |Δ(P, P) −ΔH(P, P)| < L2/10 + L2/10 = L2/5 . ⊓⊔ Lemma 8 (Decrease in infinity norm) Let P, P ∈Πn with dist(P, P) = 2. then it holds supp(P) ⊆supp(P). For any
v ∈Rn, if ∥Pv −v∥2 < ∥Pv −v∥2, then it holds ∥Pv −v∥∞≤∥Pv −v∥∞. Proof Let i ∈[n] be the index such that |e⊤
i (Pv−v)| = ∥Pv−v∥∞. If e⊤
i P = e⊤
i P,
then immediately we have Lemma 8 (Decrease in infinity norm) Let P, P ∈Πn with dist(P, P) = 2. For any
v ∈Rn, if ∥Pv −v∥2 < ∥Pv −v∥2, then it holds ∥Pv −v∥∞≤∥Pv −v∥∞. Proof Let i ∈[n] be the index such that |e⊤
i (Pv−v)| = ∥Pv−v∥∞. If e⊤
i P = e⊤
i P,
then immediately we have ∥Pv −v∥∞≥|e⊤
i (Pv −v)| = |e⊤
i (Pv −v)| = ∥Pv −v∥∞. If e⊤
i P ̸= e⊤
i P, assume ℓ∈[n] is the index such that e⊤
i P = e⊤
ℓP. Since
dist(P, P) = 2, it holds e⊤
ℓP = e⊤
i P. Denote i+ := πP(i) and ℓ+ := πP(ℓ). Because e⊤
i+ = e⊤
i P = e⊤
ℓP and e⊤
ℓ+ = e⊤
ℓP = e⊤
i P, it holds i+ = πP(ℓ) and
ℓ+ = πP(i). As a result, we have ∥Pv −v∥2 −∥Pv −v∥2 = (vℓ−vi+)2 + (vi −vℓ+)2 −(vi −vi+)2 −(vℓ−vℓ+)2 . By the assumption that ∥Pv −v∥2 < ∥Pv −v∥2, we have By the assumption that ∥Pv −v∥2 < ∥Pv −v∥2, we have By the assumption that ∥Pv −v∥2 < ∥Pv −v∥2, we have (vℓ−vi+)2 + (vi −vℓ+)2 −(vi −vi+)2 −(vℓ−vℓ+)2 < 0 . (A.9) (A.9) Let us denote L := ∥Pv −v∥∞= |e⊤
i (Pv −v)| = |vi −vℓ+|. In what follows, we
discuss different cases depending upon the ordering of the values of vi, vi+, vℓand 123 Linear regression with partially mismatched data… vℓ+. Without loss of generality, we can assume vi+ ≥vi. Then there are 12 cases of
the ordering in vi, vi+, vℓand vℓ+. In the following, the first 6 cases correspond to
when vℓ+ ≥vℓ, and the last 6 cases correspond to when vℓ+ ≤vℓ. vℓ+. Without loss of generality, we can assume vi+ ≥vi. Then there are 12 cases of
the ordering in vi, vi+, vℓand vℓ+. In the following, the first 6 cases correspond to
when vℓ+ ≥vℓ, and the last 6 cases correspond to when vℓ+ ≤vℓ. +
p
+
Case1:vi+ ≥vℓ+ ≥vi ≥vℓ.Thenwehave∥Pv−v∥∞≥|vi−vi+| ≥|vi−vℓ+| = L. Case 2: vi+ ≥vi ≥vℓ+ ≥vℓ. This is a contradiction to (A.9). So this case cannot appear. then it holds supp(P) ⊆supp(P). Let a = vi+ −vi, b = vi −vℓ+ and c = vℓ+ −vℓ. Then (vi −vℓ+)2 + (vℓ−vi+)2 −(vi −vi+)2 −(vℓ−vℓ+)2
= b2 + (a + b + c)2 −a2 −c2 ≥2b2 ≥2L2 > 0 . This is a contradiction to (A.9). So this case cannot appear. This is a contradiction to (A.9). So this case cannot appear. This is a contradiction to (A.9). So this case cannot appear. Case3:vi+ ≥vℓ+ ≥vℓ≥vi.Thenwehave∥Pv−v∥∞≥|vi−vi+| ≥|vi−vℓ+| = L. Case 4: vℓ+ ≥vi+ ≥vℓ≥vi. Let a = vℓ+ −vi+, b = vi+ −vℓand c = vℓ−vi. Then Case3:vi+ ≥vℓ+ ≥vℓ≥vi.Thenwehave∥Pv−v∥∞≥|vi−vi+| ≥|vi−vℓ+| = L. Case 4: vℓ+ ≥vi+ ≥vℓ≥vi. Let a = vℓ+ −vi+, b = vi+ −vℓand c = vℓ−vi. Then (vi −vℓ+)2 + (vℓ−vi+)2 −(vi −vi+)2 −(vℓ−vℓ+)2
= (a + b + c)2 + b2 −(b + c)2 −(a + b)2 = 2ac ≥0 . This is a contradiction to (A.9). So this case cannot appear. This is a contradiction to (A.9). So this case cannot appear. This is a contradiction to (A.9). So this case cannot appear. Case 5: vℓ+ ≥vℓ≥vi+ ≥vi. Let a = vℓ+ −vℓ, b = vℓ−vi+ and c = vi+ −vi. Then (vi −vℓ+)2 + (vℓ−vi+)2 −(vi −vi+)2 −(vℓ−vℓ+)2
= (a + b + c)2 + b2 −a2 −c2 ≥0 . This is a contradiction to (A.9). So this case cannot appear. This is a contradiction to (A.9). So this case cannot appear. Case 6: vℓ+ ≥vi+ ≥vi ≥vℓ. Then ∥Pv −v∥∞≥|vℓ−vℓ+| ≥|vi −vℓ+| = L. Case 7: vi+ ≥vℓ≥vi ≥vℓ+. Then ∥Pv −v∥∞≥|vℓ−vℓ+| ≥|vi −vℓ+| = L. Case 8: vi+ ≥vi ≥vℓ≥vℓ+. Let a = vi+ −vi, b = vi −vℓ, c = vℓ−vℓ+. Then (vi −vℓ+)2 + (vℓ−vi+)2 −(vi −vi+)2 −(vℓ−vℓ+)2
= (b + c)2 + (a + b)2 −a2 −c2 ≥0 . This is a contradiction to (A.9). So this case cannot appear. This is a contradiction to (A.9). So this case cannot appear. Case 9: vi+ ≥vℓ≥vℓ+ ≥vi. Then ∥Pv −v∥∞≥|vi −vi+| ≥|vi −vℓ+| = L. Case 10: vℓ≥vi+ ≥vℓ+ ≥vi. Then ∥Pv −v∥∞≥|vi −vi+| ≥|vi −vℓ+| = L. Case 11: vℓ≥vℓ+ ≥vi+ ≥vi. Let a = vℓ−vℓ+, b = vℓ+ −vi+ and vi+ −vi. Then Case 9: vi+ ≥vℓ≥vℓ+ ≥vi. Then ∥Pv −v∥∞≥|vi −vi+| ≥|vi −vℓ+| = L. Case 10: vℓ≥vi+ ≥vℓ+ ≥vi. Then ∥Pv −v∥∞≥|vi −vi+| ≥|vi −vℓ+| = L. Case 11: vℓ≥vℓ+ ≥vi+ ≥vi. A.2 Proof of lemma 1 Without loss of generality we assume P∗= In, (otherwise, we work with P(P∗)−1
in place of P and P(P∗)−1 in place of P). For any k ∈[n], let k+ := πP(k). Let i
be the index such that (yi+ −yi)2 = ∥P y −y∥2
∞. With out loss of generality, we can
assume yi+ > yi. Denote i0 = i and i1 = i+. By the structure of a permutation, there
exists a cycle that i0
P
−→i1
P
−→· · ·
P
−→it
P
−→· · ·
P
−→iS = i0
(A.10) (A.10) where q1
P
−→q2 means q2 = πP(q1). By moving from yi to yi+, we “upcross”
the value
yi+yi+
2
. Since the cycle (A.10) finally returns to i0, there exists one step
where we “downcross” the value
yi+yi+
2
. In other words, there exists j ∈[n] with
( j, j+) ̸= (i, i+) such that y j+ < y j and
yi+yi+
2
∈[y j+, y j]. Define P as follows: πP(i) = j+,
πP( j) = i+,
πP(k) = πP(k) ∀k ̸= i, j . We note that dist(P, P) = 2 and supp(P) ⊆supp(P). Since yi+ −yi = ∥P y −y∥∞≥y j −y j+ there are 3 cases depending upon the relative ordering yi, yi+, y j, y j+, as considered
below. there are 3 cases depending upon the relative ordering yi, yi+, y j, y j+, as considered
below. Case 1: y j ≥yi+ ≥y j+ ≥yi. In this case, let a = y j −yi+, b = yi+ −y j+ and there are 3 cases depending upon the relative ordering yi, yi+, y j, y j+, as considered
below. Case 1: y j ≥yi+ ≥y j+ ≥yi. In this case, let a = y j −yi+, b = yi+ −y j+ and
c = y j+ −yi. Then a, b, c ≥0, and below. Case 1: y j ≥yi+ ≥y j+ ≥yi. In this case, let a = y j −yi+, b = yi+ −y j+ and
c = y j+ −yi. Then a, b, c ≥0, and Case 1: y j ≥yi+ ≥y j+ ≥yi. In this case, let a = y j −yi+, b = yi+ −y j+ and
c = y j+ −yi. then it holds supp(P) ⊆supp(P). Let a = vℓ−vℓ+, b = vℓ+ −vi+ and vi+ −vi. Then (vi −vℓ+)2 + (vℓ−vi+)2 −(vi −vi+)2 −(vℓ−vℓ+)2
= (b + c)2 + (a + b)2 −a2 −c2 ≥0 . This is a contradiction to (A.9). So this case cannot appear. This is a contradiction to (A.9). So this case cannot appear. (
)
pp
Case 12: vℓ≥vi+ ≥vi ≥vℓ+. Then ∥Pv −v∥∞≥|vℓ−vℓ+| ≥|vi −vℓ+| = L. In view of all these cases, we have ∥Pv −v∥∞≥L = ∥Pv −v∥∞. ⊓⊔ Case 12: vℓ≥vi+ ≥vi ≥vℓ+. Then ∥Pv −v∥∞≥|vℓ−vℓ+| ≥|vi −vℓ+| = L. In view of all these cases, we have ∥Pv −v∥∞≥L = ∥Pv −v∥∞. ⊓⊔ Case 12: vℓ≥vi+ ≥vi ≥vℓ+. Then ∥Pv −v∥∞≥|vℓ−vℓ+| ≥|vi −vℓ+| = L. In view of all these cases, we have ∥Pv −v∥∞≥L = ∥Pv −v∥∞. ⊓⊔ ⊓⊔ 123 R. Mazumder, H. Wang A.2 Proof of lemma 1 Then a, b, c ≥0, and ∥P y −y∥2 −∥P y −y∥2
= (yi −yi+)2 + (y j −y j+)2 −(yi −y j+)2 −(y j −yi+)2
= (b + c)2 + (a + b)2 −c2 −a2
= 2b2 + 2ab + 2bc . Since
yi+yi+
2
∈[y j+, y j], we have b = yi+ −y j+ ≥yi+ −
yi+yi+
2
=
yi+−yi
2
, and hence
∥P y −y∥2 −∥P y −y∥2 ≥2b2 ≥(yi+ −yi)2/2 = (1/2)∥P y −y∥2
∞. Case 2: yi+ ≥y j ≥yi ≥y j+. In this case, let a = yi+ −y j, b = y j −yi and
c = yi −y j+. Then a, b, c ≥0, and Case 2: yi+ ≥y j ≥yi ≥y j+. In this case, let a = yi+ −y j, b = y j −yi and
c = yi −y j+. Then a, b, c ≥0, and ∥P y −y∥2 −∥P y −y∥2
= (yi −yi+)2 + (y j −y j+)2 −(yi −y j+)2 −(y j −yi+)2
= (a + b)2 + (b + c)2 −c2 −a2
= 2b2 + 2ab + 2bc . = 2b2 + 2ab + 2bc . 123 123 Linear regression with partially mismatched data… Since
yi+yi+
2
∈[y j+, y j], we have b = y j −yi ≥
yi+yi+
2
−yi =
yi+−yi
2
, and hence
∥P y −y∥2 −∥P y −y∥2 ≥2b2 ≥(yi+ −yi)2/2 = (1/2)∥P y −y∥2
∞. Case 3: yi+ ≥y j ≥y j+ ≥yi. In this case, let a = yi+ −y j, b = y j −y j+ and
c = y j+ −yi. Then a, b, c ≥0, and ∥P y −y∥2 −∥P y −y∥2
= (yi −yi+)2 + (y j −y j+)2 −(yi −y j+)2 −(y j −yi+)2
= (a + b + c)2 + b2 −c2 −a2
= 2b2 + 2ab + 2bc + 2ac . = 2b2 + 2ab + 2bc + 2ac . Note that ∥P y −y∥2
∞= (yi −yi+)2 = (a + b + c)2. Because
yi+yi+
2
∈[y j+, y j], we
have Note that ∥P y −y∥2
∞= (yi −yi+)2 = (a + b + c)2. A.2 Proof of lemma 1 Because
yi+yi+
2
∈[y j+, y j], we
have a = yi+ −y j ≤y j −yi = b + c,
and c = y j+ −yi ≤yi+ −y j+ = a + b , a = yi+ −y j ≤y j −yi = b + c,
and c = y j+ −yi ≤yi+ −y j+ = a + b , a = yi+ −y j ≤y j −yi = b + c,
and c = y j+ −yi ≤yi+ −y j+ = a
which implies a ≤(a + b + c)/2 and c ≤(a + b + c)/2. So we have
∥P y −y∥2 −∥P y −y∥2 ≥w∥P y −y∥2
∞ which implies a ≤(a + b + c)/2 and c ≤(a + b + c)/2. So we have ∥P y −y∥2 −∥P y −y∥2 ≥w∥P y −y∥2
∞ where w := min
2b2 + 2ab + 2bc + 2ac
(a + b + c)2
: a, b, c ≥0; a, c ≤(a + b + c)/2
. This is equivalent to This is equivalent to w = min
2b2 + 2ab + 2bc + 2ac : a, b, c ≥0; a, c ≤1/2; a + b + c = 1
= min
2b + 2ac : a, b, c ≥0; a, c ≤1/2; a + b + c = 1
= min
2(1 −a −c) + 2ac : a, c ≥0; a, c ≤1/2
= min
2(1 −a)(1 −c) : a, c ≥0; a, c ≤1/2
= 1/2 . Combining Cases 1, 2 and 3, completes the proof of (3.5). 2 Combining Cases 1, 2 and 3, completes the proof of (3.5). 2 For the proof of (3.6), note that if ∥P y −P∗y∥0 ≤m, then ∥P y −P∗y∥2 ≤
m∥P y −P∗y∥2
∞. Using (3.5) we have: For the proof of (3.6), note that if ∥P y −P∗y∥0 ≤m, then ∥P y −P∗y∥2 ≤
m∥P y −P∗y∥2
∞. Using (3.5) we have: ∥P y −P∗y∥2 −∥P y −P∗y∥2 ≥(1/2)∥P y −P∗y∥2
∞≥(1/(2m))∥P y −P∗y∥ which completes the proof of (3.6). 123 R. Mazumder, H. Wang A.3 Proof of lemma 2 3 Proof of lemma 2 To prove Lemma 2, we first prove the following proposition: Proposition 2 Under the assumptions of Lemma 2, it holds ∥P(t) y −P∗y∥∞≥L for
all 0 ≤t ≤k. Proof As ∥P(k) y −P∗y∥∞≥L, let i ∈[n] be the index such that |e⊤
i (P(k) y −
P∗y)| ≥L. We can assume that there exists j ≤k −1 such that e⊤
i P( j) ̸= e⊤
i P(k) but
e⊤
i P(t) = e⊤
i P(k) ∀j + 1 ≤t ≤k . (A.11)
since otherwise e⊤
i P(t) = e⊤
i P(k) for all 0 ≤t ≤k and hence ∥P(t) y −P∗y∥∞≥
|e⊤
i (P(t) y −P∗y)| ≥L for all 0 ≤t ≤k, i.e., the conclusion of Proposition 2 holds
true. e⊤
i P( j) ̸= e⊤
i P(k) but
e⊤
i P(t) = e⊤
i P(k) ∀j + 1 ≤t ≤k . (A.11)
since otherwise e⊤
i P(t) = e⊤
i P(k) for all 0 ≤t ≤k and hence ∥P(t) y −P∗y∥∞≥
|e⊤
i (P(t) y −P∗y)| ≥L for all 0 ≤t ≤k, i.e., the conclusion of Proposition 2 holds
true. e⊤
i P( j) ̸= e⊤
i P(k) but
e⊤
i P(t) = e⊤
i P(k) ∀j + 1 ≤t ≤k . (A.11) e⊤
i P( j) ̸= e⊤
i P(k) but
e⊤
i P(t) = e⊤
i P(k) ∀j + 1 ≤t ≤k . (A.11)
since otherwise e⊤
i P(t) = e⊤
i P(k) for all 0 ≤t ≤k and hence ∥P(t) y −P∗y∥∞≥
|e⊤
i (P(t) y −P∗y)| ≥L for all 0 ≤t ≤k, i.e., the conclusion of Proposition 2 holds
true. (A.11) Below we prove Proposition 2 under the assumption (A.11). By (A.11) we know
that e⊤
i P( j) ̸= e⊤
i P( j+1). For any t ≥0 let Q(t) := P(t)(P∗)−1. Then we have e⊤
i Q( j+1) = e⊤
i P( j+1)(P∗)−1 ̸= e⊤
i P( j)(P∗)−1 = e⊤
i Q( j) . A.3 Proof of lemma 2 Since dist( Q( j), Q( j+1)) = dist(P( j), P( j+1)) = 2, there must exist an index ℓ∈[n]
such that Since dist( Q( j), Q( j+1)) = dist(P( j), P( j+1)) = 2, there must exist an index ℓ∈[n]
such that Since dist( Q( j), Q( j+1)) = dist(P( j), P( j+1)) = 2, there must exist an index ℓ∈[n]
such that π Q( j+1)(i) = π Q( j)(ℓ), π Q( j+1)(ℓ) = π Q( j)(i), π Q( j+1)(w) = π Q( j)(w) ∀w ̸= i, ℓ. In the following, denote i+ := π Q( j)(i) and ℓ+ := π Q( j)(ℓ) and let y∗:= P∗y. Then
we have π Q( j+1)(i) = π Q( j)(ℓ), π Q( j+1)(ℓ) = π Q( j)(i), π Q( j+1)(w) = π Q( j)(w) ∀w ̸= i, ℓ. In the following, denote i+ := π Q( j)(i) and ℓ+ := π Q( j)(ℓ) and let y∗:= P∗y. Then
we have ∥P( j+1) y −P∗y∥2 −∥P( j) y −P∗y∥2
= ∥( Q( j+1) −In)y∗∥2 −∥( Q( j) −In)y∗∥2
= (y∗
i −y∗
ℓ+)2 + (y∗
ℓ−y∗
i+)2 −(y∗
i −y∗
i+)2 −(y∗
ℓ−y∗
ℓ+)2 . (A.12)
Since ℓ+ = π Q( j)(ℓ) = π Q( j+1)(i), we have
(y∗
i −y∗
ℓ+)2 = (e⊤
i (P∗y −Q( j+1) P∗y))2 = (e⊤
i (P∗y −P( j+1) y))2 . (A.13) ∥P( j+1) y −P∗y∥2 −∥P( j) y −P∗y∥2
= ∥( Q( j+1) −In)y∗∥2 −∥( Q( j) −In)y∗∥2
= (y∗
i −y∗
ℓ+)2 + (y∗
ℓ−y∗
i+)2 −(y∗
i −y∗
i+)2 −(y∗
ℓ−y∗
ℓ+)2 . (A.12) Since ℓ+ = π Q( j)(ℓ) = π Q( j+1)(i), we have (y∗
i −y∗
ℓ+)2 = (e⊤
i (P∗y −Q( j+1) P∗y))2 = (e⊤
i (P∗y −P( j+1) y))2 . (A.13 By the definition of j and equality (A.13), we have (y∗
i −y∗
ℓ+)2 = (e⊤
i (P∗y −P(k) y))2 ≥L2 . (A.14) (A.14) Since ∥H P( j+1) y∥≤∥H P( j) y∥, using Lemma 7 with P = P( j+1) and P =
P( j), we have ∥
P( j), we have ∥P( j+1) y −P∗y∥2 −∥P( j) y −P∗y∥2
< ∥H P( j+1) y∥2 −∥H P( j) y∥2 + L2/5 ≤L2/5. A.3 Proof of lemma 2 123 Linear regression with partially mismatched data… Linear regression with partially mismatched data… This leads to This leads to This leads to (y∗
i −y∗
ℓ+)2 + (y∗
ℓ−y∗
i+)2 −(y∗
i −y∗
i+)2 −(y∗
ℓ−y∗
ℓ+)2 < L2/5 which when combined with (A.14) leads to: (y∗
i −y∗
i+)2 + (y∗
ℓ−y∗
ℓ+)2 > (y∗
i −y∗
ℓ+)2 + (y∗
ℓ−y∗
i+)2 −(1/5)L2 ≥(4/5)L2. As a result, we have As a result, we have ∥P( j) y −P∗y∥2
∞≥max{(y∗
i −y∗
i+)2, (y∗
ℓ−y∗
ℓ+)2} > (2/5)L2
(A.15)
y Lemma 1, there exists P( j)∈Πn such that dist(P( j), P( j)) ≤2, supp(P( j)(P∗)−1)
supp(P( j)(P∗)−1) and ∥P( j) y −P∗y∥2
∞≥max{(y∗
i −y∗
i+)2, (y∗
ℓ−y∗
ℓ+)2} > (2/5)L2
(A.15) (A.15) By Lemma 1, there exists P( j)∈Πn such that dist(P( j), P( j)) ≤2, supp(P( j)(P∗)−1)
⊆supp(P( j)(P∗)−1) and By Lemma 1, there exists P( j)∈Πn such that dist(P( j), P( j)) ≤2, supp(P( j)(P∗)−1)
⊆supp(P( j)(P∗)−1) and ∥P( j) y −P∗y∥2 −∥P( j) y −P∗y∥2 ≤−(1/2)∥P( j) y −P∗y∥2
∞. (A.16) We now make use of the following claim: We now make use of the following claim: Claim. P( j) ∈NR(In). (A.17) Claim. For
each t ≤k −1, By Lemma 1, there exists P(t) ∈Πn such that dist(P(t), P(t)) ≤2,
supp(P(t)(P∗)−1) ⊆supp(P(t)(P∗)−1) and ∥P(t) y −P∗y∥2 −∥P(t) y −P∗y∥2 ≤−(1/2)∥P(t) y −P∗y∥2
∞≤−L2/2
(A ∥P(t) y −P∗y∥2 −∥P(t) y −P∗y∥2 ≤−(1/2)∥P(t) y −P∗y∥2
∞≤−L2/2
(A.19) where the second inequality is by Proposition 2. With the same arguments as in the
proof of Claim (A.17), we have P(t) ∈NR(In), hence ∥H P(t+1) y∥2 ≤∥HP(t) y∥2. Using Lemma 7 again with P = P(t) and P = P(t+1), we have where the second inequality is by Proposition 2. With the same arguments as in the
proof of Claim (A.17), we have P(t) ∈NR(In), hence ∥H P(t+1) y∥2 ≤∥HP(t) y∥2. Using Lemma 7 again with P = P(t) and P = P(t+1), we have ∥P(t+1) y −P∗y∥2 −∥P(t) y −P∗y∥2
≤∥H P(t+1) y∥2 −∥HP(t) y∥2 + L2/5 ≤L2/5 . (A.20) (A.20) Combining (A.19) and (A.20) we have Combining (A.19) and (A.20) we have ∥P(t+1) y −P∗y∥2 −∥P(t) y −P∗y∥2 ≤L2/5 −L2/2 < −L2/5 which completes the proof of the Lemma 2. which completes the proof of the Lemma 2. Claim. (A.17) Proof of Claim (A.17): Note that if j
≤
R/2 −1, then dist(In, P( j))
≤
dist(In, P( j)) + dist(P( j), P( j)) ≤R. Otherwise, from the statement of Lemma
2, we know that supp(P∗) ⊆supp(P( j)). Using Lemma 6 with P = P( j), Q = P∗
and P = P( j) we have supp(P( j)) ⊆supp(P( j)). Since P( j) ∈NR(In), we also
have supp(P( j)) ∈NR(In). The proof of Claim (A.17) is complete. ( j) Because P( j) ∈NR(In), by the update step in the local search algorithm, we know
∥H P( j+1) y∥≤∥HP( j) y∥. Using Lemma 7 again with P = P( j+1) and P = P( j),
we have ∥P( j+1) y −P∗y∥2 −∥P( j) y −P∗y∥2
≤∥H P( j+1) y∥2 −∥HP( j) y∥2 + L2/5 ≤L2/5
(A.18) (A.18) Combining (A.16), (A.18) and (A.15) we have Combining (A.16), (A.18) and (A.15) we have
∥P( j+1) y −P∗y∥2 −∥P( j) y −P∗y∥2 ≤−(1/2)∥P( j) y −P∗y∥2
∞+ L2/5 < 0 Combining (A.16), (A.18) and (A.15) we have
∥P( j+1) y −P∗y∥2 −∥P( j) y −P∗y∥2 ≤−(1/2)∥P( j) y −P∗y∥2
∞+ L2/5 < 0 ∥P( j+1) y −P∗y∥2 −∥P( j) y −P∗y∥2 ≤−(1/2)∥P( j) y −P∗y∥2
∞+ L2/5 < 0 which is equivalent to which is equivalent to ∥Q( j+1) y∗−y∗∥2 −∥Q( j) y∗−y∗∥2 < 0 . ∥Q( j+1) y∗−y∗∥2 −∥Q( j) y∗−y∗∥2 < 0 . We now use Lemma 8 with P = Q( j+1), P = Q( j) and v = y∗, to obtain ∥P( j) y −P∗y∥∞= ∥Q( j) y∗−y∗∥∞
≥∥Q( j+1) y∗−y∗∥∞= ∥P( j+1) y −P∗y∥∞≥L . ∥P( j) y −P∗y∥∞= ∥Q( j) y∗−y∗∥∞ ≥∥Q( j+1) y∗−y∗∥∞= ∥P( j+1) y −P∗y∥∞≥L . 123 R. Mazumder, H. Wang By the arguments above we have proved that ∥P( j) y −P∗y∥∞≥L. Recall that
we also have ∥P(t) y −P∗y∥∞≥L for all j + 1 ≤t ≤k, so we know that
∥P(t) y −P∗y∥∞≥L for all j ≤t ≤k. We can just replace k by j and repeat the
arguments above to obtain ∥P(t) y −P∗y∥∞≥L for all 0 ≤t ≤k. This completes
the proof of Proposition 2. ⊓⊔ With Proposition 2 at hand, we are ready to wrap up the proof of Lemma 2. A.4 Proof of lemma 3 We will prove that for any u ∈Bm (cf definition (3.1)), ∥Hu∥2 = ∥X(X⊤X)−1X⊤u∥2 ≤δn,m∥u∥2 . (A.21) (A.21) Take tn :=
3b2 log(2d/τ)/(n Σ2). By assumption in the statement of Lemma
3 we have tn Σ2 ≤γ/2. From Lemma 5 and some simple algebra we have: (1/n)X⊤X −Σ2 ≤tn Σ2
(A.22) (A.22) with probability at least 1 −τ. By Weyl’s inequality, |λmin(X⊤X)/n −λmin(Σ)| is
bounded by the left hand side of (A.22). So we have λmin(X⊤X)/n ≥λmin(Σ) −tn Σ2 ≥γ −γ/2 = γ/2
where, we use tn Σ2 ≤γ/2. Hence we have λmax((X⊤X)−1) ≤2/(nγ ) and λmin(X⊤X)/n ≥λmin(Σ) −tn Σ2 ≥γ −γ/2 = γ/2
where, we use tn Σ2 ≤γ/2. Hence we have λmax((X⊤X)−1) ≤2/(nγ ) and λmin(X⊤X)/n ≥λmin(Σ) −tn Σ2 ≥γ −γ/2 = γ/2 where, we use tn Σ2 ≤γ/2. Hence we have λmax((X⊤X)−1) ≤2/(nγ ) and X(X⊤X)−12 =
λmax((X⊤X)−1) ≤
2/(nγ ) . (A.23) (A.23) 123 123 12 123 Linear regression with partially mismatched data… Let Bm(1) := {u ∈Bm : ∥u∥≤1}, and let u1, . . . , uM be an (
δn,m/2)-net of Bm(1),
that is, for any u ∈Bm(1), there exists some u j such that ∥u j −u∥≤
δn,m/2. Since
the (
δn,m/2)-covering number of Bm(1) is bounded by (6/
δn,m)mn
m
, we can take M ≤(6/
δn,m)m
n
m
≤(3n)mnm = (3n2)m where the second inequality is from our assumption that
δn,m ≥2/n. By Hoeffding
inequality, for each fixed j ∈[M], and for all k ∈[d], we have where the second inequality is from our assumption that
δn,m ≥2/n. By Hoeffding
inequality, for each fixed j ∈[M], and for all k ∈[d], we have P
1
√n
e⊤
k X⊤u j > t
≤2 exp
−
nt2
2∥u j∥2V 2
. A.4 Proof of lemma 3 Using a union bound for k ∈[d] to the inequality above, we have that for any ρ > 0,
with probability at least 1 −ρ, the following inequality holds for all k ∈[d] Using a union bound for k ∈[d] to the inequality above, we have that for any ρ > 0,
with probability at least 1 −ρ, the following inequality holds for all k ∈[d] e⊤
k X⊤u j /√n ≤
2 log(2d/ρ)/nV ∥u j∥≤V
2 log(2d/ρ)/n , where the second inequality is because each u j ∈Bm(1). As a result, where the second inequality is because each u j ∈Bm(1). As a result, 1
√n ∥X⊤u j∥=
d
k=1
|e⊤
k X⊤u j|/√n
2 1/2
≤V
2d log(2d/ρ)/n . Take ρ = τ/M, then by the union bound, with probability at least 1 −τ, it holds ∥X⊤u j∥/√n ≤V
2d log(2dM/τ)/n
∀j ∈[M] . (A.24) (A.24) Combining (A.24) with (A.23), we have that for all j ∈[M], Combining (A.24) with (A.23), we have that for all j ∈[M], ∥X(X⊤X)−1X⊤u j∥≤ X(X⊤X)−1 2 ·∥X⊤u j∥
≤2V
(d/nγ ) log(2dM/τ). (A.25) (A.25) Recall that M ≤(3n2)m, so we have Recall that M ≤(3n2)m, so we have Recall that M ≤(3n2)m, so we have 2V
(d/nγ ) log(2dM/τ) ≤2V
d
nγ log(2d/τ) + dm
nγ log(3n2)
1/2
=
δn,m
2 where the last equality follows the definition of δn,m. Using the above bound in (A.25),
we have where the last equality follows the definition of δn,m. Using the above bound in (A.25),
we have ∥X(X⊤X)−1X⊤u j∥≤
δn,m/2. ∥X(X⊤X)−1X⊤u j∥≤
δn,m/2. 123 R. Mazumder, H. Wang For any u ∈Bm(1), there exists some j ∈[M] such that ∥u−u j∥≤
δn,m/2, hence For any u ∈Bm(1), there exists some j ∈[M] such that ∥u−u j∥≤
δn,m/2, hence ∥X(X⊤X)−1X⊤u∥≤∥X(X⊤X)−1X⊤u j∥+ ∥X(X⊤X)−1X⊤(u −u j)∥
≤
δn,m/2 + ∥u −u j∥2 ≤
δn,m. (A.26) (A.26) (A.26) Since both (A.22) and (A.24) have failure probability of at most τ, we know that (A.26)
holds with probability at least 1 −2τ. This proves the conclusion for all u ∈Bm(1). For a general u ∈Bm, u/∥u∥∈Bm(1), hence we have Since both (A.22) and (A.24) have failure probability of at most τ, we know that (A.26)
holds with probability at least 1 −2τ. This proves the conclusion for all u ∈Bm(1). A.4 Proof of lemma 3 For a general u ∈Bm, u/∥u∥∈Bm(1), hence we have ∥Hu∥= ∥X(X⊤X)−1X⊤u∥≤
δn,m∥u∥ which is equivalent to what we had set out to prove (A.21). which is equivalent to what we had set out to prove (A.21). A.5 Proof of lemma 4 To prove (3.47), using Bernstein inequality we have √n
To prove (3.47), using Bernstein inequality we have √
To prove (3.47), using Bernstein inequality we have P(|∥ϵ∥2/n −σ 2| > tσ 2) ≤2 exp(−Cn(t2 ∧t)) for a universal constant C. Taking t =
log(4/τ)/(Cn) in the inequality above, and
note that t ≤1 (because of the assumption
log(4/τ)/(Cn) + 2d log(4d/τ)/n ≤
1/4), we have P(|∥ϵ∥2/n −σ 2| >
log(4/τ)/(Cn)σ 2) ≤2 exp(−Cnt2) = τ/2. (A.31) P(|∥ϵ∥2/n −σ 2| >
log(4/τ)/(Cn)σ 2) ≤2 exp(−Cnt2) = τ/2. (A.31)
As a result with probability at least 1
τ/2 P(|∥ϵ∥2/n −σ 2| >
log(4/τ)/(Cn)σ 2) ≤2 exp(−Cnt2) = τ/2. (A.31)
As a result, with probability at least 1 −τ/2
2
2
2
2 (A.31) As a result, with probability at least 1 −τ/2 As a result, with probability at least 1 −τ/2 ∥ϵ∥2 ≥nσ 2 −nσ 2
log(4/τ)/(Cn) ≥(3/4 −
log(4/τ)/(Cn) )nσ 2 ∥ϵ∥2 ≥nσ 2 −nσ 2
log(4/τ)/(Cn) ≥(3/4 −
log(4/τ)/(Cn) )nσ 2 where the second inequality makes use of the assumption that σ 2 ≥(3/4)σ 2. On
the other hand we know that with probability at least 1 −τ/2 it holds ∥Hϵ∥≤
σ
2d log(4d/τ). As a result, with probability at least 1 −τ we have where the second inequality makes use of the assumption that σ 2 ≥(3/4)σ 2. On
the other hand we know that with probability at least 1 −τ/2 it holds ∥Hϵ∥≤
σ
2d log(4d/τ). As a result, with probability at least 1 −τ we have ∥Hϵ∥2 = ∥ϵ∥2 −∥Hϵ∥2 ≥(3/4 −
log(4/τ)/(Cn) −2d log(4d/τ)/n)nσ 2 ≥nσ 2/2 , where the last inequality uses the assumption
log(4/τ)/(Cn) + 2d log(4d/τ)/n ≤
1/4. where the last inequality uses the assumption
log(4/τ)/(Cn) + 2d log(4d/τ)/n ≤
1/4. A.6 Proof of claim (3.22) To prove Claim (3.22), we just need to prove that P(k) ∈NR(In), i.e. dist(P(k), In) ≤
R. If k ≤R/2 −1, because dist(P(t+1), P(t)) ≤2 for all t ≥0 and P(0) = In, we
have dist(P(k), In) ≤2k ≤R −2. Hence To prove Claim (3.22), we just need to prove that P(k) ∈NR(In), i.e. dist(P(k), In) ≤
R. If k ≤R/2 −1, because dist(P(t+1), P(t)) ≤2 for all t ≥0 and P(0) = In, we
have dist(P(k), In) ≤2k ≤R −2. Hence dist(P(k), In) ≤dist(P(k), P(k)) + dist(P(k), In) ≤R . Otherwise, by Proposition 1, it holds supp(P∗) ⊆supp(P(k)). Then from Lemma 6,
we have supp(P(k)) ⊆supp(P(k)), therefore P(k) ∈NR(In). A.5 Proof of lemma 4 Since ϵi ∼subG(σ 2), by Chernoff inequality, for all t > 0, we have P(|ϵi| > t) ≤2 exp(−t2/(2σ 2)) . As a result, for all t > 0, P(∥ϵ∥∞> t) ≤n
i=1 P(|ϵi| > t) ≤2n exp(−t2/(2σ 2)). Therefore with probability at least 1 −τ/3 we have ∥ϵ∥∞≤σ
2 log(6n/τ).
For any i ∈[n], since ∥e⊤
i H∥2 ≤1, so it is easy to check that e⊤
i Hϵ is also
sub-Gaussian with variance proxy σ 2. Similar to the arguments in the last paragraph,
with probability at least 1 −τ/3, we have ∥Hϵ∥∞≤σ
2 log(6n/τ). So we have
proved that with probability at least 1 −2τ/3, the inequalities in (a) hold. ⊤
⊤ Let X =
¯U D ¯V
⊤be the SVD of X with ¯U ∈Rn×d satisfying ¯U
⊤¯U = Id;
¯V ∈Rd×d satisfying ¯V
⊤¯V = Id; and D ∈Rd×d being diagonal. Then we have ∥(X⊤X)−1X⊤ϵ∥= ∥¯V D−1 ¯U
⊤ϵ∥= ∥D−1 ¯U
⊤ϵ∥≤D−1 2
√
d∥¯U
⊤ϵ∥∞
(A.27)
and ∥(X⊤X)−1X⊤ϵ∥= ∥¯V D−1 ¯U
⊤ϵ∥= ∥D−1 ¯U
⊤ϵ∥≤D−1 2
√
d∥¯U
⊤ϵ∥∞
(A 27) and and ∥Hϵ∥= ∥¯U ¯U
⊤ϵ∥= ∥¯U
⊤ϵ∥≤
√
d∥¯U
⊤ϵ∥∞
(A.28) (A.28) (A.28) Note that for any j ∈[d], one can verify that e⊤
j ¯U
⊤ϵ is sub-Gaussian with variance
proxy σ 2. Hence, for any t > 0, we have roxy σ 2. Hence, for any t > 0, we have P(∥¯U
⊤ϵ∥∞> t) ≤2d exp(−t2/(2σ 2)). As a result, with probability at least 1 −τ/3 we have ∥¯U
⊤ϵ∥∞≤σ
2 log(6d/τ),
and hence
∥(X⊤X)−1X⊤ϵ∥≤D−1 2 σ
2d log(6d/τ) ,
(A.29) As a result, with probability at least 1 −τ/3 we have ∥¯U
⊤ϵ∥∞≤σ
2 log(6d/τ),
and hence
∥(X⊤X)−1X⊤ϵ∥≤D−1 2 σ
2d log(6d/τ) ,
(A.29) (A.29) and 12 Linear regression with partially mismatched data… ∥Hϵ∥≤σ
2d log(6d/τ) . (A.30) (A.30) Note that D−12 =
1
√n λ−1/2
min ( 1
n X⊤X), so this completes the proofs of (b) and (c). To prove (3.47), using Bernstein inequality we have Note that D−12 =
1
√n λ−1/2
min ( 1
n X⊤X), so this completes the proofs of (b) and (c Note that D−12 =
1
√n λ−1/2
min ( 1
n X⊤X), so this completes the proofs of (b) and (c). Combining (A.33), (A.34) and (A.32) we have Combining (A.33), (A.34) and (A.32) we have |⟨Hz, Hϵ⟩| ≤2η∥z∥2/w + w ¯σ 2/(2η) + d ¯σ 2/2 . (A.35) (A.35) From ρn R ≤L2/(90U 2) we know that ρn ≤0.01. Therefore by Assumption 1 (3)
we have ∥Hz∥2 ≥(1 −ρn)∥z∥2 ≥0.99∥z∥2. So ∥z∥2 ≤1.02∥Hz∥2 and combines
it with (A.35) we have |⟨Hz, Hϵ⟩| ≤2.04η∥Hz∥2/w + w ¯σ 2/(2η) + d ¯σ 2/2
≤4.08η∥HP(k) y∥2/w + 4.08η∥H P(k) y∥2/w + w ¯σ 2/(2η) + d ¯σ 2/2 , where the second inequality uses the definition of z and the Cauchy-Schwarz inequal-
ity. Note that 4.08/w = 4.08/33 ≤1/8 and w/2 = 33/2 ≤17, so we have |⟨Hz, Hϵ⟩| ≤η∥HP(k) y∥2/8 + η∥H P(k) y∥2/8 + 17η−1 ¯σ 2 + d ¯σ 2/2 . (A.36) rom Assumption 2 (2), we have ¯σ 2/σ 2 ≤min{n/(660R2), n/(5Rd)}, which implies 17η−1 ¯σ 2 ≤(1/2)ηnσ 2 ≤η∥Hϵ∥2, and d ¯σ 2/2 ≤(1/2)ηnσ 2 ≤η∥Hϵ∥2. (A.37) As a result, by (A.36) and (A.37) we have As a result, by (A.36) and (A.37) we have |⟨Hz, Hϵ⟩| ≤η∥HP(k) y∥2/8 + η∥H P(k) y∥2/8 + 2η∥Hϵ∥2 . (A.38) |⟨Hz, Hϵ⟩| ≤η∥HP(k) y∥2/8 + η∥H P(k) y∥2/8 + 2η∥Hϵ∥2 . (A.38) (A.38) Multiplying 2 in both sides of (A.38) we complete the proof. A.7 Proof of claim (3.19) Note that Note that
|⟨Hz, Hϵ⟩| ≤|⟨Hz, Hϵ⟩| + |⟨z, ϵ⟩| . (A.32)
Let w = 33. Since only two coordinates of z are non-zero, we have
|⟨z, ϵ⟩| ≤∥z∥
√
2 ¯σ ≤η∥z∥2/w + w ¯σ 2/(2η) ,
(A.33)
123 Note that
|⟨Hz, Hϵ⟩| ≤|⟨Hz, Hϵ⟩| + |⟨z, ϵ⟩| . (A.32)
Let w = 33. Since only two coordinates of z are non-zero, we have
|⟨z, ϵ⟩| ≤∥z∥
√
2 ¯σ ≤η∥z∥2/w + w ¯σ 2/(2η) ,
(A.33)
123 Note that
|⟨Hz, Hϵ⟩| ≤|⟨Hz, Hϵ⟩| + |⟨z, ϵ⟩| . (A.32)
Let w = 33. Since only two coordinates of z are non-zero, we have
|⟨z, ϵ⟩| ≤∥z∥
√
2 ¯σ ≤η∥z∥2/w + w ¯σ 2/(2η) ,
(A.33)
123 |⟨Hz, Hϵ⟩| ≤|⟨Hz, Hϵ⟩| + |⟨z, ϵ⟩| . (A.32) Let w = 33. Since only two coordinates of z are non-zero, we have R. Mazumder, H. Wang where the second inequality uses Cauchy-Schwarz inequality. Using Assumption 1
(3) and (4) we have where the second inequality uses Cauchy-Schwarz inequality. Using Assumption 1
(3) and (4) we have |⟨Hz, Hϵ⟩| ≤∥Hz∥∥Hϵ∥≤√ρn∥z∥
√
d ¯σ ≤ρn∥z∥2/2 + d ¯σ 2/2 . By Assumption 1 (3), we have ρn R ≤L2/(90U 2) ≤1/73 ≤1/(aw), hence ρn/2 ≤
1/(2aRw) = η/w. As a result, |⟨Hz, Hϵ⟩| ≤∥Hz∥∥Hϵ∥≤√ρn∥z∥
√
d ¯σ ≤ρn∥z∥2/2 + d ¯σ 2/2 . By Assumption 1 (3), we have ρn R ≤L2/(90U 2) ≤1/73 ≤1/(aw), hence ρn/2 ≤
1/(2aRw) = η/w. As a result, |⟨Hz, Hϵ⟩| ≤∥Hz∥∥Hϵ∥≤√ρn∥z∥
√
d ¯σ ≤ρn∥z∥2/2 + d ¯σ 2/2 . on 1 (3), we have ρn R ≤L2/(90U 2) ≤1/73 ≤1/(aw), hence ρn/2 ≤
η/w. As a result, |⟨Hz, Hϵ⟩| ≤η∥z∥2/w + d ¯σ 2/2 . (A.34) (A.34) Combining (A.33), (A.34) and (A.32) we have Combining (A.33), (A.34) and (A.32) we have References 1. Abid, A., Poon, A., Zou, J.: Linear regression with shuffled labels. arXiv preprint arXiv:1705.01342
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and institutional affiliations. 123
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English
| null |
A METHOD OF COMPUTING SQUARE ROOT
|
School science and mathematics
| 1,916
|
public-domain
| 440
|
SCHOOL SCIENCE AND MATHEMATICS SCHOOL SCIENCE AND MATHEMATICS 750 MICHIGAN COPPER A RECORD BREAKER. The complete
reports
from
Michigan copper
mines
received by
the
United States Geological Survey show the production to be the largest
in
the
history
of
the
industry. The
total
refined
copper
produced
amounted to 23’8,95’6,410 pounds according to B. S. Butler, as compared
with 158,009,748 pounds in 1914 and with 231,112,2’28 pounds in 1912, the
record production previous to
1915. At an average
price of
1(7.5
cents per pound,
the copper output of
Michigan had a value of about $41,800,000’ for 1915. The copper mines produced
585,933 ounces of
silver
in
1915, valued
at $297,068.- There were mined and milled in the Lake Superior district 12,334,699
tons of ore-producing concentrates containing 265,283,378 pounds of copper,
or a recovery of
slightly above one per cent of copper from the
ore. A portion of the concentrates produced was not smelted in 1915. BY MRS. M. W. ARLEIGH,
San Francisco, Cal. BY MRS. M. W. ARLEIGH,
San Francisco, Cal. Two
years
ago
while
studying
a table
of
squares,
I
saw
that
the
difference of the squares of any two successive integers
is equal to the
sum of the integers. This property struck me with peculiar force, though
I ma}^ have seen it before, and I determined to find, if possible, a simple
way of using
it to compute square
root. The method
finally worked
out is based on the following:
L t
d
b
b Let x and y be any numbers. ^-y^(x-y) (x+y). ^^y^^-y)
(,x+y). When xy^l, ^=y^-{-l (x-\-y). When xy == 2,
a’2 =y2-}-2 (-r+.y). . When xy == .1, .r^y+.l 0+y), and so on. An example will show the method. Complete the square root of 5317. 702 = 4900
70+71
=-. 141
712 = 5041
72+71 ==
143
722 = 5184
73+72
=^
145
732 = 5329
.1 (73.1+73) =
14.61
73.13 = 5343.6.1
2.9244
73.12 == 5346.5344 As the amount to be added can
usually be found mentally, it need not
be
indicated
on
paper. After
some
practice, many
additions may be
avoided by inspection. Thus, in this example one could go from 702 .to 732
by adding 3 (70+73). I
i
h
hild
l
hi
th d
dil b
i i
t bl
f I find that children learn this method readily by writing out a table of
squares and seeing that 22!2 = 2+1,
3222 = 3+2,
4232 = 4+3, and
so on. I find that children learn this method readily by writing out a table of
squares and seeing that 22!2 = 2+1,
3222 = 3+2,
4232 = 4+3, and
so on.
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English
| null |
A topologically chiral catenane that is not topologically chiral
| null | 2,022
|
cc-by
| 3,963
|
SUMMARY To consider the topology of a molecule, its structure is reduced to labelled vertices (the atoms) and edges
(bonds between them) to generate a molecular graph1. If only covalent bonding interactions are included2,3,
such graphs are like the structural diagrams commonly employed by chemists atomic geometry is irrelevant;
most molecules can be represented as a two-dimensional network and most stereoisomers are topologically
identical. In 1960, Wasserman synthesized a molecule in which two rings are held together like in a chain,
which is a topological isomer of the separated rings, highlighting that molecules could be topologically non-
trivial4,5. This insight has found wider implications in biochemistry6 but it also led to the assumption that the
stereochemistry of catenanes that are chiral due to the orientation of their rings is inherently topological in
nature7,8. We show this is incorrect by synthesizing an example whose stereochemistry is Euclidean. Thus,
we can unite the stereochemistry of catenanes with that of their topologically trivial cousins, the rotaxanes,
paving the way for a more unified approach to their discussion. A topologically chiral catenane that is not topologically chiral Noel Pairault,= Federica Rizzi,= David Lozano, Ellen M. G. Jamieson, Graham J. Tizzard, Stephen M. Goldup*
Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ (UK), *s.goldup@soton.ac.uk Noel Pairault,= Federica Rizzi,= David Lozano, Ellen M. G. Jamieson, Graham J. Tizzard, Stephen M. Goldup*
Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ (UK), *s.goldup@soton.ac.uk Keywords: License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License A topologically chiral catenane that is not topologically chiral
Noel Pairault,= Federica Rizzi,= David Lozano, Ellen M. G. Jamieson, Graham J. Tizzard, Stephen M. Goldup*
Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ (UK), *s.goldup@soton.ac.uk INTRODUCTION Topology and topological are often misapplied in chemistry to mean “shape”, perhaps through confusion with
topography and topographical respectively1. Formally, chemical topology finds its roots in mathematical
topology9, the study of the properties of networks, surfaces, and objects under topologically allowed
transformations. One of the first applications of topology in chemistry was to enumerate the available isomers
of higher order alkanes (CnH2n+2)10 and there is continued interest in how molecular topology can be used to
digitize and analyze chemical structures and their properties11. Conversely, the proposal by Lord Kelvin that
atoms were knotted vortices in the aether motivated Tait to develop a systematic categorization of knots12. The key difference between a chemical topologist’s graph and a chemist’s structural diagram is that
the former does not consider molecular rigidity or geometry; when considering its topology, a molecular graph
can be distorted arbitrarily provided the bonds are not broken or pass through one another – all other
transformations are valid, including the stretching of bonds and distortion of bonding geometry to an
unphysical extent. Using this approach, we can distinguish between the topological properties of different classes of molecular isomers using the terms “homeomorphic” (same atomic connectivity) and “isotopic”
(same molecular graph). Constitutional isomers (e.g., of pentane, Fig. 1a) are neither homeomorphic (different
connectivity) or isotopic (graphs cannot be manipulated to become identical). Stereoisomers that are
distinguished by differences in their arrangement of atoms in space due the geometric properties of atoms
(Figure 1b) are homeomorphic (same connectivity) and isotopic (can be deformed into one another) – they
are topologically identical as the difference between the isomers is lost once their Euclidean properties are
relaxed. Figure 1. Example structures and molecular graphs exemplifying key concepts in chemical topology. Molecular
structures and the molecular graphs of a. the constitutional isomers of pentane and b. the enantiomers of 2-butanol. c. Schematic molecular graphs of two non-interlocked rings and the topologically isomeric [2]catenane. d. Schematic
molecular graph of an oriented ring (Cnh symmetry; n = 1) and the canonical chiral catenane it gives rise to in which
stereochemistry is determined by atom sequence and thus topological in nature. e. Schematic molecular graph of a ring
that is oriented (Cnh symmetry; n = 1) by exocyclic bond geometry and the non-canonical chiral catenane it gives rise to
whose stereochemistry is analogous to that shown in d. but unambiguously Euclidean. INTRODUCTION 2
In 19615, Wasserman and Frisch recognized that some molecular structures cannot be reduced to a
planar molecular graph, in that there is no two-dimensional projection of their three-dimensional structure in
which no edges cross one another, and that such structures have isomers to which they are homeomorphic
(same atomic connectivity) but not isotopic (different topology). This observation was inspired by
Wasserman’s synthesis of a molecular catenane4 in which two molecular rings are joined like links in a chain,
which are topological isomers of the separated components (Fig. 1c). Other classes of molecules that display
topological isomerism have since been reported, including Mobius structures13,14,15, knots16 and covalent
systems in which bond crossings are enforced17,18. Wasserman and Frisch also highlighted that catenanes 2 could exist as topological enantiomers if both rings are “oriented” due the sequence of distinguishable atoms
embedded in their molecular graph (Fig. 1d)5. Such catenanes have since been referred to simply as
“topologically chiral”, alongside Mobius ladders19 and some knots,20,21 and interlocked molecules containing
multiple crossing points22,23, which display topological enantiomerism independent of their constitution. Although it is clearly correct to discuss catenanes in terms of their topology, perhaps for this reason, no one
has to our knowledge questioned whether the stereochemistry of catenanes containing two oriented rings is
inherently topological in nature. In their discussion5, and in all subsequent reported synthetic examples7,8,24,25,26,27,28, Wasserman and
Frisch envisaged the stereogenic unit in a topologically chiral catenane as arising due to a defined sequence
of atoms in their rings (Fig. 1d). In this case the stereogenic unit is indeed topological in nature; to generate
the other enantiomer, one ring must pass through the other or the order of atoms in one ring must be changed. Focusing on the symmetry of the rings, we see that the stereogenic unit arises because the sole improper
symmetry element of one ring (the mirror plane parallel to the macrocycle) cannot be made congruent with
that of the other and so no improper symmetry elements remain in the catenane structure. Thus, the
“topologically chiral” stereogenic unit of catenanes can be defined as arising when two Cnh rings (principal
axis perpendicular to the ring plane) are interlocked. This analysis suggests that any structural feature that
results in a Cnh ring will be suitable for the construction of a chiral catenane. INTRODUCTION Here we present a chiral catenane whose rings are both oriented, conforming to the definition of the
“topologically chiral” catenane stereogenic unit, but whose stereochemistry is Euclidean because the Cnh
symmetry of one ring arises due to the geometric properties of an exocyclic double bond (Fig. 1e). RESULTS 3
Stereoselective synthesis of chiral catenane 5. Catenane 3 was synthesized (Fig. 2a) using an active
template29 Cu-mediated alkyne-azide cycloaddition reaction between azide/alkyne pre-macrocycle alkene (Z)-
1 and chiral bipyridine macrocycle (S)-2 (98% ee)30. 1H NMR analysis confirmed that catenane 3 was isolated
as a single diastereomer (>96% de), consistent with the covalent stereochemistry of (S)-2 efficiently controlling
the relative orientation of the bipyridine and alkene containing macrocycles30. Catenane 3 also contains a co-
conformational geometric stereogenic unit31, the configuration, Eco-c, of which is controlled by the geometry of
the starting pre-macrocycle, (Z)-1, and fixed in the interlocked product due to the steric bulk of the styrene Stereoselective synthesis of chiral catenane 5. Catenane 3 was synthesized (Fig. 2a) using an active and silyl ether units which prevent shuttling of the bipyridine macrocycle between the two triazole-containing
compartments. The pendant covalent chiral auxiliary unit of catenane 3 was removed following a simple
chemical sequence (steps iii v) followed by cleavage of the silyl ether unit (step vi) to give catenane 5 which
contains no covalent stereogenic unit. Catenanes 3, 4 and 5 were characterized in full (see ESI for details). Samples of catenanes 3, 4 and 5 were also synthesized starting from (R)-2 (98% ee) and rac-2. The solid-
state structure obtained by single crystal x-ray diffraction analysis of intermediate catenane 4 derived from
rac-2 (Fig. 2b) contains the rac-(S,Smp,Eco-c)-4 stereoisomer and thus the absolute stereochemistry of
catenanes 3 and 4 derived from (S)-2 can be assigned as (S,Rmp,Eco-c)-3 and (S,Smp,Eco-c)-4. The absolute
stereochemistry of the expected major enantiomers of catenane 5 derived from (S)-2 as and (R)-2 can thus
be assigned as (Rmp)-5 and (Smp)-5 respectively (see Supplementary Information section 9 for a detailed
discussion on stereochemical assignment in such systems). Figure 2. Synthesis of catenane 5 and solid-state structure of intermediate catenane 4. a. Synthetic steps used to
produce catenane 5. Reagents and conditions: i. [Cu(MeCN)4]PF6, NiPr2Et, CH2Cl2, 35 °C; ii. Me3SiCHN2, THF-MeOH
(1 : 1), rt; iii. LiAlH4, THF, 0 °C; iv. (COCl)2, Me2SO, NEt3, CH2Cl2, – 78 °C to rt; v. piperidinium acetate, THF-H2O (9 : 1),
70 °C; vi. nBu4NF, THF, 0 °C (yields over six steps from macrocycle 2: Rmp-5 = 6%, Smp-5 = 5%, rac-5 = 7%). b. Solid-
state structure of rac-(S,Smp,Eco-c)-4 in sticks representation with bipyridine-triazole C-H•••N H-bonds indicated (colors as
in a. with the exception of O [red], H [white] and N [dark blue]; the CH2OSiMe2tBu has been omitted for clarity). Stereochemical analysis of catenane 5. Care had to be taken throughout the synthesis of catenanes 5 t St
h
i
l
l
i
f
t
5 C
h d t
b
t k
th Stereochemical analysis of catenane 5. Care had to be taken throughout the synthesis of catenanes 5 to Stereochemical analysis of catenane 5. Care had to be taken throughout the synthesis of catenanes 5 to
ensure the double bond geometry, which defines the orientation of the triazole-containing macrocycle, was
maintained; the electron rich alkene unit is prone to acid-mediated isomerization. Stereoselective synthesis of chiral catenane 5. Catenane 3 was synthesized (Fig. 2a) using an active Pleasingly, chiral stationary
phase HPLC (CSP-HPLC) analysis confirmed that catenanes 5 were formed in excellent stereoselectivity Stereochemical analysis of catenane 5. Care had to be taken throughout the synthesis of catenanes 5 to (98% ee, Fig. 3a) and that the samples derived from (R)-2 and (S)-2 produce mirror image circular dichroism
(CD) spectra, whereas the sample derived from rac-2 did not produce a CD response (Fig. 3b). 1H NMR
analysis of the isolated samples of catenane 5 revealed that the bipyridine macrocycle is in slow exchange
between the triazole stations (Fig. 3c); although the Eco-c isomer was isolated and characterized, this slowly
evolved in solution (CD2Cl2, 303 K) to an equilibrium mixture containing both co-conformations (~80 : 20 Eco-c
: Zco-c, Supplementary section 7 for a detailed discussion). This process takes place by shuttling of the
bipyridine macrocycle around the triazole-containing macrocycle, past the benzylic alcohol unit, and
demonstrates that the double bond of catenane 5 has no defined geometry. Thus, the only fixed stereogenic
unit of catenane 5 results from the relative orientation of the two rings, as depicted in its highest symmetry
representation (Fig. 3d). Figure 3. Analytical data for catenane 5 and the stereoisomerization processes observed. a. Chiral stationary
phase HPLC chromatograms of catenanes (Smp)-5, (Rmp)-5, rac-5, and the partially racemized sample obtained after
heating (Rmp)-5. b. Circular dichroism spectra of catenanes (Smp)-5 and (Rmp)-5 demonstrating that they are enantiomeric. c. The observed co-conformational exchange process between as-synthesized Eco-c-5 and Zco-c-5. d. A schematic
molecular graph of catenane 5 in its highest symmetry representation with the motion that leads to the exchange of co-
conformational isomers highlighted. e. The enantiomerization process between (Rmp-Eco-c)-5 and (Smp-Eco-c)-5 that takes
place over time and confirms the non-topological nature of the stereogenic unit. f. A schematic molecular graph of
catenane 5 in its Eco-c co-conformation with motions that lead to the enantiomerization of this species indicated. 5
Finally, CSP-HPLC analysis revealed a second stereoisomerization process; alongside the
isomerization between Eco-c and Zco-c, a slower racemization process was also observed (Fig. 3e). Thus, over
time in CD2Cl2 solution, the stereopurity of (Rmp)-5 decreased from 98% ee to 84% ee (Fig. 3a). Based on the
apparent protonation of catenane 5 over time and the behavior of an analogous rotaxane model (Supplementary section 8), we tentatively propose that the enantiomerization of 5 may take place by reversible
protonation of the electron rich double bond. Stereoselective synthesis of chiral catenane 5. Catenane 3 was synthesized (Fig. 2a) using an active This process is the chemical equivalent of the topologically
allowed isomerization of the catenane stereogenic unit by a double bond isomerization process accompanied
by shuttling of the bipyridine ring around the triazole-containing ring combined (Fig. 3f). CONCLUSIONS Our results confirm that catenane 5 exists as a pair of enantiomers whose stereochemistry results from the
mechanical bond and whose configuration is defined by the exocyclic double bond. This is consistent with our
proposal that this molecule contains a mechanical stereogenic unit analogous to previously reported
catenanes labelled simply as “topologically chiral”, but it should also be clear that in this case the
stereochemistry is not topological in nature, as confirmed by the racemization of 5 in solution through double
bond isomerization. Given that the highest symmetry representations of both rings of catenane 5 are Cnh (n =
1), as is the case with all previously reported topologically chiral catenanes, we do not think it is reasonable
to consider catenane 5 as a new class of chiral mechanically interlocked molecule. It is much simpler to
acknowledge that there is a single catenane stereogenic unit when two oriented Cnh rings are combined but
that, depending on the structure of the rings, the stereochemistry can be either topological or Euclidean (Fig. 4a vs Fig. 4b). Figure 4. Schematic examples of mechanically planar chiral catenanes and rotaxanes. a. Catenanes composed of two
rings that are oriented as a consequence of a sequence of atoms in the ring are topologically mechanically planar chiral
whereas the corresponding rotaxane is simply mechanically planar chiral. b. If the ring orientation of one of the
macrocycles arises due to bond geometry the corresponding catenane and rotaxane are both simply mechanically planar
chiral. 6 This allows us to unify the stereochemistry of rotaxanes and catenanes composed of oriented
components. The stereochemistry of rotaxanes (which are topologically trivial) and catenanes (which are not)
is often considered independently but is clearly related; the schematic representation of a topologically chiral
catenane can be converted to that of a mechanically planar chiral rotaxane by the notional process of ring
opening and stoppering (Fig. 4a). Given that we have demonstrated that the “topological” stereogenic unit of
catenanes is not inherently topological in nature, we propose that their relationship could be made clearer if
both stereogenic units were united under the same name, mechanically planar chiral. In the case of
catenanes, some examples will be topologically mechanically planar chiral (all previously reported examples),
and others will simply be mechanically planar chiral (catenane 5). SUPPLEMENTAL INFORMATION Supplemental Information includes experimental procedures and characterization data for all novel
compounds. CONCLUSIONS The unification of the mechanical planar chiral stereogenic units of rotaxanes and catenanes, as we
have recently demonstrated in the context of an analogous mechanical axial stereogenic unit32, will facilitate
future work in the field by highlighting that similar synthetic concepts are likely to be of use for both rotaxanes
and catenanes. Furthermore, as the pantheon of mechanical stereogenic units expands,33 alongside recent
developments in covalent stereochemistry34,35, and applications of stereochemistry in molecular machines36,37,
it is essential that the field remains on a firm theoretical footing. Finally, our results suggest that the focus
should shift away from the topological properties of the catenanes; although chemical topology is an intriguing
topic, we are not aware of any property of catenanes that has been unambiguously linked to their topologically
non-trivial nature. Alternatively, perhaps by separating their stereochemistry into topological and Euclidean
varieties, as we have done here, it will now be possible to demonstrate a unique property of the former. DATA AVAILABILITY Raw characterization data will be available upon publication through the University of Southampton data
repository. Crystallographic data can be accessed through the Cambridge Crystallographic database
(accession numbers: 2207578, 2207579). 7 AUTHOR CONTRIBUTIONS S.M.G. secured project funding. S.M.G. and E.M.J.G. conceived the study. F.R. carried out the initial synthesis
of catenane 3 and characterized the intermediates leading to this structure. N.P. optimized the synthesis of
catenane 3 and its conversion catenane 5. N.P. and D.L.M. completed the synthesis of the final compounds
and their characterization. N.P. performed the isomerization studies on catenane 5, including the synthesis
and analysis of model compounds. N.P. obtained single crystals of catenane 4 and model rotaxane S* for x-
ray analysis, which was performed by G.J.T. N.P. led the preparation of the Supplementary Information,
including the stereochemical analysis of all interlocked products. S.M.G. wrote the manuscript. All authors
contributed to the reviewing and editing of the manuscript and Supplementary Information. ACKNOWLEDGMENTS S.M.G thanks the European Research Council (Consolidator Grant Agreement no. 724987) and the Royal
Society for a Research Fellowship (RSWF\FT\180010) DECLARATION OF INTERESTS The authors declare no competing interests. The authors declare no competing interests. 9. Flapan, E. When Topology Meets Chemistry. (Cambridge University Press, 2012). REFERENCES 1. Walba, D. M. Topological Stereochemistry. Tetrahedron 41, 3161-3212 (1985). 1. Walba, D. M. Topological Stereochemistry. Tetrahedron 41, 3161-3212 (1985). 1. Walba, D. M. Topological Stereochemistry. Tetrahedron 41, 3161-3212 (1 2. It has been proposed that a more complete description would include interactions between atoms (eg. H-bonds) by including weighting in the edges linking atoms. See ref. 3. 2. It has been proposed that a more complete description would include interactions between atoms (eg. H-bonds) by including weighting in the edges linking atoms. See ref. 3. 3. Mislow, K. On the Classification of Pairwise Relations Between Isomeric Structures. Bull. Soc. Chim. Belg. 86, 595-601 (2010). 3. Mislow, K. On the Classification of Pairwise Relations Between Isomeric Structures. Bull. Soc. Chim. Belg. 86, 595-601 (2010). 4. Wasserman, E. The Preparation of Interlocking Rings: a Catenane. J. Am. Chem. Soc. 82, 4433-4434
(1960). 4. Wasserman, E. The Preparation of Interlocking Rings: a Catenane. J. Am. Chem. Soc. 82, 4433-4434
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chiral molecular objects. Chirality 10, 125-133 (1998). 8. Yamamoto, C., Okamoto, Y., Schmidt, T., Jager, R. & Vogtle, F. Enantiomeric resolution of
cycloenantiomeric rotaxane, topologically chiral catenane, and pretzel-shaped molecules: Observation of
pronounced circular dichroism. J. Am. Chem. Soc. 119, 10547-10548 (1997). 9. Flapan, E. When Topology Meets Chemistry. (Cambridge University Press, 2012). 8 10. Cayley, E. Ueber die analytischen Figuren, welche in der Mathematik Bäume genannt werden und
ihre Anwendung auf die Theorie chemischer Verbindungen. Ber. Dtsch. Chem. Ges. 8, 1056-1059
(2006). 11. Devillers, J. & Balaban, A. T. Topological Indices and Related Descriptors in QSAR and QSPR. (CRC
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426, 819-821 (2003). 15. Segawa, Y. REFERENCES et al. Synthesis of a Möbius carbon nanobelt. Nat. Synth. 1, 535-541 (2022). 16. Fielden, S. D. P., Leigh, D. A. & Woltering, S. L. Molecular Knots. Angew. Chem. Int. Ed. 56, 11166-
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hook and ladder approach to molecular knots: synthesis of a topologically chiral cyclized hook and
ladder. J. Am. Chem. Soc. 114, 6259-6260 (1992). 20. Zhang, G. et al. Lanthanide Template Synthesis of Trefoil Knots of Single Handedness. J. Am. Chem. Soc. 137, 10437-10442 (2015). 21. Carpenter, J. P. et al. Controlling the shape and chirality of an eight-crossing molecular knot. Chem 7,
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Synthesis of Topologically Chiral Catenanes. Chem 5, 1512-1520 (2019). 28. We recently challenged this orthodoxy through the synthesis of a co-conformationally chiral molecule
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Synthesis of Both Mechanically Planar Chiral Rotaxanes and Catenanes in Excellent 9 Enantiopurity. ChemRxiv (2022). doi:10.26434/chemrxiv-2022-3f8zm This content is a preprint and has
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catenanes and noncanonical mechanically axially chiral rotaxanes. Nat. Chem. (2022). 33. Caprice, K. et al. Diastereoselective Amplification of a Mechanically Chiral [2]Catenane. J. Am. Chem. Soc. 143, 11957-11962 (2021). 34. Canfield, P. J. et al. A new fundamental type of conformational isomerism. Nat. Chem. 10, 615-624
(2018). 35. Reisberg, S. H. et al. Total synthesis reveals atypical atropisomerism in a small-molecule natural
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(2022). 10 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SSE4checkcif.pdf
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The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016
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Ciência rural
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Qiangqiang Zhang1,2
Feilong Weng1,2 Fanji Shi3 Liqun Shao1,2 Xuexi Huo1,2* Qiangqiang Zhang1,2
Feilong Weng1,2 Fanji Shi3 Liqun Shao1,2 Xuexi Huo1,2* 1College of Economics and Management, Northwest A&F University, Yangling 712100, China. E-mail: xuexihuo@nwsuaf.edu. *Corresponding author. 2Center of Western Rural Development, Northwest A&F University, Yangling, China. 3Institute of Geographic Sciences and Natural Resources Research, University of Chinese Academy of Sciences, Beijing, China. ABSTRACT: Based on the apple acreage and output data of 23 provinces in China, the LMDI decomposition method and the barycenter
analysis model were used to systematically analyze the spatiotemporal characteristics of apple production in China from 1978 to 2016. The
results showed that the apple acreage and output continued to increase, and the apple production layout has moved to south-westward;
Shaanxi, Shandong, Henan, Shanxi, Gansu, Hebei, Liaoning, and Xinjiang were the main contributors to the apple output increase in China;
yield contributed more to apple output increase, the increase of yield was a significant contributing factor to the apple output growth in
17 provinces, whereas the expansion of apple acreage was a significant contributing factor in the other 6 provinces; the barycenter of
apple acreage and output respectively moved to the southwest by 506.63 kilometers and 574.12 kilometers, and the barycenter of apple
production gradually shifted to the Loess Plateau. To stabilize the effective supply of apple and maintain industrial security, the policymakers
should attach importance to the fundamental role of technological progress in the development of the apple industry, and bring into play the
technological progress, economic, social, and environmental effects of apple industry agglomeration by optimizing the apple production layout
and strengthening policy guidance and regulatory measures. g
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Key words: apple, production layout, evolutionary characteristics, LMDI decomposition, barycenter model, China. The Evolutionary Characteristics of Apple
Ciência Rural, Santa Maria, v.51:6, e20200688, 2021 The Evolutionary Characteristics of Apple
Ciência Rural, Santa Maria, v.51:6, e20200688, 2021 a from 1978 to 2016. 1
ISSNe 1678-4596
http://doi.org/10.1590/0103-8478cr20200688
CROP PRODUCTION As características evolutivas do layout de produção da maçã na
China de 1978 a 2016 RESUMO: Com base nos dados de área cultivada e produção de maçã de 23 províncias na China, o método de decomposição do LMDI e o
modelo de análise de barcenter foram utilizados para analisar sistematicamente as características espaço-temporais da produção de maçã na
China entre 1978 e 2016. Os resultados mostraram que a área cultivada de maçã e a produção continuou a aumentar, e o layout da produção
da maçã mudou para o Sudoeste; Shaanxi, Shandong, Henan, Shanxi, Gansu, Hebei, Liaoning e Xinjiang foram os principais contribuintes
para o aumento da produção de maçãs na China; o rendimento contribuiu mais para o aumento da produção de maçã, o aumento do
rendimento foi fator de contribuição significativo para o crescimento da produção de maçã em 17 províncias, enquanto a expansão da
área cultivada de maçã foi fator de contribuição significativo nas outras 6 províncias; o baricentro da área cultivada e a produção de maçãs
se deslocaram para o Sudoeste em 506,63 quilômetros e 574,12 quilômetros respectivamente. O baricentro da produção de maçãs mudou
gradualmente para o platô de Loess. Para estabilizar o fornecimento efetivo de maçã e manter a segurança industrial, os formuladores de
políticas devem atribuir importância ao papel fundamental do progresso tecnológico no desenvolvimento da indústria da maçã e colocar em
jogo o progresso tecnológico, os efeitos econômicos, sociais e ambientais da indústria da maçã aglomeração, otimizando o layout da produção
da maçã e fortalecendo a orientação política e as medidas regulatórias. ç
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p
g
Palavras-chave: maçã, layout de produção, características evolucionárias, decomposição de LMDI, modelo de baricentro, China Ci
Received 07.23.20 Approved 11.16.20 Returned by the author 12.31.20
CR-2020-0688.R1 INTRODUCTION (2017) used the Slacks-based Measure (SBM) model
and the Malmquist index to find that the production
efficiency of China’s main apple production areas had
a spatial agglomeration effect; YUAN et al. (2017)
studied the evolution and driving mechanism of
China’s apple industry from the micro perspective of
farmers’ decision-making, and found that the pattern
of China’s apple industry is showing a “westward”
trend; ZHANG et al. (2018) studied the changes
in China’s apple production regions and found
that the barycenter of apple production in China is
moving westward obviously, but the characteristic of
“northward expansion” is not apparent. ,
p
y (
,
)
However, with the increase in income
and living standards of urban and rural residents,
consumers are increasingly focusing on the quality
and safety of agricultural products, dietary health,
and nutritional balance, resulting in the accelerated
upgrading of the apple market demand structure, and
the existing apple production and supply model is
difficult to adapt to changes in market demand in the
new period. The development of the apple industry
is faced with the imbalance between the supply
and the quality structure (ZHANG et al., 2019), it
is mainly manifested by the disconnection between
the source of production and the market demand,
the oversupply of middle and low grade apples and
the shortage of high-quality apples, and the high-
quality and low-priced imported apples impacting
the domestic market. In some major apple producing
areas, especially in non-suitable producing areas,
the problem of low apple prices damaging farmers’s
interests frequently occurs. In essence, the root cause
of the above phenomenon is that apple production
layout is unreasonable, the specificity of apple assets
strengthens the “locking effect” of apple production
in non-suitable and sub-suitable areas, the high sunk
cost of apple production hinders the optimization of
apple production layout, and has an extrusion effect
on other suitable industries in the region, which
further reduces the efficiency of regional resource
allocation, resulting in outstanding problems in
the uncoordinated and uneven distribution of apple
production regions. The pressure of structural reform
on the supply side of the apple industry highlights
the necessity and urgency of optimizing the layout of
apple production. In summary, the existing studies focus on
analyzing the characteristics of the development and
spatial distribution of apple production in China at
a certain stage, that is, focus on the spatial changes
of China’s apple production in the short term. INTRODUCTION sign of this is that the area and output of apples
increased since the rural reform. According to
the statistics of the National Bureau of Statistics of
China (NBSC), China’s area under apple production
and output increased by 2.42 times and 18.29 times
from 1978 to 2016 (NBSC, 1985-2017). Specifically,
China’s apple production has experienced four stages
of steady development (1978-1984), rapid growth
(1985-1996), rapid decline (1997-2003), and slow
growth (2004-2015). In 2016, the apple acreage With the advancement of agricultural
market-oriented reforms, the trend of product
substitution
and
integration,
and
industry
substitution and integration within China’s major
agricultural regions and between is obvious (YU
et al., 2011). In this process, the comparative
income has driven the rapid development of the
apple industry with high-value attributes. The main Ci
Received 07.23.20 Approved 11.16.20 Returned by the author 12.31.20
CR-2020-0688.R1 Zhang et al. 2 began to decline slowly, but China remains the largest
apple producer and consumer in the world (LIU et al.,
2012; YUAN et al., 2017; MA, 2016; INOUYE et al.,
2019). The United States Department of Agriculture
(USDA) estimated that China’s fresh apple output and
consumption accounted for 52.84% and 54.12% of
the world total in 2016, respectively (USDA, 2019). Therefore, the government and academia
have paid close attention to the changes in the layout
of apple production and its possible impacts. To guide
the regional development and spatial layout of the
apple industry, the Ministry of Agriculture and Rural
Affairs (MARA) of the People’s Republic of China
promulgated The Advantageous Regional Layout Plan
for Apple (2003-2007) in 2003, The Advantageous
Regional Development Plan for Apple (2008-2015)
in 2009 and The Regional Layout Plan for Special
Agricultural Products (2013-2020) in 2014. LIU et
al. (2012) found that China’s apple production has
gradually shifted to the two dominant areas of the
Bohai Bay and the Loess Plateau, and has shown
the characteristics of “north-westward expansion” in
the region; FENG et al. (2015) included non-point
source pollution into apple total factor productivity,
through the analysis of the global and local spatial
autocorrelation of apple total factor productivity, they
found that China’s apple total factor productivity
showed a significant positive spatial correlation
both in the global and local scales; ZHANG et al. Ciência Rural, v.51, n.6, 2021. INTRODUCTION Limited by statistical data, the fruits
discussed in this study are garden fruits, including
apples, oranges, pears, grapes, and bananas. between apple supply and demand, how to promote
the supply-side structural reform of the apple industry
requires an accurate grasp of the evolution pattern
of apple production layout, an innovative analysis
of the mechanism and methods of the evolution
rule of apple production layout, and the promotion
of government ability to predict and make changes
in production layout. For these reasons, this paper
studied the characteristics of apple production in
China since 1978, from the perspective of the apple
production area and yield changes at the national
and provincial scales. First, this study analyzed the
time series characteristics of apple production in
China. Second, this study used the statistical analysis
method of spatial barycenter and provincial data to
comprehensively analyze the spatial distribution
characteristics of apple production in China, to make
up for the shortcomings of existing studies in terms of
space-time scale and methods. INTRODUCTION However, due to the large differences in the economic
environment and policy support for the apple industry
development in each stage, it is difficult to reveal the
characteristics of the spatial distribution of apple
production nationwide and its evolution regularity
since 1978. Moreover, due to the comparative
gains in apple production across industries and
regions, the changes in the external environment,
the regional changes in apple production layout
are complex and irreversible (YANG et al., 2011). Therefore, identifying and grasping the evolution
characteristics of the apple production layout has
become an important issue in optimizing the layout
of apple industry and coordinating the relationship
between apple production and sales, that is, in
the context of a significant shift in the relationship The
development
of
China’s
apple
industry has entered a new historical stage, and the
relationship between apple supply and demand has
undergone major changes. Apple production must
adapt to market demand. Changes in the layout of
China’s apple production not only affect the situation
of domestic apple production and sales, but also the
pattern of international apple production and sales
pattern. In particular, the apple industry is also the
major agricultural industry in the main producing
areas and the main source of farmers’ income
(MARA, 2007). Ciência Rural, v.51, n.6, 2021. The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016. 3 (NBSC, 1985-2017) and New China Agricultural
Statistics for 60 Years (NBSC, 2009). The dataset
used in the present study covered 23 apple-
producing provinces, cities, and autonomous
regions (including Shandong, Hebei, Liaoning,
Tianjin, Beijing in the Bohai Bay production area,
Shaanxi, Gansu, Shanxi, Ningxia, Qinghai in the
Loess Plateau production area, Henan, Anhui,
Jiangsu in the Yellow River old road production
area, Sichuan, Chongqing, Yunnan, Guizhou, Tibet
in the southwest cold and highland production area,
and Inner Mongolia, Heilongjiang, Jilin, Hubei,
and Xinjiang in special production area) of China
Mainland (hereinafter China for convenience) for
thirty-nine consecutive years (1978–2016), with
a total of 858 observations (Figure 1). In 1997,
Chongqing became a direct-controlled municipality,
therefore, its statistics have been calculated since
1997. The geometric centers of the administrative
region of each province are considered as their
barycenter. The coordinate data and the vector
data of the map were obtained from the National
Geomatics Center of China (NGCC, http://www. ngcc.cn/ngcc/). Ciência Rural, v.51, n.6, 2021. LMDI decomposition method =
100%
a
Pa
C
P
∆
×
∆
(6)
=
100%
y
Py
C
P
∆
×
∆
(7) The contribution rate of increasing output
is measured by the proportion of the increase of apple
output in a certain region to the total increase of
apple output in that year. The calculation method is
as follows: ( )
where
a
C and
y
C denote the contribution rate of
apple acreage expansion and yield increase to apple
output increase in a region, and
=100%
a
y
C
C
+
. This
study divided
a
C and
y
C into more significant [50%
< max (
a
C ,
y
C ) ≤ 60%], significant [60% < max (
a
C ,
y
C ) ≤ 80%] and extremely significant [max (
a
C ,
y
C ) > 80%] three levels (FENG et al., 2016). ( )
where
a
C and
y
C denote the contribution rate of
apple acreage expansion and yield increase to apple
output increase in a region, and
=100%
a
y
C
C
+
. This
study divided
a
C and
y
C into more significant [50%
< max (
a
C ,
y
C ) ≤ 60%], significant [60% < max (
a
C ,
y
C ) ≤ 80%] and extremely significant [max (
a
C ,
y
C ) > 80%] three levels (FENG et al., 2016). 1
i
i
k
i
i
P
C
P
=
=
∑
(1)
1
i
i
k
i
i
P
C
P
=
∆
∆
=
∆
∑
(2) (1) (2) ( )
where
i
C and
i
C
∆
denote the apple output contribution
rate and the increased output contribution rate of
province i ,
iP and
iP
∆ denote the apple output
and increased output in provinces i , k denotes the
number of apple producing provinces, =1
23
k
L
，
. Barycenter analysis model The barycenter can accurately represent the
spatial distribution characteristics of the barycenter
of factors (LIN et al., 2014), resources (SUN et al.,
2018), industry (NIE et al., 2015; ZHANG et al.,
2018; LIU et al., 2018, LI et al., 2017) and economic
development (MENG et al., 2017; XU et al., 2014). This study used the barycenter theory and method to
analyze the evolution characteristics of the barycenter
of apple production, and reveal the movement of the
regional equilibrium point of the apple acreage and
the changing pattern of the regional distribution of
the apple industry. Assuming that the provincial areas
are the homogeneous plane, and the geometric center
point coordinates of the provincial administrative
region are selected as their geographic barycenter
coordinates, thus, the barycenter of apple production
refers to the moment equilibrium point of the apple
acreage or output in the provincial region distribution
in a certain period. The specific calculation method is
as follows: Changes in apple output are mainly the
result of a combination of changes in acreage and
yield. To compare the contribution of acreage and
yield changes to the increase of apple output, this
study adopted the Logarithmic Mean Division Index
(LMDI) method (ANG et al, 2007), because some
studies have shown that the LMDI method is superior
to the AMDI method in terms of theoretical basis,
applicability, simplicity, and interpretability of results
(DING et al., 2017; WANG et al., 2018). Apple
output increase can be decomposed into the sum of
the output increase contributed by acreage expansion
(referred to as “acreage contribution”) and the output
increase contributed by yield increase (referred
to as “yield contribution”) (ZHANG et al., 2019). Therefore, the increase of apple output in a certain
region can be calculated by the following methods: 1
1
N
N
t
it
i
it
i
i
X
M X
M
=
=
= ∑
∑
1
1
N
N
t
it
i
it
i
i
Y
M Y
M
=
=
= ∑
∑
(8) P
Pa
Py
∆
= ∆
+ ∆
(3) (3) where Pa
∆
and
Py
∆
denote the increase of apple
output in a certain region contributed by the acreage
expansion and yield increase. Data Sources Data on apple acreage and output at the
national and provincial scales were derived from
China Rural Statistical Yearbook (1985-2017) Ciência Rural, v.51, n.6, 2021. Figure 1 - Apple production areas in China. Figure 1 - Apple production areas in China. Figure 1 - Apple production areas in China. Zhang et al. 4 Ciência Rural, v.51, n.6, 2021. Barycenter analysis model The specific output
contribution can be obtained by decomposing Pa
∆
and Py
∆
: (8) where
t
X and
t
Y denote the longitude and latitude
values of the geographic location where the barycenter
of China’s apple production is located in the t th year,
i
X and
iY denote the longitude and latitude values
of the geometric center point of the province i;
it
M
denotes the apple acreage or output of the province i
in the t th year, and N denotes the number of apple
producing provinces. y
=
ln
ln
ln
t
b
t
t
b
b
P
P
a
Pa
P
P
a
−
∆
−
(4)
=
ln
ln
ln
t
b
t
t
b
b
P
P
y
Py
P
P
y
−
∆
−
(5) (4) (5) where
b
P and
tP denote the apple output of a region where
b
P and
tP denote the apple output of a region
at the beginning and end of the period,
b
a and
ta
denote the apple acreage of a region at the beginning
and end of the period,
b
y and
ty denote the apple
i ld f
i
h b
i
i
d
d f h
i d where
b
P and
tP denote the apple output of a region
at the beginning and end of the period,
b
a and
ta
denote the apple acreage of a region at the beginning
and end of the period,
b
y and
ty denote the apple
yield of a region at the beginning and end of the period. The moving distance of the barycenter of
China’s apple production can be calculated: pp
p
(
)
(
)
2
2
T
t
T
t
t T
D
E
X
X
Y
Y
−
=
×
−
+
− (
)
(
)
2
2
T
t
T
t
t T
D
E
X
X
Y
Y
−
=
×
−
+
−
(9) (9) The contribution ratio of apple acreage and
yield to apple output increase can be calculated: where
t T
D − denotes the moving distance of the denotes the moving distance of the Ciência Rural, v.51, n.6, 2021. The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016. Barycenter analysis model 5 four phases: the stable period (1978-1991), the rapid
growth period Ⅰ (1992-1996), the buffer period (1997-
2002), and the rapid growth period Ⅱ (2003-2016)
(Figure 3). Factors, such as technology progress, and
varieties improvement, boosted apple yield and led
to the asynchronous changes between apple acreage
and output. barycenter of China’s apple production from the t
th year to the T th year, (
)
,
t
t
X
Y
and (
)
,
T
T
X
Y
denote the latitude and longitude coordinates of the
barycenter of China’s apple production from the t
th year to the T th year, E is a plane conversion
coefficient, which denotes the value corresponding to
the transformation of geographic coordinates into a
plane distance, the value is usually 111.13 kilometers
(ZHANG et al., 2018; XIAO et al., 2018). p
From the perspective of relative volume
change, affected by the comparative returns of
other fruits and the structural adjustment of the fruit
industry, the proportion of apple acreage and output to
the total fruit acreage and output generally declined. Constrained by arable land resources and market
demand, there is a clear substitution relationship
between apple and other fruit (ROURKE, 2019). The
main reason is that since the rural reform, China’s
fruit industry took the lead in entering market-
oriented reforms. The daily consumption of fruit by
residents, such as apples, oranges, pears, grapes, and
bananas, has developed rapidly (ZHU et al., 2013). The relative price of apples in suitable production
areas was relatively high and had spillover effects,
coupled with policy support for industrial poverty
alleviation, rational differences among farmers, and
other factors, which induce the expansion of apple
acreage in non-suitable and sub-suitable areas. The
proportion of apple acreage and output in non-suitable
and sub-suitable areas has risen, but the quality is
relatively poor, leading to the decline in the overall
development quality of China’s apple industry, and Ciência Rural, v.51, n.6, 2021. RESULTS AND DISCUSSION From 1978 to 2016, the apple acreage in
China increased from 0.68 million ha to 2.32 million
ha, an increase of 1.65 million ha, with an average
annual growth rate of 3.29%; apple output increased
from 2.28 million tons to 43.83 million tons, an
increase of 41.70 million tons, with an average annual
growth rate of 8.10%. From the perspective of absolute volume
change, the changes in apple acreage since 1978 has
experienced four stages of the stable period (1978-
1984), the rapid increase period (1985-1996), the rapid
decline period (1997-2003), and the slow increase
period (2004-2015) (LIU et al., 2012; ZHANG et al.,
2018). In 2016, the apple acreage decreased slightly,
from 2.33 million ha in 2015 to 2.32 million ha in 2016,
a decrease of 0.19% (Figure 2). The changes in apple
output during the thirty-nine years can be divided into Ciência Rural, v.51, n.6, 2021. Figure 2 - Changes in apple acreage from 1978 to 2016 in China. Ciência Rural, v.51, n.6, 2021. Figure 2 - Changes in apple acreage from 1978 to 2016 in China. Figure 2 - Changes in apple acreage from 1978 to 2016 in China. Figure 2 - Changes in apple acreage from 1978 to 2016 in China. Figure 2 - Changes in apple acreage from 1978 to 2016 in China. Ciência Rural, v.51, n.6, 2021. Ciência Rural, v.51, n.6, 2021. 6
Zhang et al. Figure 3 - Changes in apple output from 1978 to 2016 in China. Zhang et al. 6 Figure 3 - Changes in apple output from 1978 to 2016 in China. Whereas the contribution of technological progress
to apple output increase was not obvious, that is, the
acreage contribution rate was greater than 1, while
the yield contribution rate was negative (LIU, 2005). On the other hand, driven by factors, such as variety
improvement, cultivation technology innovation,
orchard equipment and infrastructure upgrades,
and improvement of fruit farmers’ quality, the
contribution rate of yield changes has been increasing
year by year since 1994. After 1998, some traditional
producing areas accelerated the improvement of old
and low-efficiency orchards, and apple production
entered a phase of shifting from quantity to quality-
efficient, the apple acreage tended to be stable and
reasonable, the contribution of technological progress
in apple output was prominent. As a result, the yield
contribution rate has continued to be higher than
the acreage contribution rate since 2002. RESULTS AND DISCUSSION Generally,
the apple yield increased from 3,351.17 kg/ha to
18,883.72 kg/ha during thirty-nine years, an increase
of 4.63 times, indicating that technological progress
actuating yield improvement is the main driving force
for increased apple output in China. the amplification of the natural and market risks
of apple production, which in turn led to frequent
fluctuations in the apple market. Besides, the
development of alternative fruits, such as oranges,
pears, grapes, and bananas, the comparative
advantage of apples has been affected, resulting in
the proportion of apples in the total fruit acreage
fell from 40.98% in 1978 to 17.90% in 2016, and
the proportion of apple output in total fruit output
decreased from 34.63% in 1978 to 24.22% in 2016. From 1978 to 2016, the change in apple
acreage contributed 17.2997 million tons of increased
apple output, accounting for 41.58% of the total
increased apple output nationwide. The change in
apple yield contributed 24.3073 million tons of
increased apple output, accounting for 58.42% of the
total increased apple output nationwide (Table 1). Therefore, the contribution rate of apple yield change
to the increase of apple output was higher than that of
apple acreage change. Specifically, the contribution
rate of apple acreage and yield change from 1978 to
1992 fluctuated greatly due to the difference in the
trend and direction of apple acreage and yield change
(ZHU et al., 2013), that is, the contribution rate of
apple acreage and yield was positive or negative. From 1986 to 1991, the increase in apple output
nationwide was mainly driven by rapid acreage
increase, the apple acreage nationwide increased
from 1.17 million ha to 1.66 million ha during this
period, with an average annual growth rate of 7.20%. From the analysis of apple acreage
changes, apple acreage in Hubei, Chongqing, and
Qinghai decreased slightly from 1978 to 2016,
and apple acreage in other provinces increased in
varying degrees. Especially, Hebei and Shanxi in
North China, Liaoning in the Northeast, Shandong
in East China, Henan in Central South, Sichuan Ciência Rural, v.51, n.6, 2021. The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016. 7 Table 1 - Contributions of cumulative acreage and yield to increased apple output in China from 1978 to 2016. RESULTS AND DISCUSSION Year
AC(104 tons)
YC(104 tons)
ACR (%)
YCR (%)
Year
AC(104 tons)
YC(104 tons)
ACR
(%)
YCR
(%)
1978
—
—
—
—
1998
1,082.52
638.08
62.92
37.08
1979
21.73
37.67
36.59
63.41
1999
1,070.70
782.00
57.79
42.21
1980
20.91
-12.11
237.62
-137.62
2000
992.62
822.98
54.67
45.33
1981
17.83
55.27
24.39
75.61
2001
908.04
865.96
51.19
48.81
1982
14.10
1.40
90.94
9.06
2002
833.69
862.91
49.14
50.86
1983
19.26
107.34
15.21
84.79
2003
870.10
1,012.60
46.22
53.78
1984
28.21
38.39
42.36
57.64
2004
928.92
1,211.08
43.41
56.59
1985
70.23
63.67
52.45
47.55
2005
944.62
1,228.98
43.46
56.54
1986
151.80
-45.60
142.94
-42.94
2006
1,003.30
1,375.10
42.18
57.82
1987
238.28
-39.38
119.80
-19.80
2007
1,083.77
1,474.74
42.36
57.64
1988
286.11
-79.21
138.28
-38.28
2008
1,153.20
1,604.00
41.83
58.17
1989
297.46
-75.06
133.75
-33.75
2009
1,233.45
1,707.15
41.95
58.05
1990
279.90
-75.50
136.94
-36.94
2010
1,326.28
1,772.52
42.80
57.20
1991
293.41
-66.91
129.54
-29.54
2011
1,422.83
1,948.17
42.21
57.79
1992
419.36
8.74
97.96
2.04
2012
1,523.59
2,098.01
42.07
57.93
1993
588.80
90.70
86.65
13.35
2013
1,580.72
2,160.08
42.26
57.74
1994
767.95
117.45
86.74
13.26
2014
1,636.17
2,228.63
42.34
57.66
1995
949.01
224.29
80.88
19.12
2015
1,696.67
2,337.14
42.06
57.94
1996
1,086.66
390.54
73.56
26.44
2016
1,729.97
2,430.73
41.58
58.42
1997
1,056.20
438.20
70.68
29.32
—
—
—
—
—
Note: AC denotes acreage contribution, YC denotes yield contribution, ACR denotes acreage contribution rate, YCR denotes yield
contribution rate. “—” means no available data. Table 1 - Contributions of cumulative acreage and yield to increased apple output in China from 1978 to 2016. and Yunnan in the Southwest, and Shaanxi and
Gansu in the Northwest saw larger increases in
apple acreage. Beijing and Tianjin in North China,
Heilongjiang and Jilin in the Northeast, Anhui in
Eastern China, and Chongqing and Tibet in the
Southwest have remained stable in apple acreage. From the analysis of the spatial pattern of apple
production, Hebei and Shanxi in North China,
Liaoning in the Northeast, Shandong in East China,
Henan in Central South, Sichuan and Yunnan in the
Southwest, and Shaanxi in the Northwest are the
main apple producing provinces in their respective
regions (Figure 4). its apple output increased by 10.91 million tons,
accounting for 26.22% of the total increase in apple
production nationwide. Shandong’s apple output
increased by 8.93 million tons, accounting for
21.46 % of the total increase in apple production
nationwide. Note: AC denotes acreage contribution, YC denotes yield contribution, ACR denotes acreage contribution rate, YCR denotes yield
contribution rate. “—” means no available data. RESULTS AND DISCUSSION Shaanxi and Shandong are the main
provinces for apple output increase in the two
advantage areas (XU et al., 2015). In addition, other
provinces that increased apple output by more than
1 million tons include Henan, Shanxi, Gansu, Hebei,
Liaoning, and Xinjiang (Table 2). From the analysis of the apple output
increased extent, Shaanxi had the largest increase
extent in apple output during thirty-nine years,
with an increase in apple output of 10,996.59%,
which is 6.01 times the nationwide average increase
(1,828.76%) in apple output (Table 2). Gansu,
Ningxia, and Heilongjiang were the provinces that
increased their apple output by more than 5,000%. Among the 23 apple-producing provinces, the apple
output increase extent of 13 provinces was higher From the analysis of the apple output
increased volume, due to the expansion of acreage
and increase in yield, the output of 23 apple-
producing provinces increased to varying degrees,
but the apple production areas are mainly distributed
in the Loess Plateau and the Bohai Bay advantaged
areas (BAI et al., 2015). Among them, Shaanxi’s
apple output increase ranked first nationwide, Ciência Rural, v.51, n.6, 2021. Zhang et al. 8 Figure 4 - Changes in apple acreage by region from 1978 to 2016. Figure 4 - Changes in apple acreage by region from 1978 to 2016. output increase, there are 17 provinces (in turn,
Chongqing, Hubei, Qinghai, Tianjin, Jilin, Anhui,
Liaoning, Henan, Shandong, Sichuan, Jiangsu,
Shanxi, Inner Mongolia, Hebei, Heilongjiang,
Xinjiang, and Yunnan) that had relatively higher yield
contribution rates. Particularly, the yield contribution
of Chongqing, Hubei, and Qinghai exceeded 120%. Moreover, there are 6 provinces (in turn, Tibet,
Beijing, Gansu, Ningxia, Guizhou, and Shaanxi) that
have relatively higher acreage contribution rates. than the national average (that is, the super-growth
type), that of 10 provinces below the national
average (that is, the lagging-growth type). Apple
output in Sichuan, Yunnan, Shaanxi, and Gansu
increased by more than 30 years. From 1978 to 2016, apple production in
20 provinces achieved a “double increase” in output
and acreage. Except for Beijing and Tibet, there are
21 provinces with increased apple yield. In terms of
the contribution rate of acreage and yield to apple Ciência Rural, v.51, n.6, 2021. The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016. 9 Table 2 - Apple output increase at provincial scale in China from 1978 to 2016. RESULTS AND DISCUSSION Province
Output
Increase
Volume (104
tons)
Output
Increase
Rate (%)
Acreage
Contribution
Volume (104
tons)
Yield
Contribution
Volume (104
tons)
Acreage
Contribution
Rate (%)
Yield
Contribution
Rate (%)
Beijing
2.46
51.29
4.21
-1.75
171.06
-71.06
Tianjin
4.68
719.91
0.21
4.47
4.53
95.47
Hebei
348.35
2,021.79
166.19
182.16
47.71
52.29
Shanxi
419.84
4,781.72
149.62
270.21
35.64
64.36
Inner Mongolia
16.94
3,196.34
7.99
8.95
47.14
52.86
Liaoning
192.55
300.60
24.87
167.68
12.92
87.08
Jilin
12.22
857.20
0.66
11.55
5.44
94.56
Heilongjiang
14.68
5,437.48
7.08
7.60
48.24
51.76
Jiangsu
53.78
2,045.01
15.88
37.91
29.52
70.48
Anhui
36.29
3,269.44
3.25
33.04
8.95
91.05
Shandong
892.93
1,048.03
181.82
711.10
20.36
79.64
Henan
422.87
2,691.73
69.01
353.86
16.32
83.68
Hubei
1.01
382.60
-1.30
2.32
-128.33
228.33
Chongqing
0.07
16.40
-0.42
0.49
-603.32
703.32
Sichuan
59.05
1,604.53
16.90
42.15
28.62
71.38
Guizhou
5.79
3,737.74
3.29
2.50
56.82
43.18
Yunnan
40.85
3,280.87
20.08
20.77
49.16
50.84
Tibet
0.36
133.96
0.71
-0.35
196.94
-96.94
Shaanxi
1,090.86
10,996.59
597.23
493.63
54.75
45.25
Gansu
355.82
8,294.21
205.77
150.05
57.83
42.17
Qinghai
0.26
149.66
-0.06
0.33
-24.39
124.39
Ningxia
56.15
5,504.87
31.93
24.22
56.87
43.13
Xinjiang
132.53
3,268.27
63.97
68.56
48.27
51.73
Nationwide
4,160.71
1,828.76
1,730.01
2,430.70
41.58
58.42 Table 2 - Apple output increase at provincial scale in China from 1978 to 2016. Especially, the acreage contribution rate of Tibet and
Beijing was greater than 100% (Table 2). Overall,
due to the technological progress, management
innovation, and scale operation, the increase in apple
yield was the main driving force for apple output
increase nationwide. southwest, the provinces with extremely significant
apple acreage contribution were Xinjiang in the
northwest and Beijing in North China. From the analysis of spatial position
changes, the apple acreage barycenter changed
between 110.56°E-115.61°E and 36.58°N-37.53°N
from 1978 to 2016. Based on the analysis of the
movement trajectory, the apple acreage barycenter
changed from 115.23°E and 37.01°N (located
in Julu County of Hebei province) in 1978 to
110.69°E and 36.59°N (located in Yonghe County
of Shanxi province) in 2016 (Table 3). Therefore,
the apple acreage barycenter generally moved to
the southwest and is expected to move to Yanchuan
County of Shaanxi province. It is worth noting that
the distance moved by the apple acreage barycenter From the analysis of regional distribution,
the provinces that have the most significant yield
contribution to apple output increase mainly
distributed in the northern region. RESULTS AND DISCUSSION The provinces
that have a significant yield contribution mainly
distributed in the eastern region, namely Shandong
and Jiangsu. The provinces that have an extremely
significant contribution mainly distributed in the
central region and the northeast. The provinces
with significant apple acreage contribution mainly
distributed in the northwest and Guizhou in the Ciência Rural, v.51, n.6, 2021. The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016. 10 in the east-west direction was greater than the
distance moved in the north-south direction,
which indicates that the apple acreage in the east-
western direction has changed more (ZHANG et
al., 2018), that is, the apple acreage increased in
the Loess Plateau is greater than other producing
areas. From the perspective of moving distance, the
apple acreage barycenter moved 506.63 kilometers
to the southwest from 1978 to 2016, indicating
that the apple acreage barycenter gradually shifted
from the Bohai Bay to the Loess Plateau. The main
reason is that the Loess Plateau belongs to the apple
eugenic region, and the Loess Plateau production
area is richer in land resources than the Bohai Bay
production area, according to the statistics of NBSC,
the volume of agricultural lands in the Loess Plateau
was 96.0486 million ha in 2016, which is 2.53 times
that in the Bohai Bay (NBSC, 2017). Therefore, the
shift of the apple acreage barycenter to the Loess
Plateau is in line with the national apple industry
layout plan and industrial strategy. From the perspective of space, the apple
output barycenter changed between 111.91°E-117.23°E
and 36.32°N-37.84°N from 1978 to 2016. From
the perspective of the movement trajectory, the
apple output barycenter changed from 116.91°E and
37.83°N (located in Nanpi County of Hebei Province)
in 1978 to 111.91°E and 36.54°N (located in Lingshi
County of Shanxi Province) in 2016, that is, the apple
output barycenter generally moved to the southwest
(Table 4). From the perspective of moving distance, the
apple output barycenter moved 574.12 kilometers to the
southwest from 1978 to 2016, indicating that the apple
output barycenter gradually shifted from the Bohai
Bay to the Loess Plateau. The main reason is that
apple output and quality are greatly affected by natural
conditions, the Loess Plateau is the production center
of the high-quality apple, and its resource endowment
for developing the apple industry is superior to the
Bohai Bay. Ciência Rural, v.51, n.6, 2021. Note: “—” means no available data. RESULTS AND DISCUSSION Therefore, the shift of the apple output
barycenter to the Loess Plateau fitted the advantages
of resource endowment. Table 3 - Movement of apple acreage barycenter in China from 1978 to 2016. Table 3 - Movement of apple acreage barycenter in China from 1978 to 2016. Year
Longitude
(°E)
Latitude
(°N)
Moving
Direction
Moving
Distance
(km)
Year
Longitude
(°E)
Latitude
(°N)
Moving
Direction
Moving
Distance
(km)
1978
115.23
37.01
—
—
1998
113.34
36.86
northwest
6.61
1979
115.18
37.17
northwest
18.58
1999
113.31
36.89
northwest
4.97
1980
115.23
37.22
northeast
8.40
2000
113.27
36.90
northwest
4.77
1981
115.36
37.34
northeast
19.33
2001
113.11
36.91
northwest
17.48
1982
115.41
37.40
northeast
8.91
2002
112.97
36.90
southwest
15.98
1983
115.50
37.46
northeast
12.11
2003
112.68
36.81
southwest
33.59
1984
115.62
37.53
northeast
15.20
2004
112.46
36.77
southwest
24.75
1985
115.50
37.43
southwest
17.08
2005
112.37
36.76
southwest
10.17
1986
115.34
37.22
southwest
29.30
2006
111.97
36.73
southwest
44.37
1987
114.92
37.11
southwest
48.58
2007
111.62
36.68
southwest
39.11
1988
114.56
37.06
southwest
40.51
2008
111.38
36.68
northwest
26.59
1989
114.27
36.97
southwest
33.83
2009
111.04
36.69
northwest
37.91
1990
114.11
37.06
northwest
21.05
2010
110.79
36.75
northwest
28.88
1991
114.04
37.08
northwest
7.24
2011
110.63
36.70
southwest
18.50
1992
114.09
36.97
southeast
12.87
2012
110.56
36.65
southwest
9.69
1993
113.87
36.89
southwest
25.54
2013
111.00
36.62
southeast
49.23
1994
113.72
36.78
southwest
21.28
2014
110.93
36.61
southwest
8.49
1995
113.62
36.78
southwest
10.92
2015
110.84
36.62
northwest
9.67
1996
113.51
36.80
northwest
12.28
2016
110.69
36.59
southwest
17.21
1997
113.40
36.84
northwest
13.11
—
—
—
—
— The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016. 11 Table 4 - Movement of apple output barycenter in China from 1978 to 2016. RESULTS AND DISCUSSION Year
Longitude
(°E)
Latitude
(°N)
Moving
Direction
Moving
Distance
(km)
Year
Longitude
(°E)
Latitude
(°N)
Moving
Direction
Moving
Distance
(km)
1978
116.91
37.83
—
—
1998
114.46
36.64
southwest
18.98
1979
117.24
37.59
southeast
45.75
1999
114.27
36.51
southwest
25.03
1980
116.80
37.69
northwest
50.21
2000
114.10
36.46
southwest
20.35
1981
116.77
37.49
southwest
21.87
2001
114.02
36.42
southwest
9.45
1982
116.37
37.56
northwest
44.28
2002
113.78
36.48
northwest
27.04
1983
116.62
37.49
southeast
29.02
2003
113.77
36.50
northwest
3.39
1984
116.28
37.65
northwest
42.53
2004
113.72
36.44
southwest
8.72
1985
115.42
37.43
southwest
98.89
2005
113.60
36.45
northwest
13.54
1986
115.43
37.34
southeast
9.97
2006
113.36
36.39
southwest
27.67
1987
115.48
37.21
southeast
15.55
2007
113.18
36.33
southwest
21.55
1988
115.16
37.20
southwest
34.69
2008
113.04
36.36
northwest
15.99
1989
115.06
37.16
southwest
12.04
2009
112.88
36.40
northwest
17.75
1990
115.04
37.37
northwest
23.69
2010
112.73
36.40
northwest
17.09
1991
114.73
37.10
southwest
44.60
2011
112.69
36.47
northwest
8.75
1992
115.00
37.12
northeast
29.79
2012
112.59
36.51
northwest
12.60
1993
114.90
36.96
southwest
21.41
2013
112.52
36.56
northwest
8.87
1994
114.64
36.75
southwest
36.72
2014
112.28
36.52
southwest
27.40
1995
114.60
36.68
southwest
9.06
2015
112.15
36.53
northwest
14.28
1996
114.65
36.66
southeast
6.25
2016
111.91
36.54
northwest
27.32
1997
114.62
36.70
northwest
5.84
—
—
—
—
— Table 4 - Movement of apple output barycenter in China from 1978 to 2016. Note: “—” means no available data. CONCLUSION guidance and market selection incentives promoted
the apple production distribution to predominant
areas and special production areas, the regional
concentration of the apple industry increased, and
the regional scale economies of the apple industry
were significant. FENG X. et al. Total factor productivity of apple industry in China
considering non-point source pollution and its spatial concentration
analysis. Transactions of the Chinese Society of Agricultural FENG X. et al. Total factor productivity of apple industry in China
considering non-point source pollution and its spatial concentration
analysis. Transactions of the Chinese Society of Agricultural
Engineering (Transactions of the CSAE), v.31, n.18, p.204-211,
2015. Available from: <http://d.wanfangdata.com.cn/periodical/
nygcxb201518029>. Accessed: Apr. 19, 2018. doi: 10.11975/j. issn.1002-6819.2015.18.029. (4) China’s apple production barycenter
has gradually shifted to and concentrated on the Loess
Plateau and the Bohai Bay predominant areas since
1978, of which Shaanxi and Shandong were the two
major provinces for apple production. The barycenter
of apple production showed the characteristics of
“moving westward and expanding southward”. The
barycenter of apple acreage and output moved 506.63
kilometers and 574.12 kilometers to the southwest
and shifted to the Loess Plateau. This evolutionary
trend was in line with the national apple industry
layout plan and industry policy objective, and laid
the foundation for the effective use of agricultural
resources advantages in the eugenic and special
production areas. Engineering (Transactions of the CSAE), v.31, n.18, p.204-211,
2015. Available from: <http://d.wanfangdata.com.cn/periodical/
nygcxb201518029>. Accessed: Apr. 19, 2018. doi: 10.11975/j. issn.1002-6819.2015.18.029. FENG Z. et al. Study on the spatiotemporal patterns and
contribution factors of China’s grain output increase during 2003-
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ileName=ZRZX201606001&DbName=CJFQ2016>. Accessed:
Nov. 19, 2017. ISBN/ISSN: 1000-3037. INOUYE A. et al. Fresh Deciduous Fruit Annual-China. USDA
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10.3390/su9020313. This study only discussed the evolutionary
characteristics of apple production spatial distribution
in China from 1978 to 2016 using the LMDI
decomposition method and barycenter analysis
model, thus providing some insights concerning
where practice and research may evolve in the future. CONCLUSION (2) Analysis at both the national and
provincial scales showed that the expansion of apple
acreage and the increase in yield were the main
driving factors for increasing apple output in China. The contribution of yield was greater than the acreage
contribution, indicating that variety improvement,
cultivation
technology
innovation,
orchard
equipment and infrastructure upgrade, technological
advancements, the improvement of fruit farmers’
quality were the main reasons for the apple output
increase in China. In this study, the LMDI decomposition
method, GIS technology and the apple acreage, output
and yield data at the national and provincial scales from
1978 to 2016 were used to comprehensively analyze
the characteristics of apple output increase, regional
distribution, and barycenter movement in China. The
findings were presented as follows: In this study, the LMDI decomposition
method, GIS technology and the apple acreage, output
and yield data at the national and provincial scales from
1978 to 2016 were used to comprehensively analyze
the characteristics of apple output increase, regional
distribution, and barycenter movement in China. The
findings were presented as follows: (1) From 1978 to 2016, the apple acreage
and output in China increased by 1.64 million ha
and 41.61 million tons, but the proportion of apple
acreage in total fruit acreage decreased from 40.98%
in 1978 to 17.90% in 2016, the proportion of apple
output accounted for fruit total output dropped from
34.63% in 1978 to 24.22% in 2016. This showed that
the contribution of technological progress to China’s
apple output growth ranked first, that is, technological
progress driving higher yield leads to a slower decline
in the proportion of apple output than a decrease in
the proportion of apple acreage. (3) From 1978 to 2016, apple output in 23
apple-producing provinces showed varying degrees
of growth. Among them, the apple output increase
in 8 provinces exceeded 1 million tons, accounted
for 92.67% of the total increase in apple output
nationwide. There were 13 provinces with a super-
growth increase in apple output, and 10 provinces
with a lagging-growth increase, 4 provinces have
increased apple output for more than 30 years. It
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690, 2014. Available from: <https://kns.cnki.net/KCMS/detail/
detail.aspx?FileName=ZRZY201404003&DbName=CJFQ2014>. Accessed: Jan. 19, 2018. ISBN/ISSN: 1007-7588. Ministry of Agriculture and Rural Affairs of the People’s Republic
of China (MARA). China Apple Industry Development Report:
1995-2005. China Agricultural Press: Beijing, China. 2007. Available from: <https://spu.jd.com/1079722397.html>. Accessed:
Jan. 19, 2015. ISBN: 9787109116269. XU Y et al. 2015. Analysis on the pattern change and influence factors
of apple production in China. Information Engineering Research
Inst: Newark, USA. ISBN: 2160-1070-978-1-61275-070-5. National Bureau of Statistics of China (NBSC). New China
Agricultural Statistics for 60 Years. China Statistics Press:
Beijing, China. 2009. Available from: <https://navi.cnki.net/knavi/
YearbookDetail?pcode=CYFD&pykm=YXZGE>. Accessed: Jan. 19, 2012. ISBN: 7-5037-3402-7/F.1286. YANG W. et al. Studies on the spatial distribution changing of
China’s rice production and its influencing factors. Economic
Geography, v.31, n.12, p.2086-2093, 2011. Available from:
<https://kns.cnki.net/KCMS/detail/detail.aspx?FileName=JJDL2
01112025&DbName=CJFQ2011>. Accessed: Jan. 19, 2019. doi:
10.15957/j.cnki.jjdl.2011.12.025. National Bureau of Statistics of China (NBSC). Chinese
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?pcode=CYFD&pykm=YZGNT>. Accessed: Jan. 19, 2018. ISBN:
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agricultural policy output since 1978. Journal of Geographical
Sciences, v.21, n.3, p.475-488, 2011. Available from: <https://link. springer.com/article/10.1007/s11442-011-0858-1>. Accessed: Jan. 19, 2018. doi: 10.1007/s11442-011-0858-1. National Bureau of Statistics of China (NBSC). China Rural
Statistical Yearbook. China Statistics Press: Beijing, China,
1985-2017. Available from: <https://navi.cnki.net/knavi/Yearbook
Detail?pcode=CYFD&pykm=YMCTJ>. Accessed: Jan. 19, 2018. ISBN: 978-7-5037-9033-1. YUAN B. et al. Spatial pattern evolution and its driving factors
for apple industry in China: Based on micro perspective of
farmers’ decision making. Journal of Arid Land Resources
and Environment, v.31, n.6, p.32-37, 2017. Available from:
<https://kns.cnki.net/KCMS/detail/detail.aspx?FileName=GHZH
201706006&DbName=CJFQ2017>. Accessed: Jan. 19, 2018. doi:
10.13448/j.cnki.jalre.2017.175. YUAN B. et al. Spatial pattern evolution and its driving factors
for apple industry in China: Based on micro perspective of
farmers’ decision making. Journal of Arid Land Resources
and Environment, v.31, n.6, p.32-37, 2017. Available from:
<https://kns.cnki.net/KCMS/detail/detail.aspx?FileName=GHZH
201706006&DbName=CJFQ2017>. Accessed: Jan. 19, 2018. doi:
10.13448/j.cnki.jalre.2017.175. NIE L. et al. The evolution analysis of the grain production
concentration in China. Research of Agricultural Modernization,
v.36, n.3, p.380-386, 2015. Available from: <https://kns.cnki. net/KCMS/detail/detail.aspx?FileName=NXDH201503010&D
bName=CJFQ2015>. Accessed: Jan. 19, 2016. doi：10.13872
/j.1000-0275.2015.0044. ZHANG C. et al. Analysis on the layout of China’s apple production
transition. Economic Geography, v.38, n.8, p.141-151, 2018. Available from: <http://kns.cnki.net/kcms/detail/detail.aspx?File
Name=JJDL201808018&DbName=DKFX2018>. Accessed: Jan. 19, 2019. The Evolutionary Characteristics of Apple Production Layout in China from 1978 to 2016. Evolvement analysis of the economic gravity center
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and economic development gravity center in the regions of South Ciência Rural, v.51, n.6, 2021.
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Seismic Performance Detection in Retrofitted Concrete Spillway by FEM
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Keywords –Shotcrete, Retrofitting, Modal Analysis, Concrete Spillway, Seismic Performance Shotcrete;
concrete
consisting
of
cement,
aggregate, water and some necessary additives; It
is a type of concrete coating obtained as a result of
spraying the concrete on the surface to be coated at
a certain speed with the help of a special hose and
equipment. Shotcrete is a working method based
on very fast freezing. When looking at the usage
phase in the tunnel, it is excavated immediately
after the steel shoring and steel mesh are thrown
away. A dense-cement design concrete (injection
as it is known in the field) and the instant setter are
held in two same positions, then poured with the
help of machine pumps to the place where the steel
shoring and steel mesh are laid. The thinnest point
in spillage; is that the instant setter and the
injection come in separate channels up to the
mouth of the pump that makes the injection. Their
complete mixing occurs at the mouth point of the
concrete pump. The sprayed concrete solidifies *furkan.gunday@giresun.edu.tr Abstract – Earthquakes damage not only buildings but also infrastructure and superstructure facilities. Therefore, the seismic performance of all structure types is of vital importance. It is known that seismic
performance losses occur in various types of structures due to the earthquake effect. Various retrofitting
methods are available for such structures to regain their former seismic performance. Appropriate
retrofitting techniques should be applied according to the type of structure, its damage and the conditions
of the region. In addition, the retrofitting process can be done to the entire structure or to some sections or
parts of the structures. It is known that environmental and forced vibrations, especially in spillways,
increase the destructive effects by activating the water in the spillway. In this type of situation, stiffness
losses for spillways increase even more. Their collapse becomes inevitable under the influence of the next
forced and environmental vibrations. For this reason, this study focused on the technique of retrofitting
concrete spillways with shotcrete. A sample concrete spillway model was created using the finite element
method and modal analysis was performed. Then, the same model was retrofitted with shotcrete and
modal analysis was performed. When the results obtained were compared, it was seen that retrofitting
with shotcrete had a positive effect on the rigidity of the concrete spillway. In light of all these findings,
shotcrete can be an option for retrofitting concrete spillways. Keywords –Shotcrete, Retrofitting, Modal Analysis, Concrete Spillway, Seismic Performance All Sciences Proceedings
http://as-proceeding.com/
3rd International Conference on
Innovative Academic Studies
September 26-28, 2023 : Konya, Turkey
https://www.icias.net/
© 2023 Published by All Sciences Proceedings All Sciences Proceedings
http://as-proceeding.com/
3rd International Conference on
Innovative Academic Studies
September 26-28, 2023 : Konya, Turkey
https://www.icias.net/
© 2023 Published by All Sciences Proceedings All Sciences Proceedings
http://as-proceeding.com/ https://www.icias.net/ 3rd International Conference on
Innovative Academic Studies III. FINDINGS AND DISCUSSION The concrete spillway model and concrete
spillway retrofitted model were modal analysed
using SAP2000 software. The period and mode
shapes obtained for both models are obtained for
each mode. I. INTRODUCTION Many types of structures are damaged as a result
of natural disasters (such as floods, landslides,
earthquakes). According to the degree of damage
received, loss of life and property occurs with the
collapse. In some structures, collapse does not
occur as a result of disasters, but damage is
observed [1], [2], [3], [4]. Various reinforcement
methods are applied in such structures. Thus, the
structures are restored to their former bearing
strength and stiffness, thus avoiding possible
collapse. It is known that the destructive effects of
natural disasters also damage structures, thus
damage may occur much more than expected. Concrete spillways are also exposed to these
destructive effects. Various proven retrofit methods
are available to solve such problems. The use of
shotcrete is one of these retrofit methods. 316 within seconds and interrupts the interaction of the
ground and the air in the gradual tunnel
construction. In cases where shotcrete is used and
the interaction of the ground and air is not
interrupted, the mirror dries as the day progresses,
spills, bursts and collapses may occur [5], [6]. Researchers have conducted experimental and
theoretical studies [7], [8], [9], [10], [11], [12],
[13], [14], [15] about shotcrete which also
contributed to this study. method, the SAP2000 software, which is used in
the field of academic and engineering applications
all over the world, was used. In this study, [25],
[26], [27] studies in which the finite element
method was used for retrofit were used. A. Description of Concrete Spillway Model The model concrete spillway is dimensions;
width is 5 m, wall heights are 2 m and length are
25 m, wall thickness is 0.20 m. The mechanical
parameters of concrete spillway in model are;
poisson's ratio: 0.2, modulus of elasticity: 30 GPa,
density: 24 kN/m3. The concrete spillway finite
element model was created using the SAP2000
software. The finite element model of the concrete
spillway is given in fig. 1. y
A spillway is a structure used to provide the
controlled release of water downstream from a dam
or levee, typically into the riverbed of the dammed
river itself. Spillways ensure that water does not
damage parts of the structure not designed to
convey water. Spillways have been used for over
3000 years [16]. Despite being superseded by more
modern engineering techniques such as hydraulic
jumps in the mid twentieth century, since around
1985 [17] interest in stepped spillways and chutes
has been renewed, partly due to the use of new
construction
materials
(e.g.,
roller-compacted
concrete, gabions) and design techniques (e.g.,
embankment overtopping protection) [18], [19]. The steps produce considerable energy dissipation
along the chute [20] and reduce the size of the
required downstream energy dissipation basin [21],
[22]. Research is still active on the topic, with
newer
developments
on
embankment
dam
overflow protection systems, [22] converging
spillways [23] and small weir design [24]. Fig. 1 3D Finite element model of the concrete spillway
B. Description of Concrete Spillway Retrofitted
Model Fig. 1 3D Finite element model of the concrete spillway Fig. 1 3D Finite element model of the concrete spillway
B. Description of Concrete Spillway Retrofitted
Model Fig. 1 3D Finite element model of the concrete spillway
B Description of Concrete Spillway Retrofitted The aim of this study is to contribute to the
strengthening of concrete spillways used for many
purposes according to their modal conditions. In
this study, the effects of the shotcrete retrofit
method on concrete spillway modal periods and
mode shapes are investigated. The inner surface of the existing concrete
spillway model was coated with 0.05 m of
shotcrete to create retrofitted model. Mechanical
properties of the applied shotcrete materials;
poisson's ratio: 0.2, modulus of elasticity: 35 GPa,
density: 23 kN/m3. II. MATERIALS AND METHOD In this study, concrete spillway model was
created and modal analysis was carried out with the
finite element method. The concrete spillway
model was retrofitted with shotcrete retrofitting
and two concrete spillway model was created for
comparison. Variables on the model should be
minimized in order to better see the reinforcement
effects. Therefore, it was emphasized that the
models
should
be
designed
simply
and
symmetrically. Thus, it is aimed that the only
variable between both models is the retrofitting
method. In the application of the finite element C. Comparison of Modal Analysis Results Fig. 9 3. Mode shape (Period value = 0.077 s) Fig. 9 3. Mode shape (Period value = 0.077 s) The comparison of periods of the model non-
retrofitted and retrofitted are given in Table 1. Table 1. Comparison of period values Table 1. Comparison of period values
Mode
1
2
3
4
5
Non-
retrofitted
0.165 0.149 0.103 0.093 0.080
Retrofitted
0.155 0.144 0.077 0.069 0.058
Difference
(s)
0.010 0.005 0.026 0.024 0.022
Difference
(%)
6.06
3.36
25.24 25.81 27.50 Fig. 9 3. Mode shape (Period value = 0.077 s) Fig. 9 3. Mode shape (Period value = 0.077 s) The comparison of mode shapes of the model
non-retrofitted and retrofitted model is given in
Table 2. Fig. 10 4. Mode shape (Period value = 0.069 s) Table 2. Comparison of mode shapes
Mode Non-retrofitted
Retrofitted
1
Torsional
Torsional
2
Translational
Translational
3
Torsional
Torsional
4
Torsional
Torsional
5
Torsional
Torsional Table 2. Comparison of mode shapes A. Modal Analysis Results of Concrete Spillway
Model 11 5. Mode shape (Period value = 0.058 s) Fig. 8 2. Mode shape (Period value = 0.144 s) Fig 11 5 Mode shape (Period value = 0 058 s) Fig. 8 2. Mode shape (Period value = 0.144 s) Fig. 11 5. Mode shape (Period value = 0.058 s) Fig. 8 2. Mode shape (Period value = 0.144 s) Fig. 8 2. Mode shape (Period value = 0.144 s) C. Comparison of Modal Analysis Results A. Modal Analysis Results of Concrete Spillway
Model The modal analysis of the concrete spillway
model was performed with finite element method. The first 5 modes were taken into account in the 317 analysis. Obtained results are presented in figures
2,3,4,5,6 as periods and mode shapes. Fig. 2 1. Mode shape (Period value = 0.165 s)
Fig. 3 2. Mode shape (Period value = 0.149 s)
Fig. 5 4. Mode shape (Period value = 0.093 s)
Fig. 6 5. Mode shape (Period value = 0.080 s)
B. Modal Analysis Results of Concrete Spillway
Retrofitted Model
The modal analysis of the concrete spillway
retrofitted model was performed with finite
element method. The first 5 modes were taken into
account in the analysis. Obtained results are
presented in figures 7,8,9,10,11 as periods and
mode shapes. analysis. Obtained results are presented in figures
2,3,4,5,6 as periods and mode shapes. analysis. Obtained results are presented in figures
2,3,4,5,6 as periods and mode shapes. Fig. 5 4. Mode shape (Period value = 0.093 s) Fig. 2 1. Mode shape (Period value = 0.165 s) Fig. 5 4. Mode shape (Period value = 0.093 s) Fig. 5 4. Mode shape (Period value = 0.093 s) Fig. 5 4. Mode shape (Period value = 0.093 s) Fig. 6 5. Mode shape (Period value = 0.080 s)
B. Modal Analysis Results of Concrete Spillway
Retrofitted Model Fig. 2 1. Mode shape (Period value = 0.165 s) Fig. 2 1. Mode shape (Period value = 0.165 s) Fig. 3 2. Mode shape (Period value = 0.149 s) Fig. 6 5. Mode shape (Period value = 0.080 s) B. Modal Analysis Results of Concrete Spillway
Retrofitted Model B. Modal Analysis Results of Concrete Spillway
Retrofitted Model Fig. 3 2. Mode shape (Period value = 0.149 s) Fig. 3 2. Mode shape (Period value = 0.149 s) The modal analysis of the concrete spillway
retrofitted model was performed with finite
element method. The first 5 modes were taken into
account in the analysis. Obtained results are
presented in figures 7,8,9,10,11 as periods and
mode shapes. Fig. 4 3. Mode shape (Period value = 0.103 s) Fig. 7 1. Mode shape (Period value = 0.155 s) Fig. 4 3. Mode shape (Period value = 0.103 s) Fig. 7 1. Mode shape (Period value = 0.155 s) 318 Fig 11 5 Mode shape (Period value = 0 058 s) Fig. 8 2. Mode shape (Period value = 0.144 s)
Fig. IV. CONCLUSION Fig. 10 4. Mode shape (Period value = 0.069 s) In the mode 1, the period difference between
non-retrofitted and retrofitted status was obtained
as 0.010 s. The effect of period retrofitting with
shotcrete
retrofitting
as
a
percentage
was
determined as 6.06. In the mode 2, the period difference between
non-retrofitted and retrofitted status was obtained
as 0.005 s. The effect of period retrofitting with
shotcrete
retrofitting
as
a
percentage
was
determined as 3.36. 319 [3] Tuhta, S., Abrar, O., & Günday, F. (2019). Experimental Study on Behavior of Bench-Scale Steel
Structure Retrofitted with CFRP Composites under
Ambient Vibration. European Journal of Engineering
Research and Science, 4(5), 109-114. In the mode 3, the period difference between
non-retrofitted and retrofitted status was obtained
as 0.026 s. The effect of period retrofitting with
shotcrete
retrofitting
as
a
percentage
was
determined as 25.24. [4] Tuhta, S., & Günday, F. (2019). Application of OMA
on the Bench-Scale Aluminum Bridge Using Micro
Tremor Data. International Journal of Advance
Research and Innovative Ideas in Education, 5(5), 912-
923. In the mode 4, the period difference between
non-retrofitted and retrofitted status was obtained
as 0.024 s. The effect of period retrofitting with
shotcrete
retrofitting
as
a
percentage
was
determined as 25.81. [5] Arıoğlu, E. & Yüksel, A., Tünel ve Yer altı
Mühendislik Yapılarında Çözümlü Püskürtme Beton
Problemleri, TMMOB Maden Mühendisleri Odası,
İstanbul, 1999. In the mode 5, the period difference between
non-retrofitted and retrofitted status was obtained
as 0.022 s. The effect of period retrofitting with
shotcrete
retrofitting
as
a
percentage
was
determined as 27.50. [6] Tuhta, S. (2023). Simulation of Shotcrete in RC Tunnel
Using Finite Element Method. Presented at the 4. Baskent International Conference on Multidisciplinary
Studies, Ankara. [7] Daraei, A., Hama Ali, H. F., Qader, D. N., & Zare, S. (2022). Seismic retrofitting of rubble masonry tunnel:
evaluation of steel fiber shotcrete or inner concrete
lining alternatives. Arabian Journal of Geosciences,
15(11), 1074. With the retrofitting, some minor change in
translation and torsion was observed. It is thought
that these changes are not very important. No
negative change was observed in terms of retrofit
in mode shapes. [8] Morgan, D. R., Kazakoff, K., & Ibrahim, H. (2006). Seismic retrofit of a concrete immersed tube tunnel with
reinforced shotcrete. In Shotcrete for underground
support X (pp. 270-284). IV. CONCLUSION p
As a result of this study, it is clearly seen that the
stiffness of the concrete spillway model increases
by retrofitting the concrete spillway model with
shotcrete retrofitting. In general, it is seen that the
decrease in the periods is between 3.36% and
27.50%. In the 1st mode, in other words, there is a
decrease of 6.06 percent in the dominant period
value. It is known that this is a somewhat positive
situation in terms of retrofit. The reason for this
situation can be shown as the single layer
application of shotcrete and long of spillway
model. It is estimated that with the thicker
application of shotcrete, the period decrease will be
greater in 1st and 2nd mode. It can be said that the
period declines in the 3rd, 4th and 5th modes are
quite positive. The maximum decrease in period
values was observed in the 5th mode %27.50 and
the minimum decrease in period values was
observed in the 2nd mode %3.36. In the light of all
these results, the concrete spillway can retrofit with
the shotcrete, taking into account the existing state
of the spillway and environmental factors. [9] Morgan, D. R., & Jolin, M. (2022). Shotcrete:
Materials, Performance and Use (Vol. 22). CRC Press. [10] Eickmeier, D., Kaundinya, I., & Vollmann, G. (2017). Structural retrofitting of rescue tunnels in single shell
shotcrete construction-requirements and experiences. [11] Yang, J. M., Kim, J. K., & Yoo, D. Y. (2017). Performance of shotcrete containing amorphous fibers
for tunnel applications. Tunnelling and Underground
Space Technology, 64, 85-94. [12] Han, W., Jiang, Y., Gao, Y., & Koga, D. (2020). Study
on design of tunnel lining reinforced by combination of
pcm shotcrete and FRP grid technique. In IOP
Conference Series: Earth and Environmental Science
(Vol. 570, No. 5, p. 052035). IOP Publishing. [13] Yoggy, G. D. (2000). The history of shotcrete. Shotcrete
(American Shotcrete Association), 2(4), 28-9. [14] Wang, J., Niu, D., & Zhang, Y. (2015). Mechanical
properties, permeability and durability of accelerated
shotcrete. Construction and Building Materials, 95, 312-
328. [15] Wu, K., Shao, Z., Sharifzadeh, M., Chu, Z., & Qin, S. (2022). Analytical approach to estimating the influence
of shotcrete hardening property on tunnel response. Journal of Engineering Mechanics, 148(1), 04021127. [2] Tuhta, S. (2021). Analytical and Experimental Modal
Analysis of Model Wind Tunnel using Microtremor
Excitation. Wind & Structures, 32(6), 563-571. [1] Tuhta, S. (2018). GFRP retrofitting effect on the
dynamic characteristics of model steel structure. Steel
and Composite Structures, 28(2), 223-231.
[2] Tuhta, S. (2021). Analytical and Experimental Modal
Analysis of Model Wind Tunnel using Microtremor
Excitation. Wind & Structures, 32(6), 563-571. [1] Tuhta, S. (2018). GFRP retrofitting effect on the
dynamic characteristics of model steel structure. Steel
and Composite Structures, 28(2), 223-231. REFERENCES [16] H. Chanson (2002). Historical Development of Stepped
Cascades for the Dissipation of Hydraulic Energy. Transactions of the Newcomen Society. 71 (2): 295-
318. [17] H. Chanson (2000). Hydraulics of Stepped Spillways. Journal of Hydraulic Engineering. 126 (9): 636-637. [18] H. Chanson (1995). Hydraulic Design of Stepped
Cascades, Channels, Weirs and Spillways. Pergamon. ISBN 978-0-08-041918-3. 320 [19] H. Chanson (2002). The Hydraulics of Stepped Chutes
and Spillways. Balkema. [20] Rajaratnam, N. (1990). "Skimming Flow in Stepped
Spillways". Journal of Hydraulic Engineering. 116 (4):
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of Stepped Spillways and Downstream Energy
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Vol. 17, No. 4, pp. 223-244. [23] S.L. Hunt, S.R. Abt & D.M. Temple (2008). Hydraulic
Design of Stepped Spillways and Downstream Energy
Dissipators
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Concrete Stepped Spillways. [24] I. Meireles; J. Cabrita; J. Matos (2006). Non-Aerated
Skimming Flow Properties on Stepped Chutes over
Small Embankment Dams in Hydraulic Structures: a
Challenge to Engineers and Researchers, Proceedings of
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Tunnel Formwork Structure Retrofitted with JFRP by
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Concrete Tunnel Retrofitted with CFRP using Finite
Element Method. International Journal of Innovations in
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English
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Acknowledgment to the Reviewers of Games in 2022
|
Games
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cc-by
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Editorial Games Editorial Office
MDPI, St. Alban-Anlage 66, 4052 Basel, Switzerland
Published: 26 January 2020 The editorial team greatly appreciates the reviewers who have dedicated their considerable time
and expertise to the journal’s rigorous editorial process over the past 12 months, regardless of
whether the papers are finally published or not. In 2019, a total of 49 papers were published in the
journal, with a median time to first decision of 30.72 days and a median time from submission to
publication of 73 days. The editors would like to express their sincere gratitude to the following
reviewers for their generous contribution in 2019: Dahm, Matthias
Dalhaus, Tobias
Danese, Giuseppe
DE MARCO, Giuseppe
Deer, Lachlan
Delaney, Jason
Di, Sharon
Dimant, Eugen
Doruk, Iris
Dürsch, Peter
Ellis, Andrew
Erovenko, Igor
Espinosa, Maria Paz
FARVAQUE, Etienne
Fernández-Revelles, Andrés B. Filson, Darren
Fochmann, Martin
Fooken, Jonas
Frąckiewicz, Piotr
Fu, Jingcheng
Fujita, Toshiyuki
Galliera, Arianna
Gangl, Katharina
Ganzfried, Sam
Ghidoni, Riccardo
Goff, Sandra
Gómez-Miñambres, Joaquín
Gonzalez-Ramirez, María Jimena
Gratton, Gabriele
Grimshaw, Shaun B Abdalzaher, Mohamed S. Adriani, Fabrizio
Akhmetzhanov, Andrei R. Aleksandr, Alekseev
Alm, James
Alt, Marius
Alvarez-Bermejo, Jose Antonio
Ansink, Erik
Aragones, Enriqueta
Archetti, Marco
Arieli, Itai
Attanasi, Giuseppe
Attanasi, Giusppe
Aurell, Alexander
Badicu, Georgian
Bartholomae, Florian
Béal, Sylvain
Bilancini, Ennio
Biondo, Alessio Emanuele
Boonen, Tim J. Brownback, Andy
Bruttel, Lisa
Buchen, Clemens
Cartwright, Edward
Casajus, André
Castillo, Geoffrey
Cavalli, Fausto
Chessa, Michela
Coatney, Kalyn
Cunningham, David E. Editorial www.mdpi.com/journal/games Games 2020, 11, 7; doi:10.3390/g11010007 2 of 3 Games 2020, 11, 7
Gu, Junjian
Gueron, Yves
Gürerk, Özgür
Hanappi, Hardy
Hariskos, Wasilios
Harre, Michael
Harrison, Glenn
Hartman, Bruce
Hedges, Jules
Heine, Florian
Heintz, Christophe
Hilbe, Christian
Hochman, Guy
Hod, Shahar
Hoffer, Adam
Holzman, Ron
Howe, Lance
Hoyer, Britta
Huttegger, Simon
Ikerionwu, Charles
Jaffe, Sonia
Jajac, Nikša
Javarone, Marco Alberto
Javarone, Marco Alberto
Jin, Haiming
Johnson, David
Jung, Sungdo
Kawamura, Kohei
Kirstein, Roland
Klazar, Stanislav
Koch, Alexander
Kölle, Felix
Kolokoltsov, Vassili
Koloniari, Georgia
Kramer, Berber
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Kroumi, Dhaker
Kupferman, Orna
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Kurschilgen, Michael
Lanotte, Ruggero
Lee, Jinkwon
Leland, Jonathan Gu, Junjian
Gueron, Yves
Gürerk, Özgür
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Hariskos, Wasilios
Harre, Michael
Harrison, Glenn
Hartman, Bruce
Hedges, Jules
Heine, Florian
Heintz, Christophe
Hilbe, Christian
Hochman, Guy
Hod, Shahar
Hoffer, Adam
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Howe, Lance
Hoyer, Britta
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Jaffe, Sonia
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Rosa Garcia, Alfonso
Samu, Flóra
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Schurr, Amos
Schwanebeck, Benjamin
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© 2020 by the author. 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/). 3 of 3 © 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access
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Translating shared decision-making into health care clinical practices: Proof of concepts
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Implementation science
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BioMed Central BioMed Central Op
Study protocol
Translating shared decision-making into health care clinical
practices: Proof of concepts
France Légaré*1, Glyn Elwyn2, Martin Fishbein3, Pierre Frémont1,
Dominick Frosch4, Marie-Pierre Gagnon1, David A Kenny5,
Michel Labrecque1, Dawn Stacey6, Sylvie St-Jacques1 and Trudy van der
Weijden7 Address: 1Centre hospitalier universitaire de Québec, Hôpital St-François D'Assise, Unité de recherche évaluative, 10 rue de l'Espinay, Québec,
Québec, G1L 3L5, Canada, 2Department of Primary Care and Public Health, School of Medicine, Cardiff University, Neuadd Meirionnydd, Heath
Park CF 14 4YS, UK, 3Annenberg School for Communication, University of Pennsylvania, 3620 Walnut Street, Philadelphia, PA 19104, USA,
4UCLA Med-GIM & HSR, BOX 951736, 911 Broxton, Los Angeles, CA 90095-1736, USA, 5Department of Psychology, University of Connecticut,
406 Babbidge Road Unit 1020 Storrs, CT 06269-1020, USA, 6School of Nursing, University of Ottawa, 451 Smyth, Room RGN 3247A Ottawa, ON
K1H 8M5, Canada and 7Department of General Practice/School of Public Health and Primary Care Caphri, Maastricht University, PO Box 616,
6200 MD Maastricht, The Netherlands Email: France Légaré* - france.legare@mfa.ulaval.ca; Glyn Elwyn - elwyng@cardiff.ac.uk; Martin Fishbein - mfishbein@asc.upenn.edu;
Pierre Frémont - pierre.fremont@crchul.ulaval.ca; Dominick Frosch - dfrosch@mednet.ucla.edu; Marie-
Pierre Gagnon - david.kenny@uconn.edu; David A Kenny - marie-pierre@fsi.ulaval.ca; Michel Labrecque - michel.labrecque@mfa.ulaval.ca;
Dawn Stacey - dstacey@uottawa.ca; Sylvie St-Jacques - sylvie.st-jacques@crsfa.ulaval.ca; Trudy van der
Weijden - trudy.vanderweijden@hag.unimass.nl * Corresponding author Received: 3 December 2007
Accepted: 14 January 2008 Received: 3 December 2007
Accepted: 14 January 2008 Published: 14 January 2008 Implementation Science 2008, 3:2
doi:10.1186/1748-5908-3-2 This article is available from: http://www.implementationscience.com/content/3/1/2 © 2008 Légaré et al; licensee BioMed Central Ltd. g
;
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. Implementation Science Open Access Background situation. These results suggest that health professionals
might be selecting, a priori, certain patients for whom they
believe that SDM is feasible or functional. This is of some
concern because physicians may misjudge patients' desire
for active involvement in decision-making [17]. These
results highlight the importance of the patient's input for
successful implementation of SDM and patient decision
aids in clinical practice. Hence, the concomitant evalua-
tion of patients' and providers' perception of the decision-
making process (dyadic decision-making) remains una-
voidable for those interested in a comprehensive under-
standing of clinical decision-making [18]. g
With the increased emphasis on engagement of patients as
partners in their care, there is a need to determine effective
ways to involve patients in the process by which health-
related decisions are made in clinical settings. The health
decision-making process is complex, as it brings together
a health professional, considered a scientific content
expert, and an individual, considered an expert in his own
personal values [1]. It is in this context that there is con-
siderable interest today in the process of shared decision-
making (SDM) [2]. SDM is defined as a decision-making
process jointly shared by patients and their health care
provider [3], and is said to be the crux of patient-centered
care [4]. It relies on the best evidence about risks and ben-
efits associated with all available options (including
doing nothing) and on the values and preferences of
patients, without excluding those of health professionals
[5]. Therefore, it includes the following components:
establishing a context in which patients' views about treat-
ment options are valued and deemed necessary; reviewing
the patient's preferences for role in decision-making;
transferring technical information; making sure patients
understand this information; helping patients base their
preference on the best evidence; eliciting patients' prefer-
ences; sharing treatment recommendations; and making
explicit the component of uncertainty in the clinical deci-
sion-making process [6]. However, a recent systematic
review identified 161 conceptual definitions of SDM, thus
suggesting that SDM as a concept is still an object of ongo-
ing research [7]. In recent years, social cognitive theoretical models have
been used to improve our understanding of health care
behaviors [19,20] and health care professionals' behav-
iors [21-23]. Background At the time this research protocol was pro-
posed, most of the studies that had been conducted to
improve our understanding of the implementation of
SDM in clinical practice had no clear theoretical basis. This is of some concern because it has been acknowledged
that more attention needs to be given to the combination
of different theories that could help us understand profes-
sional behaviours [14,24] and design effective implemen-
tation strategies [25]. Nonetheless, when social cognitive
theoretical models have been used to study health care-
related behaviors, such as communication during a con-
sultation or the patient's adherence to medical advice,
groups of patients and groups of health professionals have
been studied separately as if living in separate worlds. This
is a source of concern because 'the right thing to do' may
only emerge in the course of the professional's contact
with patients or clients [26]. Considering simultaneously
both perspectives of the decision-making process is a log-
ical approach for conceptualizing SDM and its implemen-
tation in clinical practice, as well as for identifying which
aspects should be jointly evaluated by patients and their
providers [27]. Patient decision aids and decision coaching are effective
interventions to support patients to engage in SDM. When
compared to usual care, decision aids reduce patients' pas-
sivity in the decision-making process, improve patients'
knowledge about clinical options, increase realistic expec-
tations, reduce decisional conflict and the number of indi-
viduals who remain undecided, increase satisfaction with
the decision-making process, and increase congruence
between patient preferences and clinical options selected
[8]. Moreover, notwithstanding the preferred role of
patients, active participation of patients in the decision-
making process correlates with improved quality of life
measured three years after the decision [9]. However, the study of dyads poses specific conceptual as
well as methodological issues [28], and thus several chal-
lenges in advancing knowledge in this area remain,
including the lack of consensus on which aspects should
be jointly evaluated by patients and their providers; the
absence of standardized measures with established psy-
chometric properties; and the failure to take into account
the clustering of patients under health providers [29]. In
the majority of the studies pertaining to the relationship
between a patient and a health care provider, very few
have adequately addressed these methodological issues. http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 Page 2 of 6
(page number not for citation purposes) Abstract Background: There is considerable interest today in shared decision-making (SDM), defined as a decision-making
process jointly shared by patients and their health care provider. However, the data show that SDM has not been broadly
adopted yet. Consequently, the main goal of this proposal is to bring together the resources and the expertise needed
to develop an interdisciplinary and international research team on the implementation of SDM in clinical practice using
a theory-based dyadic perspective. Methods: Participants include researchers from Canada, US, UK, and Netherlands, representing medicine, nursing,
psychology, community health and epidemiology. In order to develop a collaborative research network that takes
advantage of the expertise of the team members, the following research activities are planned: 1) establish networking
and on-going communication through internet-based forum, conference calls, and a bi-weekly e-bulletin; 2) hold a two-
day workshop with two key experts (one in theoretical underpinnings of behavioral change, and a second in dyadic data
analysis), and invite all investigators to present their views on the challenges related to the implementation of SDM in
clinical practices; 3) conduct a secondary analyses of existing dyadic datasets to ensure that discussion among team
members is grounded in empirical data; 4) build capacity with involvement of graduate students in the workshop and
online forum; and 5) elaborate a position paper and an international multi-site study protocol. Discussion: This study protocol aims to inform researchers, educators, and clinicians interested in improving their
understanding of effective strategies to implement shared decision-making in clinical practice using a theory-based dyadic
perspective. Page 1 of 6
(page number not for citation purposes) (page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 3) Perform secondary analyses of existing dyadic datasets 3) Perform secondary analyses of existing dyadic datasets
One of the purposes of the workshop is to use existing
dyadic datasets to explore the research questions pre-
sented above. This will ensure that the team's discussions
are grounded in data. A dyadic dataset is defined as a data-
set that include data on both members of a dyad that is a
pair of two individuals. When only one member of the
dyad is measured, the design is termed one-sided. When
both members are measured on the same variable, the
design is termed two-sided or reciprocal. Three different
types of dyadic designs can be identified: 1) standard
dyadic design in which each individual is linked to one
and only one other individual in the sample; 2) one-with-
many design in which one individual is linked to many
other individuals; and 3) Social Relation Model design in
which each individual is paired with multiple others, and
each of these others is also paired with multiple others Other collaborators from the US are the two key invited
presenters at the two-day workshop. Together, they will
bring extensive expertise on the theoretical underpinnings
of implementing behavioral change [44-46], the study of
interpersonal influences [28] and the analysis of dyadic
data [47]. 1) Foster ongoing communication among members of this
international research network Consequently, the main goal of this new international
collaboration is to bring together the resources and the
expertise needed to develop an interdisciplinary and inter-
national research team dedicated to the study of imple-
menting SDM in clinical practice using a theory-based
dyadic perspective. Its objectives are: 1) to develop a col-
laborative research network in this area; 2) to test new
strategies to analyze dyadic data and explore the impact of
such analysis on the theoretical underpinnings guiding
the implementation of SDM in clinical practice; and 3) to
define a research agenda and best practices regarding the
implementation of SDM in clinical practice. At the outset of the project, using internet-based forum or
conference calls hosted by the group at Université Laval,
all participants discuss a similar definition of the prob-
lems and challenges with implementing SDM, including
methodological issues with analysis of dyadic data. Partic-
ipants share relevant literature within the group and start
to think about how this applies to the identified prob-
lems/challenges. Relevant collated documents are used to
create a knowledgebase that can be shared through a web-
site. An e-journal club dedicated to the critical appraisal of
relevant health-related dyadic studies is proposed. It is
possible that other issues that are truly unique to SDM
will be identified. Ongoing communication is encouraged
through a bi-weekly e-bulletin that is sent to all partici-
pants. Methods
Participants
i i p
Participants include researchers from Canada, US, UK,
and Netherlands representing medicine, nursing, psychol-
ogy, community health, and epidemiology. Team mem-
bers from Canada contribute to this project by: 1)
coordinating the proposed international collaboration; 2)
hosting the workshop; 3) providing the necessary moni-
toring and on-going support that is required for an inter-
national research group to evolve and develop; 4) hosting
the internet-based forum and collating relevant material
to be shared with the team members; 5) sharing their
experience and expertise in the development of a dyadic
approach to the implementation of SDM in clinical prac-
tice and the data management of large existing datasets; 6)
offering a unique perspective to implementing SDM in
nursing clinical practice [33,34]; and 7) providing data-
sets to be used during the workshop. 2) Provide a workshop
A
d
k h A two-day workshop in Quebec City will be based on the
previous work and expertise of participants. Each partici-
pant will be asked to prepare a short presentation outlin-
ing how they propose to address the following three
research questions: 1) What are the most appropriate the-
oretical frameworks to assess how health professionals
and patients engage in SDM, and what are the most
appropriate theoretical frameworks to guide implementa-
tion of SDM in clinical practice? 2) What are the most
appropriate measures to assess how health professionals
and patients concomitantly engage in SDM, and what is
the impact of SDM on both? 3) What are the most appro-
priate strategies and frameworks to analyze dyadic data
that are nested under health professionals? Team members from other countries contribute to this
project by: 1) providing extensive expertise in SDM at
both the conceptual and methodological levels [6,13,35-
37] and in implementation sciences [38-42]; 2) sharing
their experience in producing and conducting clinical tri-
als evaluating patient decision aids [43] and implementa-
tion strategies [38-42]; and 3) providing datasets to be
used during the workshop. http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 practice because many dyadic processes are at play:
patient-health provider, patient-family member, and
health provider-health provider, to name only a few. implementation expertise as well as on the extensive clin-
ical research background in SDM of the investigators
involved in the project, we propose to: 1) Foster ongoing communication among members of this
international research network Page 3 of 6
(page number not for citation purposes) Background The expertise, analytical strategies, and theoretical frame-
works for studying dyads that have emerged in relation-
ship studies [28,30-32] have the potential to enhance the
theoretical underpinnings and the research methods for
studying the implementation process of SDM in clinical The data show that SDM has not been broadly adopted yet
[10-13]. There are major barriers to overcome in the goal
of diffusion or dissemination of new approaches in clini-
cal practice [14,15]. In a systematic review of barriers and
facilitators to implementing SDM and patient decision
aids in clinical practice as perceived by health profession-
als [16], among 28 unique studies that had collected data
from 15 countries, the three most often reported barriers
were: time constraints, lack of applicability due to patient
characteristics, and lack of applicability due to the clinical Page 2 of 6
(page number not for citation purposes) Page 2 of 6
(page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 [47]. In this project, secondary analyses of existing dyadic
datasets with a reciprocal one-with-many design will be
favoured. published. The team will develop an international multi-
site study protocol that is based on the work accom-
plished during this project. The overarching goal of this
study is to support both health professionals and individ-
uals to engage in SDM. Based on the strong record of
research excellence of all co-investigators and on existing
dyadic data sets to be analyzed during the workshop, our
research team is firmly convinced that it will attract fund-
ing for future projects. Sources of data Previous trials and ongoing pilot trials of SDM in primary
care were selected because they include the same measures
at both the practitioner and patient levels. FL will provide
a data set of 122 primary care providers and their 923
patients [48], and a data set of about 15 family practition-
ers and 51 pregnant women facing a decision about pre-
natal testing (on-going study). FL and ML will provide a
data set of 36 to 60 family practitioners and 450 to 750
patients facing a decision about the use of antibiotics in
acute respiratory infections [49]. DF will provide a dataset
of about eight general practitioners and 164 adults facing
a decision about prostate cancer and colorectal screening
(ongoing study). Discussion
'G
d h
i 'Good theories determine what one can see and discover
in nature. Cutting-edge research methods and statistical
techniques can influence what scientists see and discover
in their data but also inform and change the way in which
scientists think theoretically'[47]. This study protocol
aims to inform researchers, educators, policy makers, and
clinicians interested in designing and/or conducting
implementation studies of SDM in clinical practice using
a theory-based dyadic perspective. Although some inter-
national collaboration has been initiated between some
of the team members, there are currently no coordinated
efforts to enhance the research capacity at the interna-
tional level to create a knowledgebase for implementing
SDM in clinical practice using a theory-based dyadic per-
spective. Also, to the best of our knowledge, the proposed
project does not duplicate other current international
research effort in the area of implementation of SDM in
clinical practice using a theory-based as well as a dyadic
perspective. Therefore, this international collaboration
addresses the many challenges associated with the system-
atic failure of implementing change in clinical practice by
ensuring that future implementation research will take
into account that the health professional's position is one
that is ultimately 'relationship-centered' [54], and thus
needs to be appraised within a dyadic perspective. Data collected and variables assessed Two datasets have data based on the Integrative Model of
Behaviour [50] including the following variables: inten-
tion, attitude, social norm, and self-efficacy regarding
engaging in SDM from the perspective of both providers
and patients. The two datasets will be pooled. Based on
the Ottawa Decision Support Framework [51,52], three
datasets have data from the Decisional Conflict Scale [53],
which was administered to both providers and patients
after a specific clinical encounter. Based on the existing lit-
erature, all constructs that will be used in the planned
analyses have excellent psychometrics in both languages
(French and English) in both providers and patients. Data analysis Existing datasets will be combined. Proper handling of
missing data will be ensured and simple descriptive statis-
tics will be computed. Diverse dyadic indexes will then be
tested between constructs assessed both in patients and
providers [47]. The Actor-Partner-Interdependence Model
(APIM) will be used to assess concomitantly in patients
and providers the relationship between constructs [31]. The deliverables of this Canadian Institute of Health
Research (CIHR) funded research initiative are many:
International and interdisciplinary group of researchers
dedicated to implementing SDM in clinical practice using
a
dyadic
perspective;
conceptual
and
analytical
approaches that will be used in future implementation of
SDM in clinical practice studies; secondary data analyses
of existing dyadic datasets; capacity building; a position
paper defining a research agenda and best practices
regarding the implementation of SDM in clinical practice;
and a protocol for an international multi-site study on the
implementation of SDM clinical practice. 4) Build capacity When and where possible, graduate students of the co-
investigators will be invited to join the think tank ses-
sions, participate in the e-journal club using the internet-
based forum, and attend the two-day workshop. If appro-
priate, graduate students will be invited to participate in
data synthesis and hypothesis testing activities. 5) Elaborate a position paper and an international multi-site study
protocol Research activities In order to develop a collaborative research network that
draws upon the extensive theoretical, methodological and Page 3 of 6
(page number not for citation purposes) Page 3 of 6
(page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 5) Elaborate a position paper and an international multi-site study
protocol In line with four of the eleven priority research themes of
the Institute of Health Services and Policy Research of the
Canadian Institute of Health Research, these deliverables
are important as they will: Provide innovative insight on
how to successfully implement change in clinical practices A position paper defining a research agenda and best prac-
tices regarding the implementation of SDM in clinical
practice using a theory-based dyadic perspective will be Page 4 of 6
(page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 using a theory-based dyadic perspective; be helpful for
future research on new models of collaborative care
within the workforce environment related to health care
provider-patient dyads; serve as a strategy to increase qual-
ity of care and patient safety; and reinforce a patient-cen-
tered care approach, one that highly values relationships
[55]. Lastly, this international research initiative is in line
with research priorities on social interactions of the Cana-
dian Institute for Advanced Research whose mission is to
'incubate ideas that go on to revolutionize the interna-
tional research community, and change the lives of people
all over the world.' In summary, the proposed initiative is
of foremost importance since it fosters a critical mass of
research activities within an international network on the
implementation of SDM in clinical practice and high-
lights a new paradigm in implementation science by
putting forward a theory-based dyadic perspective. low-up of women with breast cancer. Psychooncology 2006,
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2007–2008; DCO190GP grant # 165691-OPD-). It also receives financial
support from the Improved Clinical Effectiveness through Behavioral
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women considering hormone therapy after menopause:
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https://openalex.org/W4313475706
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https://hal.inrae.fr/hal-03991942/file/antibiotics-12-00008-v2.pdf
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English
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Multi-Criteria Decision Analysis for Assessing Social Acceptance of Strategies to Reduce Antimicrobial Use in the French Dairy Industry
|
Antibiotics
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cc-by
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Multi-Criteria Decision Analysis for Assessing Social
Acceptance of Strategies to Reduce Antimicrobial Use in
the French Dairy Industry
Diego Manriquez, Maiara Costa, Ahmed Ferchiou, Didier Raboisson,
Guillaume Lhermie
To cite this version:
Diego Manriquez, Maiara Costa, Ahmed Ferchiou, Didier Raboisson, Guillaume Lhermie.
Multi-
Criteria Decision Analysis for Assessing Social Acceptance of Strategies to Reduce Antimicrobial Use
in the French Dairy Industry. Antibiotics, 2023, 12 (1), pp.8. 10.3390/antibiotics12010008. hal-
03991942 Diego Manriquez, Maiara Costa, Ahmed Ferchiou, Didier Raboisson,
Guillaume Lhermie To cite this version: Diego Manriquez, Maiara Costa, Ahmed Ferchiou, Didier Raboisson, Guillaume Lhermie. Multi-
Criteria Decision Analysis for Assessing Social Acceptance of Strategies to Reduce Antimicrobial Use
in the French Dairy Industry. Antibiotics, 2023, 12 (1), pp.8. 10.3390/antibiotics12010008. hal-
03991942 Distributed under a Creative Commons Attribution 4.0 International License Citation: Manriquez, D.; Costa, M.;
Ferchiou, A.; Raboisson, D.; Lhermie,
G. Multi-Criteria Decision Analysis
for Assessing Social Acceptance of
Strategies to Reduce Antimicrobial
Use in the French Dairy Industry. Antibiotics 2023, 12, 8. https://
doi.org/10.3390/antibiotics12010008 Keywords: antimicrobial resistance; livestock; decision analysis; public health Article
Multi-Criteria Decision Analysis for Assessing Social
Acceptance of Strategies to Reduce Antimicrobial Use in the
French Dairy Industry ,2
, Maiara Costa 1, Ahmed Ferchiou 1
, Didier Raboisson 1
and Guillaume Lhermie 1,3,* Diego Manriquez 1,2
, Maiara Costa 1, Ahmed Ferchiou 1
, Didier Raboisson 1
and Guilla 1
CIRAD, ASTRE, Université de Toulouse, ENVT, 31300 Toulouse, France
2
Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523, USA
3
School of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4Z6, Canada
*
Correspondence: guillaume.lhermie@envt.fr 1
CIRAD, ASTRE, Université de Toulouse, ENVT, 31300 Toulouse, France
2
Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523, USA
3
School of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4Z6, Canada
*
Correspondence: guillaume.lhermie@envt.fr Abstract: To respond to the antimicrobial resistance (AMR) threat, public health entities implement
policies aiming to reduce antimicrobial use (AMU) in livestock systems, in which policy success
and sustainability might be subject to the social acceptability of the novel regulatory environment. Therefore, consistent methods that gather and synthesize preferences of stakeholder groups are
needed during the policy design. The objective of this study was to present a methodology for
evaluating the acceptability of potential strategies to reduce AMU using multi-criteria decision
analysis (MCDA) using French dairy industry as a model. Preference-ranking organization methods
for enrichment evaluations were applied to rank stakeholders’ acceptance of four different potential
AMU reduction strategies: 1. Baseline AMU regulations in France; 2. Total interdiction of AMU;
3. Interdiction of prophylaxis and metaphylaxis AMU; and 4. Subsidies to reduce AMU by 25%. A total of 15 stakeholders (consumers, n = 10; farmers, n = 2; public health representatives, n = 3)
representing the French dairy sector and public health administration participated in the acceptance
weighting of the strategies in relation with their impact on environmental, economic, social, and
political criteria. We established a MCDA methodology and result-interpretation approach that can
assist in prioritizing alternatives to cope with AMR in the French dairy industry or in other livestock
systems. Our MCDA framework showed that consumers and public health representatives preferred
alternatives that consider the restriction of AMU, whereas farmers preferred to maintain baseline
policy. 1. Introduction Academic Editor: Ulf Magnusson
Received: 8 December 2022
Revised: 16 December 2022
Accepted: 18 December 2022
Published: 21 December 2022 Academic Editor: Ulf Magnusson Academic Editor: Ulf Magnusson
Received: 8 December 2022
Revised: 16 December 2022
Accepted: 18 December 2022
Published: 21 December 2022 Antimicrobial resistance (AMR) is one of the most serious threats for public health [1]. By the year 2050, 10 million deaths could occur worldwide if the current trend of inappro-
priate and excessive use of antimicrobial agents continues [2]. Antimicrobial resistance
occurs when bacteria, virus, fungi, and parasites change over time and no longer respond
to medicines, making infections harder to treat and increasing the risks of disease spread,
illness severity, and death [3,4]. Two main factors contributing to AMR have been identi-
fied. First, the use of antimicrobials that inhibit susceptible bacteria and allow resistant
bacteria to survive, and second, the activation of dormant resistance genes due to antibiotic
pressure [3,5,6]. Therefore, ecological niches with continuous exposure to antimicrobials
can be significant drivers for AMR [7]. In this sense, animal farming is a critical component
in the emergence and transmission of AMR organisms [8–10]. HAL Id: hal-03991942
https://hal.inrae.fr/hal-03991942v1
Submitted on 16 Feb 2023 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 antibiotics antibiotics antibiotics antibiotics antibiotics Copyright:
© 2022 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/). In livestock systems, antimicrobials play a significant role in treating and preventing
disease that cause animal suffering and decrease in productivity [9,11,12]. In dairy farming,
antimicrobials are used for the control and treatment of subclinical and clinical mastitis,
retained placenta, metritis, lameness, and respiratory disorders [6,13,14]. Despite these https://www.mdpi.com/journal/antibiotics Antibiotics 2023, 12, 8. https://doi.org/10.3390/antibiotics12010008 Antibiotics 2023, 12, 8 2 of 16 benefits, practices around antimicrobial use (AMU) by farmers and veterinarians and
the development of AMR due to over-using, inappropriate dosage, incorrect treatment
duration, or drug choice are of great concern for the dairy industry and public health
institutions [14]. There is consensus that the AMR threat must be approached from a One Health per-
spective [7,15]. In this sense, the role of stakeholders implicated in the AMR development,
as well as the communities at risk and policy makers, must be clearly identified. In France,
efforts to control AMU include a national program combining measures to identify and
prevent infectious livestock disease, controlled prescriptions, farmer and veterinarian ed-
ucation, and enforcement of current national and international regulations of AMU [16]. Concerning French dairy farming, mastitis is the most commonly reason reported for
AMU [14] and the use of critically important antimicrobials (third- and fourth-generation
cephalosporins) is allowed only for sick cows and banned for prophylaxis [17,18]. Farmers might face challenges to adopt policy against AMR when regulations do
not consider their interests and needs. These challenges range from a lack of cost-benefit
analyses of using antimicrobials or not, to farmers’ experience detecting and treating
infectious diseases, and to welfare considerations of not using antimicrobials [19,20]. On the
other hand, consumer perceptions on AMR exert social pressure to improve animal farming
practices towards the reduction in AMU, which drives the demand for food produced
respecting the environment, animals, and public health. Thus, finding a balance between
the main concerns of all stakeholders is necessary to maximize the benefits to farmers, to
reduce risks to animal and human health, and to find a rational AMU in dairy farming and
in other livestock operations. In this sense, the preference ranking organization methods for
enrichment evaluation (PROMETHEE) approach, nested in the multiple-criteria decision
analysis (MCDA) spectrum, is a useful tool to assist in the decision-making process on AMU
regulations because it can include views from all potential stakeholders, rank strategies
according to several criteria and preferences, and prioritize the acceptability of current
and novel policy [21]. In consequence, our objective was to develop a MCDA framework
for assessing the social acceptability of potential strategies for reducing AMU in animal
agriculture, using the French dairy sector as model. 2. Results Nonetheless, they differed in in terms of preferences and criteria rankings due to the
separation between the stakeholders’ axis (Figure 1B). On the other hand, farmers did
not prefer the remaining three strategies at all. Finally, in the public health group, total
interdiction of AMU was the closest to the decision axis (Figure 1C), although the first-
ranked strategy was not as close to the decision axis as the strategies preferred by consumers
and farmers groups (Figure 1A,B). This might be due to more dissimilar criteria ranking
among public health stakeholders. For instance, in Figure 1C, stakeholder 3 is pointing
to the left side of the Y-axis because the baseline strategy was their first-ranked strategy. Stakeholder 1 is the closest to the decision axis, meaning that they have the best agreement
with the first ranked strategies. Stakeholders 2 and 3 were very distant in their rankings. strategy, and contrary to the other stakeholder groups, the least accepted strategy was the
subsidies to reduce AMU by 25% with a score of −0.06. strategy, and contrary to the other stakeholder groups, the least accepted strategy was the
subsidies to reduce AMU by 25% with a score of −0.06. Table 1. Acceptability ranking scores of strategies to reduce antimicrobial use in French dairy farms
by stakeholder group. by stakeholder group. Stakeholders
Weighted Ranking
Strategy
Phi
Phi+
Phi−
Consumers
1
AMU interdiction
0.23
0.58
0.22
2
Preventive AMU interdiction
0.007
0.24
0.25
3
Subsides to reduce AMU
−0.10
0.27
0.37
4
Baseline strategy
−0.19
0.19
0.32
Farmers
1
Baseline strategy
0.1
0.36
0.26
2
AMU interdiction
−0.02
0.34
0.36
3
Subsides to reduce AMU
−0.03
0.29
0.32
4
Preventive AMU interdiction
−0.05
0.28
0.33
Public health
representatives
1
AMU interdiction
0.12
0.45
0.33
2
Preventive AMU interdiction
−0.004
0.25
0.26
3
Baseline strategy
−0.03
0.34
0.35
4
Subsides to reduce AMU
−0.09
0.21
0.30
Phi: overall preference flow; Phi+: positive preference flow; Phi−: negative preference flow. Figure 1 shows the multi-criteria problem graphically, considering the acceptability
of each stakeholder and the strategies around a decision axis (red line in Figure 1). The
exact positions are defined by the stakeholder’s weights and performance of strategies
considering each criterion. The closer a stakeholder was to the decision axis, the greater is
their agreement with the first ranked strategy. 2. Results The preference weights of different criteria that gauged the impact of the potential
AMU policy scenarios were estimated based on stakeholders’ interviews with consumers,
farmers, and public health representative groups. After the interview invitations were
distributed to potential participants, a total of 10 responders from the consumers group
(residents of Toulouse, France), 2 from the farmers group (from Occitanie region, France),
and 3 from the public health representatives group (French Ministry of Agriculture, OIE,
trade union for the pharmaceutical industry and veterinary diagnostic (SIMV, France))
responded to the interview. A model of the interview is presented in Appendix A. p
p
pp
The acceptability rankings of strategies to reduce AMU were performed within each
stakeholder group using the overall performance score (phi) of each strategy, determined
by criteria weighing. The consumers’ group score ranged from −0.09 to 0.15 (Table 1). In
this group, the first ranked strategy was the total interdiction of AMU with a score of 0.15. The interdiction of prophylaxis and metaphylaxis of AMU was ranked second with a score
of 0.01, followed by the strategy subsidizing the reduction AMU by 25% with a score of
−0.08 and the baseline strategy with a score of −0.09. In the farmers group, the scores
ranged from −0.05 to 0.10 (Table 1). The first ranked was the baseline strategy with a score
of 0.10 followed by the total AMU interdiction with a score of −0.02 and the subsidies to
reduce AMU by 25% with a score of −0.03. The least accepted strategy was the interdiction
of prophylaxis and metaphylaxis AMU with a score of −0.05. On the other hand, the scores
of the public health representatives ranged from −0.06 to 0.12. In this group, the total
interdiction of AMU was in the first place of acceptation with a score of 0.12, followed by
the interdiction of preventive and metaphylactic AMU with a score of −0.01, the baseline Antibiotics 2023, 12, 8 3 of 16 strategy, and contrary to the other stakeholder groups, the least accepted strategy was the
subsidies to reduce AMU by 25% with a score of −0.06. Table 1. Acceptability ranking scores of strategies to reduce antimicrobial use in French dairy farms
by stakeholder group. 2. Results Stakeholders
Weighted Ranking
Strategy
Phi
Phi+
Phi−
Consumers
1
AMU interdiction
0.23
0.58
0.22
2
Preventive AMU interdiction
0.007
0.24
0.25
3
Subsides to reduce AMU
−0.10
0.27
0.37
4
Baseline strategy
−0.19
0.19
0.32
Farmers
1
Baseline strategy
0.1
0.36
0.26
2
AMU interdiction
−0.02
0.34
0.36
3
Subsides to reduce AMU
−0.03
0.29
0.32
4
Preventive AMU interdiction
−0.05
0.28
0.33
Public health
representatives
1
AMU interdiction
0.12
0.45
0.33
2
Preventive AMU interdiction
−0.004
0.25
0.26
3
Baseline strategy
−0.03
0.34
0.35
4
Subsides to reduce AMU
−0.09
0.21
0.30
Phi: overall preference flow; Phi+: positive preference flow; Phi−: negative preference flow. Figure 1 shows the multi-criteria problem graphically, considering the acceptability
of each stakeholder and the strategies around a decision axis (red line in Figure 1). The
exact positions are defined by the stakeholder’s weights and performance of strategies
considering each criterion. The closer a stakeholder was to the decision axis, the greater is
their agreement with the first ranked strategy. For instance, in Figure 1A, the stakeholder
10 from the consumer group agreed more strongly with the first ranked solution of this
group (total interdiction of AMU). On the other hand, stakeholders 3 and 8 were the most
distant to this strategy and they disagreed, mostly, in terms of rankings. In the consumers
group, all stakeholders were located on the right side of the Y-axis, meaning that their
overall preferences were not discordant at all. Stakeholders with longer axis have strong
decision choice power [22]. The strategy that considered total interdiction of AMU (AMU
interdiction in Figure 1A) was the closest to the decision axis, meaning that it was the
preferred strategy by the consumers group. Interdiction of prophylaxis and metaphylaxis
AMU was on the right side, since it was placed second in the ranking (Table 1). On the
contrary, the least preferred strategies, baseline and subsidies to reduce AMU, were located
on the left side of the Y-axis. A similar interpretation can be employed for preference ranking of stakeholders from
the farmer (Figure 1B) and the public health representative (Figure 1C) groups. In the
farmers’ group, the baseline strategy was closer to the decision axis; therefore, it was the
most preferred strategy. The participating farmers were not discordant with the overall
acceptability of the baseline strategy, since they were located on the right of the Y-axis. 2. Results For instance, in Figure 1A, the stakeholder
10 from the consumer group agreed more strongly with the first ranked solution of this
group (total interdiction of AMU). On the other hand, stakeholders 3 and 8 were the most
distant to this strategy and they disagreed, mostly, in terms of rankings. In the consumers
group, all stakeholders were located on the right side of the Y-axis, meaning that their
overall preferences were not discordant at all. Stakeholders with longer axis have strong
decision choice power [22]. The strategy that considered total interdiction of AMU (AMU
interdiction in Figure 1A) was the closest to the decision axis, meaning that it was the
preferred strategy by the consumers group. Interdiction of prophylaxis and metaphylaxis
AMU was on the right side, since it was placed second in the ranking (Table 1). On the
contrary, the least preferred strategies, baseline and subsidies to reduce AMU, were located
on the left side of the Y-axis. A similar interpretation can be employed for preference ranking of stakeholders from
the farmer (Figure 1B) and the public health representative (Figure 1C) groups. In the
farmers’ group, the baseline strategy was closer to the decision axis; therefore, it was the
most preferred strategy. The participating farmers were not discordant with the overall
acceptability of the baseline strategy, since they were located on the right of the Y-axis. Nonetheless, they differed in in terms of preferences and criteria rankings due to the
separation between the stakeholders’ axis (Figure 1B). On the other hand, farmers did
not prefer the remaining three strategies at all. Finally, in the public health group, total
interdiction of AMU was the closest to the decision axis (Figure 1C), although the first-
ranked strategy was not as close to the decision axis as the strategies preferred by consumers
and farmers groups (Figure 1A,B). This might be due to more dissimilar criteria ranking
among public health stakeholders. For instance, in Figure 1C, stakeholder 3 is pointing
to the left side of the Y-axis because the baseline strategy was their first-ranked strategy. Stakeholder 1 is the closest to the decision axis, meaning that they have the best agreement
with the first ranked strategies. Stakeholders 2 and 3 were very distant in their rankings. 4 of 16 Antibiotics 2023, 12, 8
Antibiotics 2023, 12, x F Figure 1. 2. Results Aggregated decision map (GAIA plane) of decision makers considered in this study. (Delta = 96.6%; 96.6% of the information is conserved in the two-dimensional representation of
this map). pact of each strategy on each criterion considering all stakeholder groups (Figure 3). Ac-
cording to the interviewees’ preferences, the baseline strategy (Figure 3A) performs well
in the production costs and in the social and political criteria; however, it performs poorly
in most economic and environmental criteria. The total interdiction of AMU (Figure 3B)
performs well in almost all economic and environmental criteria; however, it performs
poorly in production cost and in social and regulatory framework criteria. The subsidies
to reduce AMU by 25% (Figure 3C) perform badly in almost all economic, environmental,
and policy investment criteria; nonetheless, they perform more efficiently on the produc-
tion cost, regulatory framework, and social criteria. Finally, the interdiction of prophylaxis
and metaphylaxis AMU (Figure 3D) strategy performs well in farmer’s revenues and rea-
sonably well in the attributable fraction, mortality rate, and policy investments. Con-
versely, it is not efficient in the other economic criteria and in the regulatory framework. The action profile tool on the VP software was used to represent graphically the impact
of each strategy on each criterion considering all stakeholder groups (Figure 3). According
to the interviewees’ preferences, the baseline strategy (Figure 3A) performs well in the
production costs and in the social and political criteria; however, it performs poorly in
most economic and environmental criteria. The total interdiction of AMU (Figure 3B)
performs well in almost all economic and environmental criteria; however, it performs
poorly in production cost and in social and regulatory framework criteria. The subsidies to
reduce AMU by 25% (Figure 3C) perform badly in almost all economic, environmental, and
policy investment criteria; nonetheless, they perform more efficiently on the production
cost, regulatory framework, and social criteria. Finally, the interdiction of prophylaxis and
metaphylaxis AMU (Figure 3D) strategy performs well in farmer’s revenues and reasonably
well in the attributable fraction, mortality rate, and policy investments. Conversely, it is not
efficient in the other economic criteria and in the regulatory framework. To evaluate whether changes in criteria weighing could have an impact on the analysis,
a sensitivity analysis was performed using the stability intervals window of the VP software. Table 2 shows the weight stability intervals for each criterion. 2. Results Decision map (GAIA plane) of consumers (A), farmers (B), and public
makers weighted preferences. (Delta = 96.6%; 96.6% of the information is conser
mensional representation of this map). Figure 1. Decision map (GAIA plane) of consumers (A), farmers (B), and public health (C
makers weighted preferences. (Delta = 96.6%; 96.6% of the information is conserved in
dimensional representation of this map). Figure 1. Decision map (GAIA plane) of consumers (A), farmers (B), and public health (C) d
makers weighted preferences. (Delta = 96.6%; 96.6% of the information is conserved in the
mensional representation of this map). Figure 1. Decision map (GAIA plane) of consumers (A), farmers (B), and public health (C) decision
makers weighted preferences. (Delta = 96.6%; 96.6% of the information is conserved in the two-
dimensional representation of this map). Antibiotics 2023, 12, 8 5 of 16 5 of 16 To assess group acceptability, an overall decision map was developed considering the
preferences of the three groups of stakeholders. Therefore, in Figure 2, consumers, farmers,
and public health representatives were aggregated in an individual axis by averaging
their weights for each criterion. For the consumer and public health groups, the AMU
interdiction (Figure 2) was the closest to the decision axis, since this was the preferred
strategy for both groups. On the other hand, the farmers’ group had baseline strategy
closest their axis. The two strategies with the least preference for all stakeholders were on
the left side of the Y-axis (Figure 2). EVIEW
5 of 16
strategy closest their axis. The two strategies with the least preference for all stakeholders
were on the left side of the y-axis (Figure 2). Figure 2. Aggregated decision map (GAIA plane) of decision makers considered in this study. (Delta
= 96.6%; 96.6% of the information is conserved in the two-dimensional representation of this map). The action profile tool on the VP software was used to represent graphically the im-
Figure 2. Aggregated decision map (GAIA plane) of decision makers considered in this study. (Delta = 96.6%; 96.6% of the information is conserved in the two-dimensional representation of
this map). Figure 2. Aggregated decision map (GAIA plane) of decision makers considered in this study. (Delta
= 96.6%; 96.6% of the information is conserved in the two-dimensional representation of this map). The action profile tool on the VP software was used to represent graphically the im
Figure 2. 2. Results Weights outlying this intervals
could affect stakeholders’ rankings of the preferred strategies. The sensitivity analysis
showed that the stakeholders can change the weights due to policy investments from 7.53
to 100 without affecting the stakeholder’s ranking strategies. However, any variations in
the weight of the production cost, for example, beyond the range of 12.92 to 15.22 will
result in a change in the ranking, indicating that results are more sensitive to this criterion. Antibiotics 2023, 12, 8 6 of 16
mental,
roduc- Due to short stability intervals, other sensitive criteria were farmers’ revenues, culling rate,
regulatory framework, and the attributable fraction (Table 2). and metaphylaxis AMU (Figure 3D) strategy performs well in farmer s revenues and rea
sonably well in the attributable fraction, mortality rate, and policy investments. Con-
versely, it is not efficient in the other economic criteria and in the regulatory framework. Due to short stability intervals, other sensitive criteria were farmers’ revenues, culling rate,
regulatory framework, and the attributable fraction (Table 2). and metaphylaxis AMU (Figure 3D) strategy performs well in farmer s revenues and rea
sonably well in the attributable fraction, mortality rate, and policy investments. Con-
versely, it is not efficient in the other economic criteria and in the regulatory framework. Figure 3. Performance profile of strategies to reduce antimicrobial use under each criterion. The
strategies included were baseline strategy of antimicrobial use (A), total interdiction of antimicro-
bial use (B), subsides to reduce antimicrobial use by 25% (C), and interdiction of preventive and
metaphylactic antimicrobial use (D). Abbreviations. Economic dimension (blue): Production costs
(PC), farmers’ revenues (FR), culled cow price (CP), milk price (MP). Environmental dimension
(green): animal exposure level to antimicrobials (ALEA), attributable factor of antimicrobial-resistant
human infection to livestock (AF). Social dimension (red): culling rate (CR), death rate (DR). Political
dimension (pink): regulatory framework (RF), investment policies (IP). Figure 3. Performance profile of strategies to reduce antimicrobial use under each criterion. The
strategies included were baseline strategy of antimicrobial use (A), total interdiction of antimicro-
bial use (B), subsides to reduce antimicrobial use by 25% (C), and interdiction of preventive and
metaphylactic antimicrobial use (D). Abbreviations. Economic dimension (blue): Production costs
(PC), farmers’ revenues (FR), culled cow price (CP), milk price (MP). Environmental dimension
(green): animal exposure level to antimicrobials (ALEA), attributable factor of antimicrobial-resistant
human infection to livestock (AF). Social dimension (red): culling rate (CR), death rate (DR). 2. Results Political
dimension (pink): regulatory framework (RF), investment policies (IP). Table 2. Sensitivity analysis of stakeholder weighing. Criteria
Weight Stability Interval
Minimum
Maximum
Difference
Regulatory framework
9.28
11.12
1.84
Farmer’s revenues
15.84
18.1
2.26
Production cost
12.92
15.22
2.3
Culling rate
5.75
8.24
2.49
Attributable fraction 2
2.99
8.84
5.85
Product price
1.31
12.86
11.55
ALEA 1
0
11.99
11.99
Mortality rate
5.8
17.94
12.14
Price culled cow
0
12.54
12.54
Policies investments
7.53
100
92.47
1 ALEA: animal level of exposure to antimicrobials; 2 Fraction of antimicrobial-resistant human infections
attributed to animal agriculture origin. Table 2. Sensitivity analysis of stakeholder weighing. 3. Discussion We aimed to present a MCDA framework including implementation and result inter-
pretation that could be used to assess the social acceptability of a novel policy environment
against AMR in the French dairy industry. Although we performed comparisons between
strategies to reduce AMU to exemplify our MCDA approach, we did not intent to test the
viability of these strategies in real settings. Moreover, we acknowledge the limitations of
the strategy comparison due to the low response rate. Therefore, we encourage readers to
use the information presented here as a framework to perform MCDA around the AMU
policy development. Antibiotics 2023, 12, 8 7 of 16 7 of 16 The number of AMR microorganisms is rising at high levels in all parts of the
world [1,3,21]. Therefore, fighting AMR requires a multidisciplinary approach [23]. In
order to succeed, concatenated efforts involving the needs and visions of diverse members
of the society require methodologies and metrics that estimate the appropriateness of
interventions aiming to reduce AMU [23]. In this sense, MCDA can be a key component for
addressing AMR, since its conclusions assist in finding an informed balance for decision
makers, considering the interests and ethical concerns of diverse stakeholders. Moreover,
MCDA not limited to the consumers, farmers, and public health groups that we considered
to develop our approach. p
pp
In the dairy industry, antimicrobials are critical for the control and treatment of
mastitis, which produces significant economic loses at the farm level worldwide, and
other disorders that cause economic losses and animal suffering [24]. In France, mastitis
was considered responsible for a third of the economic impact related to health disorders
in dairy cows [25]. Moreover, every dairy cow receives on average the equivalent of
1.58 antimicrobial treatments for mastitis per year, which represents 70% of all the AMU
in dairy cows [26]. To respond to AMR, a more restrictive usage of antimicrobials by the
livestock industry has been suggested to prevent outbreaks of multi-resistant bacteria and
avoid the spread of resistance genes to other hosts and communities [3,5]. When public policies are formulated, the complexity of the disease, the diversity of
farm environments, a farm’s microbial ecology, and the idiosyncrasy of farmers should be
considered. Nonetheless, it is common to observe that AMU policy only considers one side
of the problem, such as total restriction, which might affect other management practices and
not be sustainable in the long-term. 3. Discussion In the context of AMR, risk analyses are useful before the implementation of
policies for assessing and managing human and animal health risks associated with the Antibiotics 2023, 12, 8 8 of 16 8 of 16 development of resistance, including appropriate communication measures. Nonetheless,
the implementation of risk analysis is costly and time consuming because it requires several
steps to be completed, including hazard identification, risk assessment, risk management,
and risk communication [34]. In this study, we used MCDA because it provides a structured
and systematic process for identifying gaps in scientific knowledge related to important
decision issues, which can be used to outline research priorities in public health. In
addition, MCDA can be adapted and potentially used with real-time decision-making
methods, and it supports individuals or groups of decision makers to classify, select, and
compare different alternatives for solving a problem [21]. Another advantage of MCDA is
that multiple comparisons are viable when there are competing and multiple evaluation
criteria. Moreover, the MCDA methods can include quantitative and qualitative data in
the analysis and, contrary to a cost-benefit analysis, they do not assign a monetary value,
which is extremely difficult to estimate for environmental and social impacts. Additionally,
MCDA allows one to include weights or perspectives from all parties involved in AMR [35]. g
p
p
p
Despite the strategies designed by the French administration, there has been an
increase in exposure to antimicrobials in dairy cows between 2017 and 2018 [16]. Therefore,
the assessment of new strategies for reducing AMU are continuously needed. In this
sense, MCDA and PROMETHEE provide a useful and simple procedure for assessing the
acceptability of innovative strategies against AMR. To the authors’ knowledge, this is the
first report that applies this framework to analyze a potential scenario of AMR regulations
applicable to the dairy industry. pp
y
y
The PROMETHEE procedure is based on the pair-wise comparison of strategies. In
this case, the deviation between the evaluations of two alternatives on one particular
criterion is considered. Small deviations indicate weak preference or no preference, as the
stakeholders will consider this deviation small or negligible. For larger deviations, higher
preference levels are expected. With PROMETHEE, preference levels are measured on
the degree of preference flow (phi) between −1 to 1, where −1 means no preference and
1 means total preference [36]. 3. Discussion In this study, we proposed to participants four potential
strategies that have not been fully implemented. However, these strategies synthesize
potential scenarios of AMU policy and allowed us to develop our MCDA framework. Regarding these strategies, no country has yet established a regulation that totally bans
AMU in animal agriculture. Nonetheless, special productive settings, such as voluntary
organic dairy production, have been already implemented posing new challenges to dairy
farmers regarding health and reproduction management [27]. Additionally, the European
Union has banned AMU as growth promoters [28,29] and has fostered the antimicrobial-free
production. In France, some regulations supervise the use of third- and fourth-generation
cephalosporine and have implemented a plan to reduce AMU by 25% during a 5-year
period [16]. Concerning the implementation of direct subsidies for farmers to encourage
AMU reduction, experiences are scarce, and the long-term effects have not been fully
explored [20]. However, few programs have been implemented. These programs usually
come from the private sector, where manufacturers pay premiums for animal products
raised without antibiotics [30]. On the other hand, in the public sector, tax systems have
been preferred to reduce antimicrobial sales [23,31]. p
The introduction of new regulations for controlling AMU affects production costs
and product prices. For instance, in the USA, it has been estimated that the interdiction of
prophylaxis AMU would cause loses of $1.8 billion USD for the beef industry [32] and, in
the dairy industry, a reduction in AMU may lead to increased morbidity and/or mortality
and reduce the production output for the milk supply chain [33]. Moreover, interventions
that consider antimicrobial prohibition or abrupt reduction in AMU must have an ethical
approach because of the impact on animal welfare and economic consequences in veterinary
care, agriculture, and relevant bio-industries. For farmers, these restrictions may threaten
the quality and quantity of animal food products. Additionally, low-income countries may
face greater challenges to meet production goals or compete with larger markets [29]. In
this sense, the assessment of AMU sustainability is necessary to advise policymakers about
the possible impact on stakeholders’ view and acceptability [20]. p
p
p
y
To perform sustainability policy assessments, decision support tools are frequently
used, in which the most common are risk analysis, cost-benefit analysis, system dynamics,
and MCDA. 4.1. Study Design
4.1. Study Design
Thi
t d This study was conducted from January to July 2020 with the general objective of
creating a MCDA framework for assessing potential scenarios of AMU policy, allowing one
to rank strategy preference from consumers, farmers, and public health representatives in
the French dairy sector. Within the MCDA methodology, the PROMETHEE approach was
used. The study started planning a general framework, which is necessary for carrying out
the PROMETHEE technique. This study was conducted from January to July 2020 with the general objective of
creating a MCDA framework for assessing potential scenarios of AMU policy, allowing
one to rank strategy preference from consumers, farmers, and public health representa-
tives in the French dairy sector. Within the MCDA methodology, the PROMETHEE ap-
proach was used. The study started planning a general framework, which is necessary for
carrying out the PROMETHEE technique. T
b
i
thi f
k
l
t d
it
i
hi h
b
d
d
i q
To begin this framework, we selected criteria which can be assessed under environ-
mental, economic, social, and political dimensions of AMR and AMU. The environment
dimension had two criteria: AMU assessed by the animal level of exposure to antimicro-
bials (ALEA) and AMR assessed by the fraction of AMR human infections attributable to
livestock. The economic dimension had three criteria: production costs, farmer’s revenues,
and product prices (meat and milk). The social dimension was assessed using animal
welfare metrics, including herd culling rate and mortality. Finally, the political dimension
criteria consisted of the number of policies and investments to fight AMR. Following this,
we identified stakeholders in AMU policies comprising three groups of consumers of dairy
products, dairy farmers, and public health representatives in the region of Occitanie, France. The proposed MCDA framework for criteria and stakeholder selection is depicted in
Figure 4. After criteria and stakeholder identification, stakeholders individually weighed
the impact that four potential strategies to reduce AMU will have on each criterion. The
strategies were: 1. Baseline AMU regulations in France; 2. Total interdiction of antimi-
crobials; 3. Interdiction of prophylaxis and metaphylaxis AMU; and 4. Subsidies to
reduce AMU by 25%. Following this step, group criteria weighing was analyzed using
PROMETHEE, in which strategy preference was compared within and between stake-
holder groups. To begin this framework, we selected criteria which can be assessed under environ-
mental, economic, social, and political dimensions of AMR and AMU. 3. Discussion Additionally, PROMETHEE relies on a preference function
indicating the degree of preference from one alternative over the other [37]. In this study,
the entire decision analysis was carried out in VP software, which greatly facilitates the
performance assessment of strategies on each criterion [21]. From our MCDA approach, we determined that the first-ranked alternative for con-
sumer and public health representative groups was related to the total interdiction of AMU
(Table 1). This agrees with other studies suggesting that consumers prefer to buy antibiotic-
free products when only the AMR issue is evaluated [38]. Therefore, it is plausible to
suggest that consumers will support antimicrobial prohibition for livestock systems. On the
other hand, public health administration is expected to improve the education efforts about
AMU and AMR, to strengthen surveillance and research, optimize AMU in human and
animal health, and develop sustainable investment policies for new medicines, diagnostic
tools, and other interventions [39]. Thus, MCDA may be useful to prioritize educational
topics for specific stakeholders. p
p
Other strategies presented in this study had lower scores and, therefore, were less
preferred by consumers and public health representatives (Table 1). In the individual
rankings, this issue was evident because only one stakeholder from these two groups
did not select AMU interdiction as the first-ranked choice. The only differences found
between the consumers and public health groups were between the third and fourth
position in the ranked strategies (Table 1). Conversely, farmers performed a ranking where
the baseline strategy was preferred. This might be explained because farmers may see their
managements and costs and benefits at risk [20]. Overall, most stakeholder weighing was
allocated to the economic dimension with an average of 43.84 points for the consumers,
37.33 for the farmers, and 59.33 for the public health representatives. This shows that this
dimension is of great importance for all stakeholder groups. However, larger number
of participants will be needed to make robust comparisons between the potential AMU
reduction strategies. Antibiotics 2023, 12, 8 9 of 16 9 of 16 The methodology presented in this study is highly flexible in incorporating more
stakeholders and blocking stakeholders by demographic features, relevant criteria, and
other productive contexts to take better decisions regarding AMU and AMR. 3. Discussion Nonetheless,
future research using our MCDA approach will require a larger number of participants, con-
siderations of demographic bias, and a comparable number of stakeholders in each group
to assure the external validity of conclusions made from MCDA in AMU policy design. VIEW
9 of 16 ALEA =
Live weight treated
(Total number o f animals ∗Weight o f adults animals or at slauther) ALEA =
Live weight treated
(Total number o f animals ∗Weight o f adults animals or at slauther) Table A1 shows criteria values for criteria for each strategy scenario. Current esti-
mations performed by ANSES (2018) [41] set ALEA at 0.27 for bovines. Therefore, this
value was attributed to ALEA in the baseline strategy for AM reduction. Additionally, in
the environmental dimension, we used the estimated attributable fraction of AMR human
infections associated with animal agriculture. The CDC (2013) [42] estimated that one out of
five AMR bacterial infections are linked to food or animals, but an accurate fraction of AMR
human infections attributable to dairy cattle is unknown. Various experts estimated that the
overall contribution was about 4% in 2000 [43]. Nonetheless, due to the complexity of the
phenomenon and the difficulty in evaluating it, this appears largely underestimated [44]. For this study, the value of 4% for the attributable fraction was assigned to the baseline
strategy. In the economic dimension, farmers’ costs and revenues and product prices
were selected as measurable criteria. The costs and revenues were measured by the milk
production costs, the revenues from the milk price, and the average price of culled cows. The product price is defined by the selling price of a liter of milk. The farmers’ costs were
estimated from the current expenses, depreciation, and additional expenses. The costs were
set at 494 euros/1000 liters of milk [45] and the revenues at 0.78 cents/liter of milk [46]. Additionally, the average price of culled cows was set at 2.4 euros/kg net [47]. These values
were assigned to the baseline strategy. In the social dimension, animal welfare was assigned
as a measurable criterion due to the impact that reducing AMU might have on animal
wellbeing and reflected by changes in mortality or live culling and morbidity. In France, the
estimated annual mortality and culling rates are 3.8% and 21.3%, respectively [48]. These
rates were used in the baseline strategy. Finally, the political dimension was evaluated
using the regulatory framework concerning AMR and policy investments. These criteria
were measured in a semi-quantitative way (null, low, moderate, high, very high). The idea
was to show stakeholders that greater regulations and investments related to strategies
for reducing AMU are also associated with increased costs to the population, in the form
of taxes. 4.1. Study Design
4.1. Study Design
Thi
t d The environment
dimension had two criteria: AMU assessed by the animal level of exposure to antimicro-
bials (ALEA) and AMR assessed by the fraction of AMR human infections attributable to
livestock. The economic dimension had three criteria: production costs, farmer’s revenues,
and product prices (meat and milk). The social dimension was assessed using animal wel-
fare metrics, including herd culling rate and mortality. Finally, the political dimension
criteria consisted of the number of policies and investments to fight AMR. Following this,
we identified stakeholders in AMU policies comprising three groups of consumers of
dairy products, dairy farmers, and public health representatives in the region of Occitanie,
France. The proposed MCDA framework for criteria and stakeholder selection is depicted
in Figure 4. After criteria and stakeholder identification, stakeholders individually
weighed the impact that four potential strategies to reduce AMU will have on each crite-
rion. The strategies were: 1. Baseline AMU regulations in France; 2. Total interdiction of
antimicrobials; 3. Interdiction of prophylaxis and metaphylaxis AMU; and 4. Subsidies to
reduce AMU by 25%. Following this step, group criteria weighing was analyzed using
PROMETHEE, in which strategy preference was compared within and between stake-
holder groups. Figure 4. Multi-criteria decision analysis flowchart used to determine criteria and stakeholder
groups involved in AMU in French dairy industry. AMU: antimicrobial use. AMR: antimicrobial
resistance. Figure 4. Multi-criteria decision analysis flowchart used to determine criteria and stakeholder groups
involved in AMU in French dairy industry. AMU: antimicrobial use. AMR: antimicrobial resistance. Figure 4. Multi-criteria decision analysis flowchart used to determine criteria and stakeholder
groups involved in AMU in French dairy industry. AMU: antimicrobial use. AMR: antimicrobial
resistance. Figure 4. Multi-criteria decision analysis flowchart used to determine criteria and stakeholder groups
involved in AMU in French dairy industry. AMU: antimicrobial use. AMR: antimicrobial resistance. Antibiotics 2023, 12, 8 10 of 16 4.2. Development and Assessment of Criteria 4.2. Development and Assessment of Criteria We translated the issues related to AMR and AMU into four dimensions with mea-
surable assessment criteria within them. In the environmental dimension, we used the
ALEA indicator as an estimation of antimicrobial sales and animal exposure to antimicro-
bials [40]. The ALEA indicator is used by French authorities to report and monitor yearly
antimicrobial sales [41]. This criterion estimates the percentage of animals treated with
antimicrobials out of a total animal population. It is calculated as follows: 4.4.3. Weighing Criteria and Criteria Group Ranking After the criteria and the strategies to be evaluated have been defined, a total of
10 residents of Toulouse as part of the consumer group, 10 dairy farmers from the Occitanie
region, and 10 public health representatives from the French Ministry of Agriculture, OIE,
the National Veterinary School of Toulouse, the French National Agency for Food, and the
Environmental and Occupational Health Safety Agency were invited to participate in the
interviews using the interview model shown in Appendix A. Following this, stakeholders
who responded to our invitation participated in the criteria weighing. Here, stakeholders
were asked to distribute a total of 100 points among the criteria in order of importance,
considering how the strategies will affect the criteria as shown in Table 3. The most
important criteria for them should receive more points. 4.4. PROMETHEE Implementation 4.4. PROMETHEE Implementation The general framework depicted in Figure 1 was applied using the PROMETHEE
method as developed by Behzadian et al. (2010) [51] and Aenishaenslin et al. (2013) [52]. To use PROMETHEE and assess societal acceptance of strategies for reducing AMU in the
French dairy sector, we carried out the following steps: 4.3. Definitions of Strategies against AMR The four strategies used in this study were created with the purpose of developing a
MCDA approach. The baseline strategy corresponds to the current situation of antimicro-
bial use in French dairy farms. The current French strategy against AMR has led to a 37%
reduction in the veterinary AMU between 2012 and 2016 [16,49]. This strategy assumes that
AMU will remain being critical for dairy cattle, especially for mastitis control and treatment
of pneumonia in calves [19,50]. The total interdiction of antimicrobials assumes an AMU
ban at all production stages in dairy farms. We assumed that no substitution treatment
or alternatives are implemented. For the interdiction of prophylaxis and metaphylaxis of
AMU, it was assumed that these uses correspond to 35% of the total AMU in French dairy
farms. Subsidies to reduce AMU by 25% consist in encouraging producers to adopt desir-
able practices to reduce AMU and receive monetary compensation. Here, the hypothesis
was that farmers will manage to reduce the use of antimicrobials by 25% and they will
receive subsidies. Antibiotics 2023, 12, 8 11 of 16 11 of 16 4.4.1. Problem Definition and Identification of Stakeholders 4.4.1. Problem Definition and Identification of Stakeholders To address the general problem of identifying, evaluating, and ranking different
strategies to decrease AMR in dairy farms in France, three groups of stakeholders (con-
sumers, farmers, and public health representatives) were invited to participate in the
weighing-criteria process. We aimed a convenience sample size of 10 responders from each
stakeholder group. A stakeholder was defined as a person representing an organization or
a group with direct responsibilities or with specific interests in AMU in dairy cattle [52]. 4.4.2. Identification of Key Decision Issues and Definition of Criteria 4.4.2. Identification of Key Decision Issues and Definition of Criteria Criteria were identified to evaluate the effectiveness of AMU strategies. These criteria
were nested into four dimensions: environmental, economic, social, and political. Defined
criteria had scaled values derived from our experience and literature to calibrate the
PROMETHEE model that might determine decision making of stakeholders under each
strategy (Table 3). Table 3. Criteria and scaled measures used in the PROMETHEE models under strategies (STRA) for
reducing antimicrobial use in French dairy farms. Table 3. Criteria and scaled measures used in the PROMETHEE models under strategies (STRA) for
reducing antimicrobial use in French dairy farms. Criteria
STRA01
STRA02
STRA03
STRA04
Environmental
ALEA 1
0.27
0
0.17
0.20
Attributable fraction 2 (%)
0.04
0
0.026
0.03
Economic
Production costs (€/1000 L)
494
684
667
617.5
Farmers’ revenues (€/1000 L)
334
473
451
417.5
Culled cow price (€/Kg)
2.4
2.64
2.4
2.4
Product price (€/L)
0.78
1.85
1.05
0.96
Social
Mortality rate (%)
3.8
4.8
4.1
4.04
Culling rate (%)
21.3
50.5
31.5
28.6
Political
Regulatory framework
Moderate
Very high
High
Moderate
Investment Policies
High
High
Moderate
Very high
1 ALEA: animal level of exposure to antimicrobials; 2 Fraction of antimicrobial-resistant human infections
attributed to animal agriculture origin; STRA01: baseline current strategy of antimicrobial use in France; STRA02:
the total antimicrobial interdiction; STRA03: interdiction of antimicrobial use as prophylaxis and metaphylaxis
management; STRA04: the implementation of subsidies to reduce antimicrobial use by 25%. 1 ALEA: animal level of exposure to antimicrobials; 2 Fraction of antimicrobial-resistant human infections
attributed to animal agriculture origin; STRA01: baseline current strategy of antimicrobial use in France; STRA02:
the total antimicrobial interdiction; STRA03: interdiction of antimicrobial use as prophylaxis and metaphylaxis
management; STRA04: the implementation of subsidies to reduce antimicrobial use by 25%. 4.4.3. Weighing Criteria and Criteria Group Ranking 4.4.3. Weighing Criteria and Criteria Group Ranking 4.4.6. Sensitivity Analysis A sensitivity analysis was performed using the Visual Stability Intervals tool in VP
for assessing the impact of a stakeholder’s weighing preferences on their individual and
group rankings. This analysis gives indications of the robustness of the results and can be
generated for each stakeholder for all criteria. 4.4.5. Interpretation of Results Each stakeholder was asked to weight each criterion after observing the impact of
each strategy on criteria (Table 3). Preference flow values were calculated using the VP
software. These values are consolidated results of the preferences of each strategy and
allow pairwise comparisons between strategies. The positive preference flow (phi+) allows
one to measure the extent of agreement of one stakeholder with a strategy versus the other
strategies and it represents a value of the strength of acceptability for one strategy. On the
other hand, the negative preference flow (phi−) measures how much other strategies are
preferred versus a particular strategy. Thus, phi−is a global measure of the weakness of a
strategy in terms of acceptability. Finally, the two parameters are combined and result in
the net flow score (phi), which provides the overall performance score for each strategy. Values greater than 0 are more preferred, conversely, values lower than 0 are less preferred. To model the way the stakeholder perceives the criterion measurement scale, the
PROMETHEE procedure requires associating a preference function with each criterion. The
V-shape preference function was used to analyze quantitative criteria [21]. This preference
function is a special case of the linear preference function where the Q indifference threshold
is equal to 0. This method is efficient for quantitative criteria when even small deviations
are accounted for [37]. 4.4.4. MCDA The Visual PROMETHEE software (VP; ULB, Brussels, Belgium) was used to perform
MCDA and obtain comparative rankings of a set of alternatives. Overall numerical scores
assigned by the participants were entered for each criterion on VP. Following this, results Antibiotics 2023, 12, 8 12 of 16 12 of 16 were visualized using a visual model performed by the GAIA (Graphical Analysis for
Interactive Aid) tool provided in VP. Group rankings of strategies yielding the best to
worst alternatives were performed using the individual stakeholder’s values expressed via
criteria weighing. Appendix A Interview example used to assess stakeholder preference for strategies to reduce
antimicrobial use in the French dairy industry: Study title: Evaluation of the societal acceptability of strategies to reduce the use of
antibiotics in the dairy industry in France. After extensive research, we have identified a number of potential strategies for
reducing antibiotic use. The strategies to be evaluated are: After extensive research, we have identified a number of potential strategies for
reducing antibiotic use. The strategies to be evaluated are: 1. Current scenario of antibiotic use in the dairy industry (STRA01). Current scenario of antibiotic use in the dairy i 2. Total interdiction of antimicrobial use (STRA02). 3. Interdiction of preventive and metaphylactic antimicrobial use (STRA03). 4. Subsidies for farmers committing to reduce the use of antibiotics by 25% (STRA04). To assess which strategies are most accepted by stakeholders, we need to compare
respondents’ preferences. To assess which strategies are most accepted by stakeholders, we need to compare
respondents’ preferences. The method consists in assigning 100 points among the criteria showed below for each
of the 4 strategies. The more points you assign, the more important the criterion is for you. g
p
y
g
p
y
Method: -
First, allocate 100 points among the 4 dimensions (Environmental, Economic, Social,
and Political) according to the degree of importance of this dimension for the class
of stakeholders you represent (in your case: Public Health—construction of public
policy). In the table, fill in the total row of each dimension. p
y
-
Second, for each dimension, distribute the total number of points allocated to all
criteria that make up this dimension. Thus, each criterion will have a score. In the
table, fill in the blank cells of the score columns. -
Repeat the process for each strategy. -
Strategy score must per add up to 100 points. A list of criteria you will need to consider when evaluating criteria is presented in
Table A1 and a description is presented below: A list of criteria you will need to consider when evaluating criteria is presented in
Table A1 and a description is presented below: •
Cost of production of food of animal origin •
Farmer income •
Cull cow price
•
Milk price •
ALEA: indicator of the level of exposure of animals to antibiotics (mass of the treated
population divided by the mass of the total population of the animal species). 5. Conclusions Our MCDA framework offers a methodology to assess potential strategies to reduce
AMR development in the French dairy industry. In our approach, we observed that the most
accepted strategy for consumers and public health representatives was total interdiction of
AMU, whereas farmers opted to maintain the baseline strategy. Although the strategies
considered in this study might not be plausible in the current dairy industry and due to the
low number of responders, this study provides a MCDA framework that can be applied
with an ample range of strategies for assessing their acceptability by relevant stakeholders
in the food-animal supply. Author Contributions: Conceptualization, D.M., M.C., D.R. and G.L.; Data curation, M.C., A.F. and
G.L.; Formal analysis, M.C. and G.L.; Investigation, M.C. and G.L.; Methodology, D.M., M.C., A.F. and G.L.; Project administration, A.F., D.R. and G.L.; Software, D.M., M.C. and A.F.; Supervision,
D.R.; Validation, G.L.; Writing—original draft, D.M.; Writing—review and editing, D.M., A.F., D.R. and G.L. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, D.M., M.C., D.R. and G.L.; Data curation, M.C., A.F. and
G.L.; Formal analysis, M.C. and G.L.; Investigation, M.C. and G.L.; Methodology, D.M., M.C., A.F. and G.L.; Project administration, A.F., D.R. and G.L.; Software, D.M., M.C. and A.F.; Supervision,
D.R.; Validation, G.L.; Writing—original draft, D.M.; Writing—review and editing, D.M., A.F., D.R. and G.L. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Datasets are available upon request to the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Antibiotics 2023, 12, 8 13 of 16 Appendix A The
higher the ALEA, the more animals in a population are treated with antimicrobials. •
Attributable fraction: antibiotic resistance in humans that is attributed to the use
of antibiotics in agriculture. The higher this fraction is, the greater is the impact of
antimicrobial use in animal farming on public health. g
p
•
Mortality rate: number of dairy cows that died in a given period. •
Cull rate: number of cows unfit for calf and/or milk production, due to aging or other
criteria, and now fit for fattening and/or slaughter. •
Regulatory framework: public policies related to antibiotic resistance. This frame can
take 4 values: weak, moderate, strong, very strong. •
Investments in public policies needed to reduce antibiotic use. Investments can take 4
values: weak, moderate, strong, very strong. Table A1 presents an example of the preference evaluation grid for the dimensions
and criteria in each strategy. The score columns are where points should be allocated. Table A1 presents an example of the preference evaluation grid for the dimensions
and criteria in each strategy. The score columns are where points should be allocated. In the example, in the production cost criterion, the weight 15 is given for the antibiotic
ban scenario and lower scores for the other strategies. The environmental dimension
received 30 points, which indicates that this dimension means a lot to the person answering
the interview the score. The social dimension received less points, indicating that this
dimension does not have much importance for the participant. Antibiotics 2023, 12, 8 14 of 16 14 of 16 Table A1. Example of the preference evaluation grid to be completed by multi-criteria decision
analysis participants. Table A1. Example of the preference evaluation grid to be completed by multi-criteria decision
analysis participants. Table A1. Example of the preference evaluation grid to be completed by multi-criteria decision
analysis participants. Appendix A Dimension
Criteria
STRA01
Score
STRA02
Score
STRA03
Score
STRA04
Score
Economic
Production costs (€/1000L)
494
9
684
15
667
14
617.5
12
Farmers’ revenues (€/1000L)
334
22
473
27
451
26
417.5
23
Culled cow price (€/Kg)
2.4
12
2.64
8
2.4
8
2.4
8
Product price (€/L)
0.78
8
1.85
19
1.05
16
0.97
16
Total
51
69
64
59
Environmental
ALEA
0.273
15
0
1
0.177
10
0.204
11
Attributable Fraction (%)
4
15
0
1
2.6
10
3
11
Total
30
2
20
22
Social
Mortality rate (%)
3.8
2
4.8
2
4.1
2
4.04
2
Culling rate (%)
21.3
3
50.5
5
31.5
5
28.6
3
Total
Total
5
7
7
5
Political
Regulatory framework
Moderate
12
Very
high
14
High
5
Moderate
10
Investment Policies
High
2
High
8
Moderate
4
Very
high
4
Total
14
22
9
14
Score Column Total
100
100
100
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50. Skjolstrup, N.K.; Nielsen, L.R.; Jensen, C.S.; Lastein, D.B. Veterinary Herd Health Consultancy and Antimicrobial Use in Dairy
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and applications. Eur. J. Oper. Res. 2010, 200, 198–215. [CrossRef] 52. Aenishaenslin, C.; Hongoh, V.; Cisse, H.D.; Hoen, A.G.; Samoura, K.; Michel, P.; Waaub, J.P.; Belanger, D. Multi-criteria decision
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2013, 13, 897. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
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Ecosystem Based Adaptation: Concept and Terminology in Strategic Adaptation Planning (Municipal and Inter-Municipal) in Portugal
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Keywords: ecosystem-based adaptation (EbA); strategic planning of adaptation; climate change;
biodiversity; ecosystem service; nature-based solution Ecosystem Based Adaptation: Concept and Terminology in
Strategic Adaptation Planning (Municipal and Inter-Municipal)
in Portugal Ana Cousiño *
and Gil Penha-Lopes Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências da Universidade de
Lisboa, Campo Grande, Bloco C2, Piso 5, 1749-016 Lisbon, Portugal; gppenha-lopes@fc.ul.pt
* Correspondence: ana.rez.cousino@gmail.com Abstract: Ecosystem-based adaptation (EbA) is a nature-based solution that has gained importance
in the context of international climate policy, such as in the EU Adaptation Strategy (2013), which
explicitly encourages its adoption and which should continue in The European Green Deal. This
study aims to analyze how the EbA concept has been adopted in strategic adaptation planning at the
municipal and intermunicipal levels in Portugal after the publication of the European adaptation
strategy in 2013. Thus, a quantitative content analysis was carried out, based on EbA keywords, of
municipal strategies and intermunicipal plans in Portugal. The term “ecosystem-based” has not been
transposed as an explicit objective at the municipal and intermunicipal levels. All strategies and
plans have included indirect references to the underlying elements of the EbA concept. This study
highlights that although the EU Adaptation Strategy explicitly encourages EbA, this does not mean
that it is adopted as a preferred adaptation approach at the local level in Portugal. The EbA seems to
be more widely understood by the research community than by municipal technicians or private
companies. It is necessary to explore how the EbA concept can be more widely accepted through the
generation of co-benefits and by synergies between topics. Citation: Cousiño, A.; Penha-Lopes,
G. Ecosystem Based Adaptation:
Concept and Terminology in Strategic
Adaptation Planning (Municipal and
Inter-Municipal) in Portugal.
Sustainability 2021, 13, 6145.
https://doi.org/10.3390/
su13116145 sustainability sustainability sustainability sustainability 1. Introduction Ecosystem-based adaptation (EbA) is defined as the use of biodiversity and ecosystem
services to help people adapt to climate change impacts [1]. EbA is closely related to
Community-based Adaptation (CbA), “a community-led process, based on communities’
priorities, needs, knowledge and capacities, which should empower people to plan for
and cope with the impacts of climate change” [2] (p. 13) and is influenced by an earlier
concept, Ecosystem-based Management (EbM), a management approach that tries to
balance objectives, such as the sustainable use of resources, guaranteeing equal access to
resources and reaching objectives conservation [3]. Received: 16 March 2021
Accepted: 26 May 2021
Published: 29 May 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. EbA approach is referred to as a flexible, economical and widely applicable ap-
proach [4], capable of dealing with the magnitude, speed and uncertainty of climate
change [5]. In addition, there are references that EbA can potentially provide multiple
economic, social and environmental co-benefits [4]. 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/). The EbA concept appears in the international political arena by the United Nations
Framework Convention on Climate Change in 2008. Since then, it has become an important
aspect in the international climate policy framework, it is the case with the EU climate
adaptation strategy [6], which explicitly encourages the adoption of ecosystem-based adap-
tation approaches. This trend will tend to continue in the new EU strategy on adaptation
to climate change as mentioned in The European Green Deal “Climate adaptation work
must continue to influence public and private investments, including solutions based on https://www.mdpi.com/journal/sustainability Sustainability 2021, 13, 6145. https://doi.org/10.3390/su13116145 Sustainability 2021, 13, 6145 2 of 13 nature” [7] (p. 5). Solutions based on nature are solutions that are inspired and supported
by nature, which are cost-effective, simultaneously provide environmental, social and
economic benefits and help build resilience. Such solutions bring nature, natural features
and processes into cities, landscapes and seascapes, through locally adapted, resource-
efficient and systemic interventions [8]. EbA is a nature-based solution to deal with the
impacts of climate change, focuses on the benefits that humans derive from biodiversity
and ecosystem services, and how these benefits can be used in the face of climate change [9]. 1. Introduction nature” [7] (p. 5). Solutions based on nature are solutions that are inspired and supported
by nature, which are cost-effective, simultaneously provide environmental, social and
economic benefits and help build resilience. Such solutions bring nature, natural features
and processes into cities, landscapes and seascapes, through locally adapted, resource-
efficient and systemic interventions [8]. EbA is a nature-based solution to deal with the
impacts of climate change, focuses on the benefits that humans derive from biodiversity
and ecosystem services, and how these benefits can be used in the face of climate change [9]. y
g
The EU adaptation strategy paved the way for the Member States and municipalities
to design and implement strategies for adapting to climate change [6]. In this sense,
several municipalities in Portugal have been designing and implementing adaptation
strategies [10] in order to better deal with the impacts of climate change at the local level. More recently, municipalities have joined freely in intermunicipal entities that have been
developing their strategic adaptation plans. Municipal planning represents a key way to integrate adaptation actions [11]. It
is at the local level that the establishment of adaptation strategies focused on dealing
with regional impacts will provide tangible benefits for local residents [12]. In this sense,
climate change adaptation strategies have been considered in local planning, for example,
in European cities [13]. However, in relation to EbA, specific references and knowledge
about its integration into local adaptation planning are still poorly documented [14,15]. In
addition, EbA is often not systematized or labeled as such [16]. Knowing the use and acceptance of the concept of EbA in local strategic planning is
essential to promote its implementation effectively. Thus, taking into account the objective
of the EU’s adaptation strategy (2013), we sought to analyze the use of the EbA concept in
municipal strategies and in inter-municipal plans for adapting to climate change, answering
the following questions: How is the EbA concept referred to in the strategies and plans? 1. How is the EbA concept referred to in the strategies and plans? p
g
p
2. What are the thematic sections of the strategies and plans that refer to the EbA concept? 2. What are the thematic sections of the strategies and plans that refer to the EbA concept? Based on this analysis, we seek to infer whether the concept of EbA advocated by
European policy has been adopted in strategic adaptation planning. 2. Materials and Methods To answer the questions raised, a quantitative content analysis [17] based on keywords
related to the EbA concept was used. The documentation used for the analysis were
municipal strategies, intermunicipal plans for adaptation to climate change in Portugal
and the European Strategy for adaptation on climate change (2013). 1. Introduction In this sequence, we
try to provide some ideas to strengthen the explicit adoption of the concept of EbA in
strategic adaptation planning at the local and sub-regional levels. 2.1. Selection of Documents to Be Analyzed The documents analyzed were the first municipal strategies (2015–2016) and the first
intermunicipal plans for adapting (2017–2019) to climate change carried out after the
publication of the EU Adaptation Strategy in 2013. This selection is the result of the policy
change in financing where the elaboration of intermunicipal plans was prioritized instead
of municipal strategies. The research focused on the analysis of municipal strategies (EMAAC, acronym in
Portuguese) and intermunicipal plans (PIAAC, acronym in Portuguese) for adaptation to
climate change in Portugal in comparison with the European adaptation strategy (2013). In this sense, the 27 municipal strategies carried out under the ClimAdapt.Local project
(2015–2016) (https://www.adapt-local.pt/, accessed on 16 February 2021) and the six
intermunicipal plans publicly available online (2017 to 2019) were selected. The consortium
responsible for ClimAdaPT.Local is led by the CCIAM/cE3c research center at the Faculty
of Sciences of the University of Lisbon and consists of Portuguese and Norwegian entities
(academics, companies, NGOs and municipalities). 3 of 13 Sustainability 2021, 13, 6145 The municipal strategies followed a basic methodology called ADAM (Support for
Decision on Municipal Adaptation) that guided its elaboration. The ADAM methodology
was fully developed within the scope of the ClimAdaPT.Local project and was specially
adapted to the Portuguese reality based on the model developed by the UKCIP (https:
//www.ukcip.org.uk/, accessed on 16 February 2021) (UK Climate Impacts Program). This
methodology is composed of six interrelated steps forming a strategic development cycle:
0. Prepare the work; 1. Identify current vulnerabilities; 2. Identify future vulnerabilities;
3. Identify adaptation options; 4. Assess adaptation options; 5. Integrate, monitor and
review. It was a pioneering project in Portugal in the area of adaptation to climate change
that involved 27 national municipalities in the creation of their strategies. The municipal strategies resulted from an interactive process between the municipal
technicians and the project’s scientific team. The municipal technical team (EMAAC
coordinating team) received specific training on the application of the methodology and all
work was monitored and supported by the external team of the ClimAdaPT.Local project. The first intermunicipal plans were presented in 2017, following the implementa-
tion of the Operational Program for Sustainability and Efficiency in the Use of Resources
(POSEUR, acronym in Portuguese) approved by the EC (2014) whose specific objective
is investment priority 5.1—o “Investment support for adaptation to climate change, in-
cluding ecosystem-based approaches” (https://poseur.portugal2020.pt/en/investment-
axes/axis-ii/, accessed on 22 February 2021). 2.1. Selection of Documents to Be Analyzed The intermunicipal plans were developed
and coordinated by consortia that include research teams (academia) and/or companies
private with the collaboration of the municipalities; not all of them have the same structure
or the same details, with differences in the level of development of those presented in 2017
to 2019, but it can be said that the ADAM methodology also influenced their construction. gy
Within the scope of municipal strategies and intermunicipal plans, workshops were
held with stakeholders in order to involve local communities with strategic documents. g
A list of the documentation that was analyzed is provided in Appendix A. 2.2. Analysis of Strategies and Plans m of reference to the EbA concept: explicit, direct references to the
d” were understood as evidence of the conscious adoption of the c
irect references (terms that describe underlying concepts such as
diversity) were understood as a conceptual understanding of the
benefits [19] (Figure 1). •
Analysis of the EbA keywords context, associating the EbA keywords to the thematic
section identified in the analyzed documents in order to identify in which context
they are used. In this sense, the quantification of the number of mentions is used for
each of the EbA keywords inserted in the thematic sections identified in the strategic
documents. The thematic sections that were identified in the documents are: general
context (framing the theme of climate change, local characterization), objectives (objec-
tives and strategic vision), climate change (cc) impact/vulnerability (projected climate
changes; observed and projected climate impacts and vulnerabilities) and adapta-
tion options (adaptation options that allow responding to vulnerabilities and climate
risks—current and future). Comparison with the EU’s adaptation strategy (2013). m of reference to the EbA concept: explicit, direct references to the
d” were understood as evidence of the conscious adoption of the c
irect references (terms that describe underlying concepts such as
diversity) were understood as a conceptual understanding of the
benefits [19] (Figure 1). Figure 1. Procedures used for content analysis, from left to right. Figure 1. Procedures used for content analysis, from left to right. edures used for content analysis, from left to rig
Figure 1. Procedures used for content analysis, from left to right. 2.2. Analysis of Strategies and Plans After selecting the documents, common sections were selected in order to analyze
their content. These sections represent thematically different (context) parts of the strategies
and plans (Figure 1). The analysis of the documents (EU’s adaptation strategy, municipal strategies and
intermunicipal plans) was carried out in two stages: •
Quantitative content analysis based on EbA keywords (words used to describe EbA
from the literature review)—ecosystem-based, ecosystem, ecosystem service, en-
vironmental service, biodiversity, biological diversity, green infrastructure, green
structure [18] and green area—and comparison with the quantitative content anal-
ysis, based on the EbA keywords, of the EU Adaptation Strategy (2013). The EU
Adaptation Strategy comprises a set of documents that were analyzed (COM216,
SWD131, SWD133, SWD136, SWD137, SWD138, COM213) (https://ec.europa.eu/
clima/policies/adaptation/what_en#tab-0-1, accessed on 22 February 2021) excluding
documents referring to technical guides and guidelines SWD134, SWD135, SWD139
and SWD132. Each document was analyzed for the presence of keywords and the
number of mentions was quantified. Knowing that the EbA concept includes components such as biodiversity, ecosystem
services and adaptation to climate change, we sought to know the degree to which the EbA
concept is used in strategic documents. Thus, the keywords were classified according to
their form of reference to the EbA concept: explicit, direct references to the term “ecosystem-
based” were understood as evidence of the conscious adoption of the concept, and implicit,
indirect references (terms that describe underlying concepts such as ecosystem services,
biodiversity) were understood as a conceptual understanding of the related objectives and
benefits [19] (Figure 1). Sustainability 2021, 13, 6145 4 of 13 •
Analysis of the EbA keywords context, associating the EbA keywords to the thematic
section identified in the analyzed documents in order to identify in which context
they are used. In this sense, the quantification of the number of mentions is used for
each of the EbA keywords inserted in the thematic sections identified in the strategic
documents. The thematic sections that were identified in the documents are: general
context (framing the theme of climate change, local characterization), objectives (objec-
tives and strategic vision), climate change (cc) impact/vulnerability (projected climate
changes; observed and projected climate impacts and vulnerabilities) and adapta-
tion options (adaptation options that allow responding to vulnerabilities and climate
risks—current and future). Comparison with the EU’s adaptation strategy (2013). f h E
3. Results is of the EbA keywords context, associating the EbA keywords to th
identified in the analyzed documents in order to identify in wh
e used. In this sense, the quantification of the number of mentions
the EbA keywords inserted in the thematic sections identified in t
The term “ecosystem-based” appears to be explicitly referred to in the EU strategy
(13% of all related mentions); however, at the national level, this expression has not been
transposed to municipal strategies, being discreetly mentioned (0.8%) in intermunicipal
plans (Figure 2). 5 of 13
3 Sustainability 2021, 13, 6145
S
t i
bilit 2021 13 6 (a)
(b)
(c)
Figure 2. Percentage of total mentions for each EbA related keywords per set of analyzed documents, (a) EU’s adaptation
strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. The EbA-key-
words with zero mentions appear absent in the graphs. Figure 2. Percentage of total mentions for each EbA related keywords per set of analyzed documents, (a) EU’s adaptation
strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. The
EbA-keywords with zero mentions appear absent in the graphs. (b) (a)
(c) (b) (a) (c) Figure 2. Percentage of total mentions for each EbA related keywords per set of analyzed documents, (a) EU’s adaptation
strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. The EbA-key-
words with zero mentions appear absent in the graphs. Figure 2. Percentage of total mentions for each EbA related keywords per set of analyzed documents, (a) EU’s adaptation
strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. The
EbA-keywords with zero mentions appear absent in the graphs. Figure 2. Percentage of total mentions for each EbA related keywords per set of analyzed documents, (a) EU’s adaptation
strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. The EbA-key-
words with zero mentions appear absent in the graphs. Figure 2. Percentage of total mentions for each EbA related keywords per set of analyzed documents, (a) EU’s adaptation
strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. The
EbA-keywords with zero mentions appear absent in the graphs. f h E
3. Results The transposition of the reference “ecosystem service”
followed a similar pattern at the intermunicipal level in relation to the EU strategy, being
absent at the municipal level (Figure 3). 6 of 13 (a)
(b)
(c)
gure 3. Percentage of total mentions for each EbA-keyword associated with the thematic sections identified in the documents,
EU´s adaptation strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. nly keywords with more than 1% representation. Figure 3. Percentage of total mentions for each EbA-keyword associated with the thematic sections identified in the
documents, (a) EU´s adaptation strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal
plans, Nkeywords = 784. Only keywords with more than 1% representation. (b) (a) (b) (c) (c) ure 3. Percentage of total mentions for each EbA-keyword associated with the thematic sections identified in the documents,
EU´s adaptation strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal plans, Nkeywords = 784. y keywords with more than 1% representation. Figure 3. Percentage of total mentions for each EbA-keyword associated with the thematic sections identified in the
documents, (a) EU´s adaptation strategy, Nkeywords = 77, (b) municipal strategies, Nkeywords = 759 and (c) intermunicipal
plans, Nkeywords = 784. Only keywords with more than 1% representation. In the analyzed strategies and plans, the term “ecosystems-based” occurs neither as
an explicit objective nor with representativeness in the contexts of climate change impacts
and adaptation options. Looking from a time perspective, six years after the publication
of the EU Adaptation Strategy (2013), there are no references to ecosystem-based adapta-
tion as an explicit objective in strategic adaptation planning at the local level (Figure 3). In relation to the other keywords, there is an explicit absence in the objectives of the
strategies and plans in relation to the EU strategy (2013). The references “biodiversity”
a d “e o y te
” ha e bee
i
luded i
the o te t of li
ate ha
e i
a t a d ada
With a focus on the differences between the municipal strategies and intermunicipal
plans, it should be noted that they result from different preparation processes and their
authors have different backgrounds. In municipal strategies (EMAACs), the term “green
area” stands out in the context of adaptation options. f h E
3. Results All strategies and plans included indirect references to the components underlying
the concept of EbA, reflected in the use of related keywords, namely “ecosystem”, “biodi-
versity” with emphasis, “green area” and “green infrastructure” with less expression. However, the term “ecosystem service”, although closely related to EbA, receives atten-
tion only in intermunicipal plans (PIAAC) and is absent from municipal strategies
(EMAACs) (Figure 2). Considering, in the context analysis, only keywords with more than 1% representa-
All strategies and plans included indirect references to the components underlying
the concept of EbA, reflected in the use of related keywords, namely “ecosystem”, “bio-
diversity” with emphasis, “green area” and “green infrastructure” with less expression. However, the term “ecosystem service”, although closely related to EbA, receives attention
only in intermunicipal plans (PIAAC) and is absent from municipal strategies (EMAACs)
(Figure 2). tion, the term “ecosystem-based” appears as an explicit objective of the EU strategy and
associated with the contexts of climate change impacts and adaptation options (Figure 3). Considering, in the context analysis, only keywords with more than 1% representa-
tion, the term “ecosystem-based” appears as an explicit objective of the EU strategy and
associated with the contexts of climate change impacts and adaptation options (Figure 3). g
p
p
p
g
In the analyzed strategies and plans, the term “ecosystems-based” occurs neither as
an explicit objective nor with representativeness in the contexts of climate change impacts
and adaptation options. Looking from a time perspective, six years after the publication of
the EU Adaptation Strategy (2013), there are no references to ecosystem-based adaptation
as an explicit objective in strategic adaptation planning at the local level (Figure 3). In relation to the other keywords, there is an explicit absence in the objectives of the
strategies and plans in relation to the EU strategy (2013). The references “biodiversity” and Sustainability 2021, 13, 6145 6 of 13 “ecosystem” have been included in the context of climate change impacts and adaptation
options, while “green area” and “green infrastructure” appear essentially associated with
the context of adaptation options. The transposition of the reference “ecosystem service”
followed a similar pattern at the intermunicipal level in relation to the EU strategy, being
absent at the municipal level (Figure 3). 6 of 13 “ecosystem” have been included in the context of climate change impacts and adaptation
options, while “green area” and “green infrastructure” appear essentially associated with
the context of adaptation options. f h E
3. Results Its preparation was coordinated,
in each municipality, by an internal team of technicians with different backgrounds but
where the disciplinary areas associated with engineering, design and planning stand out
(Figure 4). 7 of 13
tand out Sustainability 2021, 13, 6145 Figure 4. Municipal technicians by area of knowledge present in the coordinating teams in the
municipal strategies (EMAAC), n = 66. Figure 4. Municipal technicians by area of knowledge present in the coordinating teams in the
municipal strategies (EMAAC), n = 66. (Figure 4). Figure 4. Municipal technicians by area of knowledge present in the coordinating teams in the
municipal strategies (EMAAC), n = 66. Figure 4. Municipal technicians by area of knowledge present in the coordinating teams in the
municipal strategies (EMAAC), n = 66. Figure 4. Municipal technicians by area of knowledge present in the coordinating teams in the
municipal strategies (EMAAC), n = 66. Figure 4. Municipal technicians by area of knowledge present in the coordinating teams in the
municipal strategies (EMAAC), n = 66. The intermunicipal plans (PIAACs) benefited from the experience acquired by the
ClimAdapt.Local project. Its preparation was coordinated by a consortium made up of
research teams (academia) and/or consulting companies, with emphasis on the greater
number of mentions of the keyword “ecosystem service” in the plans prepared by consor-
tia that integrate the research teams (Figure 5). The intermunicipal plans (PIAACs) benefited from the experience acquired by the
ClimAdapt.Local project. Its preparation was coordinated by a consortium made up of
research teams (academia) and/or consulting companies, with emphasis on the greater
number of mentions of the keyword “ecosystem service” in the plans prepared by consortia
that integrate the research teams (Figure 5). The intermunicipal plans (PIAACs) benefited from the experience acquired by the
ClimAdapt.Local project. Its preparation was coordinated by a consortium made up o
research teams (academia) and/or consulting companies, with emphasis on the greate
number of mentions of the keyword “ecosystem service” in the plans prepared by consor
tia that integrate the research teams (Figure 5). Figure 5. Number of EbA-keywords mentions in each intermunicipal plan (PIAAC) and type of
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. Figure 5. Number of EbA-keywords mentions in each intermunicipal plan (PIAAC) and type of
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. Figure 5. 4.1. The Use of the EbA Concept in Strategic Adaptation Planning Documents The EU’s adaptation strategy (2013) explicitly recognizes the relevant role of EbA in
adapting to climate change. However, this form has not been transposed as an explicit
objective of the first municipal strategies (EMAAC) or the first intermunicipal plans (PI-
AAC). This can be due to the fact that The National Strategy for Adaptation to Climate
Change 2020, a guiding document for local adaptation strategies, does not include EbA
as an explicit objective [20]. However, most of the strategies and plans analyzed included
indirect references to the underlying elements of EbA (ecosystems, biodiversity, green
infrastructure, and green area), indicating general recognition of elements of this concept. g
g g
g
p
Regarding the underlying concept of “ecosystem service”, there has been an evolution
in the way the concept expresses itself from municipal strategies to intermunicipal plans
in relation to the EU’s adaptation strategy, which seems to result from the contribution
of PIAACs prepared by consortia that include the academia (Figure 5). This relation-
ship with academia is also seen in Germany, where, for example, Dresden’s adaptation
strategy that resulted from a research project entitled “REGKLAM” contains the most
references to ecosystem services [18]. This observation is probably the result of the concept
of “ecosystem services” being introduced by the scientific community, with great promi-
nence in 1997 with the publications of Gretchen Daily [21] and Robert Costanza [22], but
it also raises the question of the concept of “ecosystem service” being comprehensively
understood outside academia. Some reasons can be exposed, namely, the coexistence
of better-established concepts like “green infrastructure” in the planning discourse [23]. Perhaps that is why the term “ecosystem service” does not appear in municipal strate-
gies in Portugal. In addition, climate change adaptation measures are often dominated
by an infrastructure-based approach and, consequently, remain unrelated to ecosystem
services [24]. Also, the lack of national promotion of “ecosystem services” in Portugal—as
in Sweden, where “ecosystem services” were adopted by city officials, making them a
key factor for nature-based planning approaches at the local level [16,24]—can hinder
the integration of ecosystem services in spatial planning at the local level. This difficulty
is visible, for example, in the two most important legislative documents in the area of ter-
ritorial management, such as the basic law of public soil policy, land-use planning and ur-
banism (Law No. f h E
3. Results Number of EbA-keywords mentions in each intermunicipal plan (PIAAC) and type of
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. r of EbA keywords mentions in each intermunicipal p g
y
p
p
yp
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. Figure 5. Number of EbA-keywords mentions in each intermunicipal plan (PIAAC) and type of
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. Figure 5. Number of EbA-keywords mentions in each intermunicipal plan (PIAAC) and type of
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. Figure 5. Number of EbA-keywords mentions in each intermunicipal plan (PIAAC) and type of
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. Figure 5. Number of EbA-keywords mentions in each intermunicipal plan (PIAAC) and type of
PIAAC coordinating consortium (research team, research team and consulting companies), n = 6. Sustainability 2021, 13, 6145 8 of 13 4. Discussion The results of the strategies and plans were analyzed based on the objective of the
EU’s adaptation strategy for the adoption of EbA. 4.1. The Use of the EbA Concept in Strategic Adaptation Planning Documents 31/2014) (https://data.dre.pt/eli/lei/31/2014/05/30/p/dre/pt/html,
accessed on 3 March 2021) and in the instruments of territorial management in 2015 (DL nº
80/2015) (https://data.dre.pt/eli/dec-lei/80/2015/p/cons/20201002/pt/html, accessed on
3 March 2021) where there is no concrete reference to ecosystem services. The emphasis given to the term “biodiversity” probably results from the recognition
of the importance of the impact of climate change on biodiversity in Portugal (Figure 3), as
is recognized in other countries such as Germany [18]. Also, part of Portugal’s commitment
to European and international agreements on the conservation of nature and biodiversity
is an example of the European Biodiversity Strategy 2020, the United Nations Agenda for
Sustainable Development 2030 and the Convention on Biological Diversity. It is important
to add that the recognition of the importance of biodiversity by Portugal has been going
on for some time, with the ratification of the Convention on Biological Diversity in 1993,
from where the definitions of ecosystem and biodiversity that are still the most used
today emerged. y
g
The creation of the Natura 2000 Network in Portugal also contributed to reinforcing the
importance of biodiversity as it represents about 22% of its land area, average EU-28: 18%
(RCM 55/2018) (https://data.dre.pt/eli/resolconsmin/55/2018/05/07/p/dre/pt/html,
accessed on 3 March 2021), which corresponds to 71% of the municipalities with the area
classified in this scope (INE, 2016) (https://www.ine.pt/, accessed on 3 March 2021). This local expression is also visible in the strategies and plans analyzed, that is, 70% Sustainability 2021, 13, 6145 9 of 13 9 of 13 of the municipalities in the municipal strategies and 64% of the municipalities in the
intermunicipal plans have designated areas of the Natura 2000 Network. of the municipalities in the municipal strategies and 64% of the municipalities in the
intermunicipal plans have designated areas of the Natura 2000 Network. p
p
g
As for the term “green infrastructure” (GI) it appears in the EU´s context as a “strate-
gically planned network of natural and semi-natural areas with other environmental
characteristics designed and managed to provide a wide range of ecosystem services” [25]
(p. 3) and it is explicitly encouraging its adoption as an approach to adaptation to climate
change such as EbA [6]. 4.2. Strengthen Explicit Adoption of the EbA Concept in Strategic Adaptation Planning From the analyzed documents, the strategic orientation at the European level does
not seem to be sufficient for the preferential integration of EbA in strategic adaptation
planning at the local and sub-regional (municipal and intermunicipal) levels in Portugal. Thus, strong leadership will be needed to promote ecosystem-based adaptation at the local
and sub-regional levels, even without the support of higher levels of decision-making
or supporting legislation [29]. This is the case, for example, in Lomma, Sweden, where
the integration of ecosystem-based planning was made possible by local politicians who
explicitly took steps to reverse the decline in green infrastructure [16,24]. It is also crucial
to involve municipal officials committed to previous experience in integrating other issues
at the municipal level. For example, the involvement of municipalities in environmental
planning (in Germany and Sweden), their support for the concept of ecosystem services
(in Sweden) or the mitigation of climate change (in Germany), enabled the structures,
processes and instruments necessary to pave the way for the integration of ecosystem-
based adaptation at the municipal level [24]. p
p
The promotion of EbA at the local and sub-regional level could be facilitated by
synergies between topics, for example, the promotion of biodiversity can motivate the use
of ecosystem services to adapt to climate change. This is what happened in the municipality
of Helsingborg, Sweden, where the promotion of biodiversity through green infrastructure
was used to manage rainwater flows [24,30]. In addition, synergies between adaptation
and mitigation of climate change can be provided by EbA. This is the case of German cities
that recommend ecological measures, such as planting trees on the streets or green trails,
as well as installing green facades to mitigate urban heat [31]. The greening of the facades
can decrease the demand for refrigeration energy in a building [32], urban vegetation can
sequester and store carbon emission [33], thus, helping to mitigate climate change. In
addition, it provides various ecosystem services, such as new habitats for animals and
plants [34], noise reduction [35], particle filtering and absorption of air pollutants [36]. Ecosystem-based adaptation has the potential to generate co-benefits, that is, it can
help meet various environmental, social and economic objectives [37,38], in addition to
helping to overcome the challenge of climate change. 4.1. The Use of the EbA Concept in Strategic Adaptation Planning Documents This relationship with the adaptation context is visible both at the
level of municipal strategies and intermunicipal plans, which is not the case with the term
“ecosystem-based”, which may be related to the “antiquity” of the concept, GI was first
introduced in the middle of the 90s [26] and has been adopted by the various disciplines
related to design, conservation, and planning [27]. This trend seems compatible when we
consider the areas of knowledge of the municipal technicians responsible for coordinating
the EMAACs (Figure 4). Associated with the GI appears the term “green area” because
they are important components of the GI and are considered public goods that allow free
access to all citizens and represent grants of nature for all residents [28]. 4.2. Strengthen Explicit Adoption of the EbA Concept in Strategic Adaptation Planning In this sense, the use of co-benefits
could be a way of exploring to promote broader acceptance of this approach by different
stakeholders, as the evidence suggests that citizens are more likely to act on climate change
if the wider co-benefits of these actions are emphasized [39]. However, the co-benefits
approach requires going beyond its communication, that is, including co-benefits in policy
formulation and decision making [39]. In this sense, the development of tools that make
it possible to measure the co-benefits of EbA options at the local level could be a way to
explore to help support the decision and involve different actors in the implementation
of EbA. 10 of 13 10 of 13 Sustainability 2021, 13, 6145 5. Conclusions This study found that the adoption of the EbA concept and terminology in the mu-
nicipal strategies and intermunicipal plans in Portugal is influenced by the educational
background, objectives and interests of the stakeholders (municipal technicians, companies,
academics) involved in its elaboration. Although the EU’s adaptation strategy (2013) explicitly encourages EbA, this does not
mean that it is adopted as a preferred adaptation approach at the local level in Portugal, as
the strategic documents analyzed show that EbA is not an objective explicit. However, there
is recognition of the importance of “green infrastructure” and “green spaces” for a clear
adaptation to climate change and “biodiversity” and “ecosystems” in a less expressive way. p
g
y
y
p
y
The term “ecosystem service”, although closely related to EbA, receives less attention
than other terms, such as “biodiversity”, “ecosystems”, “green infrastructure” and “green
spaces”, and appears to be more widely understood by the research community than
by municipal technicians or private companies. This trend has consequences for the
understanding of the concept of EbA, which seems to accompany this same trend, which
can hinder its implementation. Author Contributions: Conceptualization, A.C. and G.P.-L.; methodology, A.C. and G.P.-L.; inves-
tigation, A.C.; supervision, G.P.-L.; writing—original draft preparation A.C.; writing review and
editing, A.C. and G.P.-L. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are openly available in the reports of
the municipal strategies and intermunicipal adaptation plans made available in Appendix A. Data Availability Statement: The data presented in this study are openly available in the reports of
the municipal strategies and intermunicipal adaptation plans made available in Appendix A. Acknowledgments: We would like to thank Fundação para a Ciência e Tecnologia for supporting
the FCT Investigator contract (IF/00940/2015) of G.P.-L. The APC was funded by the cE3c FCT Unit
funding UIDB/00329/2020. Acknowledgments: We would like to thank Fundação para a Ciência e Tecnologia for supporting
the FCT Investigator contract (IF/00940/2015) of G.P.-L. The APC was funded by the cE3c FCT Unit
funding UIDB/00329/2020. Table A1. List of documents analyzed. 5. Conclusions Strategic Document Type
Name
Year
Coordination
PIAAC
Plano Intermunicipal de Adaptação às Alterações
Climáticas da CIM-Região de Coimbra
https://www.cim-regiaodecoimbra.pt/wp-
content/uploads/2018/10/PIAAC-CIM-RC-vers%
C3%A3o-web.pdf (accessed on 8 February 2021)
2017
CEF and CEGOT from University
of Coimbra
PIAAC
Plano Intermunicipal de Adaptação às Alterações
Climáticas-Alentejo Central
https://www.cimac.pt/wp-content/uploads/2020
/12/PIAAC_AC-Relatorio-Final.pdf (accessed on
8 February 2021)
2017
CEDRU—Technical consulting
company
We Consultants (territorial
consultancy projects)
IGOT from University of Lisbon
PIAAC
Plano de Ação Intermunicipal para as Alterações
Climáticas do Douro
https://www.cimdouro.pt/adapt_clima/files/
proposta_paiac.pdf (accessed on 8 February 2021)
2017
Inflection Point, Unipessoal Lda
(Consultancy for information
management, strategic planning
and cartography production) Table A1. List of documents analyzed. 11 of 13 Sustainability 2021, 13, 6145 Table A1. Cont. Strategic Document Type
Name
Year
Coordination
PIAAC
Plano Intermunicipal de Adaptação às Alterações
Climáticas-Lezíria do Tejo
https://www.cimlt.eu/pdf-s/2486-piaaclt-plano-
intermunicipal-de-adaptacao-alteracoes-climaticas-
lt/file (accessed on 8 February 2021)
2018
PROCESL (company specialized
in consulting)
Matos, Fonseca & Associados,
Estudos e Projectos, Lda. (Environmental studies and
projects company)
PIAAC
Plano Intermunicipal de Adaptação às Alterações
Climáticas-Médio do Tejo
https://mediotejo.pt/images/CIMT/Areas_de_
Intervencao/Alteracoes-Climaticas/PIAAC_MT_
Final.pdf(accessed on 8 February 2021)
2018
Enhidrica company
(environmental engineering
consultancy)
PIAAC
Plano Intermunicipal de Adaptação às Alterações
Climaticas do Algarve
https://amal.pt/comunicacao/publicacoes/234-
plano-intermunicipal-de-adaptacao-as-alteracoes-
climaticas-piaac-amal (accessed on 8 February 2021)
2019
CCIAM/cE3c/Faculty of Sciences
University of Lisbon
CIMA/University Algarve
Bentley Systems Portugal
(software solutions for
engineering and infrastructure)
EMAAC
Estratégia Municipal de Adaptação às Alterações
climáticas de:
Amarante, Barreiro, Braga, Bragança, Castelo Branco,
Castelo de Vide, Coruche, Evora, Ferreira do
Alentejo, Figueira da Foz, Funchal, Guimarães,
Ilhavo, Leiria, Lisboa, Loulé, Mafra, Montalegre,
Odemira, Porto, São João da Pesqueira, Seia, Tomar,
Tondela, Torres Vedras, Viana do Castelo, Vila
Franca do campo. https://apambiente.pt/index.php?ref=16&subref=
81&sub2ref=118&sub3ref=395 (accessed on
8 February 2021)
2016
Municipal technicians with
support from the
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English
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Assessment of Complementary Feeding Practices and Associated Factors among Infants Aged from 6-24 months in Hargeisa, Republic of Somaliland: A Cross-Sectional Study in 2024"
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Research Square (Research Square)
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cc-by
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Assessment of Complementary Feeding Practices
and Associated Factors among Infants Aged from
6-24 months in Hargeisa, Republic of Somaliland: A
Cross-Sectional Study in 2024" Hamse Kh. Hassan Addis ababa Medical University college
Abdeta M. Ahmed
Addis ababa Medical University college
Mohamed A. Ahmed
Addis ababa Medical University college Posted Date: May 7th, 2024 Posted Date: May 7th, 2024 Research Article Keywords: Complementary feeding, maternal and child health, Somaliland, nutrition practices, infant
feeding, public health. DOI: https://doi.org/10.21203/rs.3.rs-4352402/v1 icense: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/20 Abstract Background and Objectives: The weaning process from the breast milk to intake of other foods and
drinks, during infancy is a very crucial stage whereby a child is vulnerable to malnourishment should
proper feeding practices not be put into practice. This work was essentially important to assess the
prevalence and associated factors of appropriate complementary feeding practice among infants aged
from 6-24 months in Hargeisa, Republic of Somaliland 2024. . Methods: A descriptive cross- sectional study was carried out among the primary health facilities in
Hargeisa. The multistage sampling approach was very used and recruited 370 mother-and-child pairs. The data collection was done through the pre-tested interviewer administered questionnaire with the
WHO infant and young children feeding indicators (IYCF) IBM SPSS version 23 was employed to go into
and encode quantitative data. data was presented in descriptive statistics using tables and figures by
computing the frequencies and percentages of Appropriate complementary feeding practices, Bivariate
logistic regression analyses was done to identify candidate variables for adjusted regression at p-values
of <0.05 and multivariable logistic regression were presented as p-value <0.05 with Adjusted Odds ratios
(AOR) with 95% confidence intervals. All tests were two sided to determine statistical significance Results: The analysis revealed that the overall prevalence of appropriate complementary feeding
practices was 46%. While Unemployed mothers were more likely to practice ACFP compared to
employed ones (AOR=3.5, 95% CI: 2.15-5.69, p<0.001). Larger families (≥5 members) exhibited higher
adherence to ACFP (AOR=4.193, 95% CI: 2.650-6.634, p<0.001). Avoiding bottle feeding (AOR=2.907, 95%
CI: 1.88-4.49, p<0.001), maintaining good WASH and hygiene status (AOR=4.488, 95% CI: 2.86-7.05,
p<0.001), achieving adequate dietary diversity (AOR=0.032, 95% CI: 0.05-0.068, p<0.001), meeting the
minimum acceptable diet (AOR=0.012, 95% CI: 0.005-0.027, p<0.001), and timely introduction of
complementary foods at 6 months (AOR=2.504, 95% CI: 1.636-3.832, p<0.001) were all significantly
associated with higher ACFP adherence. These findings underscore the importance of addressing socio-
demographic and dietary factors to improve infant feeding practices in the community. Conclusion and Global Health Implications: The research revealed that problematic complementary
feeding practices were in the high cases. Measurement of composite indices showed a qualitative
picture of these various practices and their components, understanding the diverse manifestations of
complementary feeding. This information may be very indispensable to obtain targeted interventions
aimed at improving the complementary feeding practices in this community. Background Page 2/20
Introduction of solid, semi-solid, or soft foods at the age of 6–8 months to child constitutes
complementary feeding. This practice intends to satisfy the increasing energy and nutrient demands of
the child. It will be quite a stretch until the child is in a position to consume a diet similar to the rest of
the family (Mitchodigni, Amoussa Hounkpatin et al. 2017). The complementary feeding period is a key
window of opportunity for the prevention of different types of malnutrition including wasting, stunting, obesity and overweight (White, Bégin et al. 2017). It is therefore due to the fact that at the age of 6
months breast milk alone is no longer able to provide all the needed nutrients and calories that such
other foods are introduced to complement the nutrients and energy needs of the infant. Lack of
complementary foods provided at the appropriate age or their inappropriate introduction may lead to the
retardation of infant's growth(Abeshu, Lelisa et al. 2016, Olatona, Adenihun et al. 2017). Complementary feeding is the addition of safe and appropriate foods to children aged six months and
above, through which the child's diet is diversified to meet his/her changing nutritional needs and
support growth, development, and overall well-being.(WHO 2008) This is a critical stage because it
coincides with a period of heightened vulnerability in children which is characterized by many growth
challenges, micronutrient deficiencies and an increased risk of diseases such as diarrhea and respiratory
infections (Ndiokwelu, Maduforo et al. 2014) Improved complementary feeding results to 6% mortality reduction However, in the developing world the
complementary foods provided to infants and young children are often bad or not enough in quantity
(WHO 2008). The Baby Friendly Initiative, the National Policy on Infant and Young Child Feeding and
other such efforts have been introduced in Nigeria, but the level of practice of appropriate
complementary feeding practices remains low, affecting less than 10% of children aged 6–23 months in
the country (Health 2010, Nutrition 2013). Globally, almost half to two-thirds of child deaths are associated with under-nutrition with one third of
child fatality stemming from low complementary feeding and sub-optimal breastfeeding. Malnutrition is
the 60 percent of the total 10.9 million annual deaths from children under five years of age worldwide. Background The majority of these deaths, more than two-thirds, occur within the first two years of life and are
commonly linked with inappropriate complementary feeding practices and infection Every year, over 10
million children who are less than five years old die of different factors, and 41% of these deaths
transpire in sub-Saharan Africa while an additional 34% transpires in South Asia. Immediately, poor
breastfeeding and complementary feeding practices are identified as leading causes of these
mortalities. It is estimated that by the end of the first 6 months of a sub-optimal complementary feeding
leads to 1.4 million death and accounts for about 10% of the morbidity in children under the age of
five (Alemu, Ahmed et al. 2017). The proportion of infants and young children having proper feeding practices has been on a decrease in
Nigeria; the percentage went down from 30% in 2008 to 10% in 2013. On 2018, overall, only 11% of the
children aged 6-24 months received minimum acceptable diet as recommended which indicates need
for persistent interventions to promote and implement appropriate infant feeding practices in the
country(Commission 2013, Demographic 2019). The rate of child malnutrition is very disconcerting at 50% in Somalia. A comprehensive studies'
summary by FSNAU concludes a nationwide 24.9 percent national stunting mean. This means around
1.4 million kids become the victims and that is more than the whole population of San Diego, California. Some areas in Somaliland have challenges with 4.2% of the children suffering stunting and 9.8% Page 3/20 Page 3/20 experiencing wasting. A stunting prevalence exceeding 20% by the World Health Organization (WHO)
becomes a major public health problem (Kinyoki, Berkley et al. 2016). The literature has used the WHO IYCF indicators to study complementary feeding and the data has
shown a low prevalence of appropriate feeding practices in various regions. In Nigeria, data spanning
from 2003 to 2013 indicates a concerning trend: The minimum dietary diversity for children aged 6-23
months declined from 26% in 2003 to 16% in 2013, while the minimum meal frequency showed an
improvement from 43% in 2003 to 56% in 2013. On the other hand, the incidence of minimum acceptable
diets decreased somewhat from 11% to 9% the same period. Likewise, in the Northern Ghana, 58.2% met
the minimum number of meals, 34.8% for the minimum diet variety, and 27.8% for the minimum
acceptable diet. Study design, area and setting The study site was a Hargeisa city local government Administration (LGA) in Republic of Somaliland. It
was a descriptive cross-sectional design study targeted mothers or caregivers of children of age ranges
6-24 months attending immunization clinics at selected PHCs in the Hargeisa city LGA. Over 50% of the
local government Administrations Primary Health Centers were selected, through a multistage sampling
approach, for 370 mother- child pairs. Background The situation in Cambodia was equally bad, as only 28% of children met the minimum
dietary requirements. The rate of minimum dietary diversity among children in Southern Ethiopia was
particularly low, with about 19% of them meeting this criterion (Kassa, Meshesha et al. 2016). Data quality management To ensure Reliability and validity were checked using pilot testing, Cronbach’s alpha. The contents of this
study were based on objectives and data was double entry validated through Epi-data software and data
collectors were trained and oriented how to avoid errors during data collection. Data analysis procedures Data were used to enter Epi-Data version 3.1, and exported to SPSS, version 23.0 windows for further
analysis. Reliability of the equipment was checking using Cornbrash’s Alpha. The data was presented in
descriptive statistics using tables and figures by computing the frequencies and percentages of
Appropriate complementary feeding practices, Bivariate logistic regression analyses was done to identify
candidate variables for adjusted regression at p-values of <0.05 and multivariable logistic regression
were presented as p-value <0.05 with Adjusted Odds ratios (AOR) with 95% confidence intervals. All tests
were two sided to determine statistical significance Study Variables The dependent variables covered complementary feeding practices such as MDD, MMF and
MAD. Covariance of the study included the mother’s level of education, income, place of work, marital
status and household size. Independent variables referred to the socio-demographic characteristics of
the children such as gender, age and so on. MDD, MMF, and MAD were delivered and calculated with the
help of WHO definitions and seizure appropriate. MMF denotes the percentage of children received complementary foods a minimum recommended
count for the last 24 hours. MDD was being calculated by recalling information of foods consumed in amount equal to four or more
varieties out of seven food groups divided by seven food groups in the previous 24-hour sample among
the children aged 6–24 months. MAD is composite indicator of MDD and MMF, where breastfed children meeting both criteria are
Moaning to have met MAD. Page 4/20 Page 4/20 The appropriate complementary feeding was assessed for a child currently breastfeeding,
complementary food at 6 months age, MMF and MDD Appropriate complementary feeding was taken to
be Appropriate, and inappropriate complementary feeding. Data Collection A pre-tested semi-structured interviewer-administered questionnaire was used to collect data on
Sociodemographic characteristics, complementary feeding practices, 24-hour diet recall and food
frequency. Three research assistants were trained on the instrument and conducted the interview along
with the principle investigator. Results and Discussion The analysis of Appropriate Complementary Feeding Practices (ACFP) reveals significant associations
with various socio-demographic factors. Notably, the age of the mother/caregiver emerges as a critical
determinant, with 12% of those under 20 years, 60% of those aged 21-29 years, and 28% of those aged
30 or older adhering to correct CF practices. This association is statistically significant with a p-value of
0.048. Furthermore, educational attainment plays a pivotal role, with 47% of individuals who attended school
practicing appropriate feeding, compared to 53% among those who did not. Family income also
influences adherence, with 33% of families earning ≤ 150 USD, 28% earning 151-250 USD, and 39%
earning ≥ 251 USD following appropriate practices. Family size emerges as another crucial variable, with 49% of families with fewer than four members
adhering better to appropriate feeding practices, compared to 51% in larger families (5 or more Page 5/20 members). The gender of the child is also significant, as 43% of girls receive appropriate complementary
feeding, compared to 57% of boys. Age of the child is a key determinant, with 27% of children aged 6-8 months receiving appropriate feeds,
contrasting sharply with 17% at 9-11 months and 56% at 12-24 months. Antenatal care visits also
demonstrate significance, with 60% of attendees following appropriate feeding guidelines compared to
40% of non-attendees. Lastly, the number of antenatal care visits correlates with feeding practices, as
54% of women attending less than four times adhere to appropriate practices, compared to 46% of those
attending four or more times. Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 Page 6/20 Variable
Appropriate complementary feeding Practice (ACFP)
Appropriate N (%)
Inappropriate N (%)
Age of the mother/caregiver
<20 years
19 (12%)
15 (7%)
21-29 years
102 (60%)
183 (92%)
≥30 years
49 (28%)
2 (1%)
Ever attend school
Yes
81 (47%)
80 (40%)
No
89 (53%)
120 (60%)
Monthly income of the family
≤ 150 USD
56 (33%)
41 (21%)
151-250 USD
48 (28%)
155 (77%)
≥ 251 USD
66 (39%)
4 (2%)
Family Size
<4
83 (49%)
160 (80%)
≥5
87 (51%)
40 (20%)
Sex of the child
Girl
73 (43%)
106 (53%)
Boy
97 (57%)
94 (47%)
Age of the child in months
6-8 Months
46 (27%)
38 (19%)
9-11 Months
28 (17%)
80 (40%)
12-24 Months
96 (56)
82 (41%)
Ante Natal Care visit
Yes
103 (60%)
147 (74%)
No
67 (40%)
53 (26%)
Number of Antenatal Care Visit
< 4 visits
91 (54%)
41 (21%) Page 7/20 The feeding practices among infants and young children across different age groups reveal notable
trends. The vast majority of children meet the recommended minimum frequency of meals, with 95% at
6-8 months, 90% at 9-11 months, and 30% at 12-24 months. There is a significant disparity in meeting the
minimum dietary diversity criteria, with only 46% at 6-8 months, 78% at 9-11 months, and 76% at 12-24
months. Similarly, there is a notable difference in meeting the minimum acceptable diet criteria, with 30% at 6-8
months, 20% at 9-11 months, and 53% at 12-24 months. The timely introduction of complementary foods
compared to the three months prior remains consistent across age groups, with 69% at 6-8 months, 66%
at 9-11 months, and 52% at 12-24 months. However, breastfeeding rates decline notably with age, showing 70% at 6-8 months, 68% at 9-11 months,
and 54% at 12-24 months. Conversely, bottle feeding practices show a significant increase, with 70% at
six to eight months, 68% at nine to eleven months, and 54% at twelve to twenty-four months. Table 2: Complementary feeding practices among Infants aged from 6-24 months Using the WHO infant
and yang child feeding Indicators by child Age in Months in Hargeisa, Republic of Somaliland 2024. Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 Page 8/20 WHO IYCF Indicator
6-8 months
9-11 months
12-24 months
P-Value
N (%)
N (%)
N (%)
Minimum meal frequency
Met
80 (95%)
96 (90%)
54 (30%)
<0.001
Not Met
4 (4%)
12 (11%)
124 (70%)
Minimum Dietary Diversity (≥ 4 food groups)
Inadequate (≤ 4)
39 (46%)
85 (78%)
136 (76%)
<0.001
Adequate (≥ 5)
45 (54%)
23 (22%)
42 (24%)
Minimum Acceptable Diet
Met
25 (30%)
21 (20%)
94 (53%)
<0.001
Not met
59 (70%)
87 (80%)
84 (47%)
Timely Introduction of complementary foods at 6 months
Yes
58 (69%)
71 (66%)
91 (52%)
0.006
No
26 (31%)
37 (34%)
87 (48%)
Bottle feeding practice
Yes
59 (70%)
74 (68%)
97 (54%)
0.013
No
25 (30%)
34 (32%)
81 (46%)
Appropriate complementary feeding (ACF)
Inappropriate
38 (45%)
80 (74%)
82 (46%)
<0.001
Appropriate
46 (55%)
28 (26%)
96 (54%) The summary of the infant and child feeding habits for different age groups revealed a set of remarkable
and indisputable observations. To begin with, infants’ breastfeeding rate drops sharply whereas at the
age of 12 24 months only 20 1% infants breastfeed rather than 35 1% at 6 8 months Then there is the The summary of the infant and child feeding habits for different age groups revealed a set of remarkable
and indisputable observations. To begin with, infants’ breastfeeding rate drops sharply whereas at the
age of 12-24 months only 20.1% infants breastfeed rather than 35.1% at 6-8 months. Then, there is the
rise of the bottle feeding fashion from 28.2% to 36.9% at 6-8 months and 12-24 months respectively. It
implies a change in the feeding techniques as the baby grows older, perhaps related with age-specific
nutrients or evolution of the mother and society as new feeding patterns arise. The summary of the infant and child feeding habits for different age groups revealed a set of remarkable
and indisputable observations. To begin with, infants’ breastfeeding rate drops sharply whereas at the
age of 12-24 months only 20.1% infants breastfeed rather than 35.1% at 6-8 months. Then, there is the
rise of the bottle feeding fashion from 28.2% to 36.9% at 6-8 months and 12-24 months respectively. It
implies a change in the feeding techniques as the baby grows older, perhaps related with age-specific
nutrients or evolution of the mother and society as new feeding patterns arise. Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 As an additional point, dietary diversity as well as food group frequency demonstrates a reduction in
proportion to age increase. The share of infants with poor Dietary Diversity Scores skyrockets in line with
the number of infants with the low Food Group Frequency Score increasing from 21.3% at 6-8 months to
69.1% at 12-24 months. The findings indicate the difficulties in achieving a diet with variety of foods and
a balance for the infants and children as they get old, this requires interventions that would promote
healthier feeding practices and dietary variety among the world population. Furthermore, the feeding Page 9/20 frequency gets f faster with the growth of the infants which could indicate a shift towards more frequent
feeding patterns involving higher energy needs and progressing developmental milestones. Generally,
these trends underscores the need for focused interventions that will promote adequate feeding
practices across infancy period and early childhood to bolster mothers to continue breastfeeding and eat
variety. frequency gets f faster with the growth of the infants which could indicate a shift towards more frequent
feeding patterns involving higher energy needs and progressing developmental milestones. Generally,
these trends underscores the need for focused interventions that will promote adequate feeding
practices across infancy period and early childhood to bolster mothers to continue breastfeeding and eat
variety. Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 Table 3: Complementary feeding practices among Infants aged from 6-24 months Using infant and child
feeding Index components in Hargeisa, Republic of Somaliland 2024 Page 10/20 ICFI Components
6-8 months
9-11 months
12-24 months
6-24 months
P-value
N (%)
N (%)
N (%)
N (%)
Current breastfeeding status
Yes
84 (35.1%)
107 (44.8%)
48 (20.1%)
239 (64.6%)
<0.001
No
0
1 (1%)
130 (99%)
131 (35.4%)
Bottle feeding practice
Yes
59 (70%)
74 (68%)
97 (56%)
230 (80.5%)
0.0013
No
25 (30%)
34 (32%)
81 (44%)
131 (19.5%)
Dietary Diversity Score
Low (0)
23 (19.2%)
14 (11.7%)
83 (69.1%)
120 (32.4%)
<0.001
Medium (1)
30 (17.9%)
69 (41.1%)
69 (41.1%)
168 (45.4%)
High (2)
31 (37.8%)
25 (30.5%)
26 (31.7%)
82 (22.2%)
Food Group frequency Score
Low (0-2)
19 (21.3%)
14 (15.7%)
56 (62.9%)
89 (24.1%)
<0.001
Medium (3-4)
30 (16.5%)
69 (37.9%)
83 (45.6%)
182 (49.2%)
High (5-6)
35 (35.4%)
25 (25.3%)
39 (39.4%)
99 (26.8%)
Feeding frequency score
Low (0)
7 (6.3%)
3 (2.7%)
101 (91.0)
111 (30.0%)
<0.001
Medium (1)
52 (43.3%)
38 (31.7%)
30 (25.0%)
120 (32.4%)
High (2)
25(18.0%)
67 (48.2%)
47 (33.8%)
139 (37.6%)
ICFI categories
Low (0-4)
48 (31.6%)
51(33.6%)
53 (34.9%
152 (41.1%)
<0.001
Medium (5-8)
5 (7.0%)
12 (16.9%)
54 (76.1%)
71 (19.2%)
High (9-12)
31 (21.1%
45 (30.6%
71 (48.3%)
147 (39.7%)
e result of the Infant and Child Feeding Index (ICFI) Score analysis shows that it is correlated with a
mber of maternal and demographic variables. maternal age emerges as a key determinant, with a
ajority of caregivers under 20 years exhibiting low ICFI scores (73.5%), while those aged 21 to 29 ar
ore likely to have high ICFI scores (46.7%). This association is statistically significant with a p-value
h The result of the Infant and Child Feeding Index (ICFI) Score analysis shows that it is correlated with a
number of maternal and demographic variables. maternal age emerges as a key determinant, with a
majority of caregivers under 20 years exhibiting low ICFI scores (73.5%), while those aged 21 to 29 are
more likely to have high ICFI scores (46.7%). This association is statistically significant with a p-value
less than 0.001. The result of the Infant and Child Feeding Index (ICFI) Score analysis shows that it is correlated with a
number of maternal and demographic variables. Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 maternal age emerges as a key determinant, with a
majority of caregivers under 20 years exhibiting low ICFI scores (73.5%), while those aged 21 to 29 are
more likely to have high ICFI scores (46.7%). This association is statistically significant with a p-value
less than 0.001. Page 11/20 Page 11/20 Education, above all else, proves to be crucial, with a considerable proportion of caregivers who attended
school showing a more balanced distribution across ICFI scores, particularly 29.2% in the high category,
compared to only 11.8% among non-attendees. Family income also correlates with ICFI scores, with a higher percentage of high ICFI scores evident for
families earning ≥251 USD (24.3%), compared to families earning ≤150 USD (9.3%). Furthermore, larger
families, i.e., those with 5 or more members, demonstrate a higher proportion of medium and high ICFI
scores. Gender-related feeding practices show disparity, with a greater proportion of boys scoring highly
on ICFI (42.9%) compared to girls (36.3%). The age of the child is another crucial factor, with younger infants (6-8 months) more likely to exhibit low
ICFI scores (57.1%) compared to older age groups, Antenatal care (ANC) attendance positively
influences feeding practices, as mothers who attended ANC have a higher proportion of medium and
high ICFI scores (82%) compared to non-attenders (18%). Moreover, the number of ANC visits further impacts feeding practices, with a higher proportion of
medium and high ICFI scores among mothers who had ≥4 visits (82%), compared to those with <4 visits
(18%). Moreover, the number of ANC visits further impacts feeding practices, with a higher proportion of
medium and high ICFI scores among mothers who had ≥4 visits (82%), compared to those with <4 visits
(18%). These findings underscore the intricate interplay of socio-demographic factors in shaping infant and
child feeding practices, emphasizing the need for tailored interventions to address specific determinants
and promote improved nutritional outcomes for infants and young children. These findings underscore the intricate interplay of socio-demographic factors in shaping infant and
child feeding practices, emphasizing the need for tailored interventions to address specific determinants
and promote improved nutritional outcomes for infants and young children. Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 Table 4: Comparison between mothers who have high, medium and low infant and child feeding index
among socio demographic variables in Hargeisa, Republic of Somaliland 2024 Table 4: Comparison between mothers who have high, medium and low infant and child feeding index
among socio demographic variables in Hargeisa, Republic of Somaliland 2024 Page 12/20 Variable
infant and child feeding index scores (ICFI)
Low ICFI
N (%)
Medium ICFI
N (%)
High ICFI
N (%)
P-value
Age of the mother/caregiver
<20 years
25 (73.5%)
3 (8.8%)
6 (17.6%)
< 0.001
21-29 years
110 (38.6%)
42 (14.7%)
133 (46.7%)
≥30 years
17 (33.3%)
26 (51.0%)
8 (15.7%)
Ever attend school
Yes
95 (59.0%
19 (11.8%
47 (29.2%)
< 0.001
No
57 (27.3%)
52 (24.9%)
100 (47.8%)
Monthly income of the family
≤ 150 USD
56 (57.7%)
9 (9.3%)
32 (33.0%)
< 0.001
151-250 USD
78 (38.4%)
27 (13.3%)
98 (48.3%)
≥ 251 USD
18 (25.7%)
35 (50.0%)
17 (24.3%)
Family Size
<4
118 (48.6%)
25 (10.3%)
100 (41.2%
< 0.001
≥5
34 (26.8%)
46 (36.2%)
47 (37.0%)
Sex of the child
Girl
96 (53.6%)
18 (10.1%)
65 (36.3%)
< 0.001
Boy
56 (29.3%)
53 (27.7%)
82 (42.9%)
Age of the child in months
6-8 Months
48 (57.1%)
5 (6.0%)
31 (36.9%)
< 0.001
9-11 Months
51 (47.2%)
12 (11.1%)
45 (41.7%)
12-24 Months
53 (29.8%)
54 (30.3%)
71 (39.9%)
Ante Natal Care visit
Yes
106 (70%)
24 (34%)
120 (82%)
< 0.001
No
46 (30%)
47(66%)
27 (18%)
Number of Antenatal Care Visit
< 4 visits
74 (48%)
31 (44%)
27 (18%) < 0.001
≥ 4 visits
78 (52%)
40 (46%)
120 (82%)
In a study analyzing factors associated with appropriate complementary feeding practices (ACFP)
among mothers in Hargeisa, Somaliland, several key variables were examined for their impact on feeding
practices. The analysis provided both Crude Odds Ratios (COR) and Adjusted Odds Ratios (AOR)
alongside 95% Confidence Intervals (CI) and p-values to ascertain the strength and significance of these
associations. Notable findings include the positive impact of being unemployed on ACFP (AOR=3.5, 95%
CI: 2.15-5.69, p<0.001), indicating that unemployed mothers are more likely to practice ACFP compared
to their employed counterparts. Large family size (≥5 members) was another significant factor, with an
AOR of 4.193 (95% CI: 2.650-6.634, p<0.001), suggesting families with more members are likelier to
adopt ACFP. Table 5: Factors Associated Appropriate Complementary feeding practices among Infants aged from 6-
24 months Clinics in Hargeisa, Republic of Somaliland Discussion The findings of the current study provide valuable insights into infant and child feeding practices (ACFP)
and underscore the importance of socio-demographic factors in shaping these practices. Comparison
with previous researches highlights both consistencies and disparities, shedding light on evolving trends
and persistent challenges in infant and child nutrition. The overall prevalence of appropriate complementary feeding practice among children aged from 6–24
months is 46% which is slightly low compared to other previous studies including several studies have
investigated the prevalence of appropriate complementary feeding practices among children aged 6–24
months, consistently revealing suboptimal rates worldwide. For instance, a study conducted in Ethiopia
found that only 25.2% of children received appropriate complementary feeding practices (Kassa,
Meshesha et al. 2016) Similarly, research in Nepal reported a prevalence of 38.3%, indicating inadequate
feeding practices among infants and young children (Kabir, Khanam et al. 2012). In contrast, a study in
Nigeria demonstrated a slightly higher prevalence of 51.3%, yet still reflecting insufficient adherence to
recommended feeding guidelines. These findings collectively underscore the global challenge of
achieving adequate complementary feeding practices and highlight the urgent need for targeted
interventions to improve child nutrition and health outcomes. In our study, the proportion of mothers/caregivers below 20 years practicing Appropriate complementary
feeding practice were 12%, contrasting with 60% among those aged 21–29 years and only 28% among
those aged 30 years or older. These figures corroborate with previous studies by (Sharma, Gernand et al. 2008) which emphasized the vulnerability of younger mothers to suboptimal feeding practices,
underlining the need for targeted interventions in this demographic group. Similarly, our findings regarding the association between level of education and adherence to safe
feeding practices align with the conclusions of studies conducted by, (White, Bégin et al. 2017, Appleton,
Laws et al. 2018) which reported higher rates of appropriate feeding among educated mothers
compared to those with lower levels of education. Our study reinforces the importance of educational
initiatives in promoting optimal nutrition among caregivers. Furthermore, the influence of family income on feeding practices echoes the findings of research by
(Green, Anderson et al. 2011), which highlighted disparities in feeding practices based on socioeconomic
status. The observed decline in breastfeeding rates and the rise in bottle feeding practices with
increasing child age are consistent with the longitudinal trends reported by) (Jones, Steer et al. 2010),
emphasizing the need for sustained efforts to support breastfeeding beyond infancy. Table 1: distribution of Complementary feeding practices by characteristics of nursing Mothers in
Hargeisa, Republic of Somaliland 2024 Avoiding bottle feeding (AOR=2.907, 95% CI: 1.88-4.49, p<0.001) and maintaining good
WASH and hygiene status (AOR=4.488, 95% CI: 2.86-7.05, p<0.001) were also associated with higher
ACFP adherence. Furthermore, dietary factors played a crucial role; adequate dietary diversity
(AOR=0.032, 95% CI: 0.05-0.068, p<0.001) and meeting the minimum acceptable diet (AOR=0.012, 95%
CI: 0.005-0.027, p<0.001) were highly indicative of appropriate feeding practices. Additionally, the timely
introduction of complementary foods at 6 months significantly favored ACFP (AOR=2.504, 95% CI: 1.636-
3.832, p<0.001). n a study analyzing factors associated with appropriate complementary feeding practices (ACFP)
among mothers in Hargeisa, Somaliland, several key variables were examined for their impact on feeding
practices. The analysis provided both Crude Odds Ratios (COR) and Adjusted Odds Ratios (AOR) Table 5: Factors Associated Appropriate Complementary feeding practices among Infants aged from 6-
24 months Clinics in Hargeisa, Republic of Somaliland Page 14/20 Variables
Appropriate complementary feeding
Practice (ACFP)
COR[95% CI]
AOR[95% CI]
Appropriate N (%)
Inappropriate N (%)
Occupational Status
Unemployed
102 (60%)
168 (84%)
1 (Ref)
1 (Ref)
Employed
68 (40%)
32 (16%)
2.25 (1.32,
3.84)*
3.5 (2.15, 5.69)**
Family Size
≤4
83 (49%)
160 (80%)
1 (Ref)
1 (Ref)
≥5
87 (51%)
40 (20%)
2.7 (1.633,
4.45)**
4.193 (2.650,
6.634)**
Bottle feeding practice
Yes
83 (49%)
147 (73%)
1 (Ref)
1 (Ref)
No
87 (51%
53 (27%)
2.1 (1.27,
3.26) *
2.907 (1.88,
4.49)**
WASH and Hygiene status
Poor
130 (77%)
84 (42%)
1 (Ref)
1 (Ref)
Good
40 (23%)
116 (58%)
3.1 (1.12,
8.38) *
4.488 (2.86,
7.05)**
Dietary Diversity Score
Inadequate ( ≥
5)
69 (41%
191 (95%)
1 (Ref)
1 (Ref)
adequate (≤ 4)
101 (59%)
9 (5%)
.017 (.005,
.060)**
.032 (0.05,
0.068)**
Minimum Acceptable Diet
Met
132 (78%)
8 (6%)
1 (Ref)
1 (Ref)
Not met
38 (22%)
192 (94%)
.021 (.008,
.053)**
.012 (.005,
.027)**
Timely Introduction of complementary foods (at 6 months)
No
89 (52%
61 (31%)
1 (Ref)
1 (Ref)
Yes
81 (48%)
139 (69%)
6.5 (2.19,
18.73)*
2.504 (1.636,
3.832)**
COR : Crude Odds Ratio, AOR: Adjusted Odds Ratio * indicate p-value less than 0.05, ** p-value less
than 0 001 Timely Introduction of complementary foods (at 6 months) COR : Crude Odds Ratio, AOR: Adjusted Odds Ratio * indicate p-value less than 0.05, ** p-value less
than 0.001 Page 15/20 Page 15/20 Conclusion The findings of our study underscore the intricate relationship between socio-demographic factors and
infant and child feeding practices. Our research highlights disparities in feeding practices based on
maternal age, education, family income, family size, and child age, emphasizing the need for tailored
interventions to address these variations. The observed decline in breastfeeding rates with increasing child age and the rise in bottle feeding
practices signal evolving trends in infant nutrition, necessitating sustained efforts to promote
breastfeeding beyond infancy. Moreover, the association between maternal age, education, and family
income with adherence to safe feeding practices underscores the importance of educational initiatives
and financial support for vulnerable populations. The Infant and Child Feeding Index (ICFI) analysis reveals correlations with maternal and demographic
variables, highlighting the complex interplay of socio-demographic factors in shaping feeding practices. These findings provide valuable insights for policymakers, healthcare providers, and community
stakeholders to develop targeted interventions that address specific determinants and promote
improved nutritional outcomes for infants and young children. Limitations The study has certain limitations that should be considered. The evaluation of children's feeding relied on
maternal recollection of the foods consumed, introducing the possibility of recall bias as some mothers
may have either exaggerated or underestimated their children's food intake, or not maintained the
necessary level of attentiveness. Additionally, it is noteworthy that not all mothers seek services from
primary health facilities, even though the participants were chosen to represent a diverse sample. Furthermore, the research was conducted in a cross-sectional manner, offering only a descriptive
snapshot of maternal complementary feeding practices during the study period. Conducting longitudinal
studies would enhance our understanding of the factors influencing these practices and their potential
impact on the nutritional status of children. Discussion The Infant and Child Feeding Index (ICFI) analysis in our study revealed correlations with maternal and
demographic variables, consistent with the findings of studies by (Davis, Clark et al. 2005) These
findings underscore the intricate interplay of socio-demographic factors in shaping feeding practices,
emphasizing the need for tailored interventions to address specific determinants and promote improved
nutritional outcomes for infants and young children. Page 16/20 Page 16/20 Page 16/20 While our study contributes to the existing body of research on infant and child feeding practices, it also
highlights areas for further exploration and intervention. Future studies could delve deeper into the
cultural and environmental factors influencing feeding practices, as well as explore innovative
approaches to address disparities and promote optimal nutrition among vulnerable populations. While our study contributes to the existing body of research on infant and child feeding practices, it also
highlights areas for further exploration and intervention. Future studies could delve deeper into the
cultural and environmental factors influencing feeding practices, as well as explore innovative
approaches to address disparities and promote optimal nutrition among vulnerable populations. Funding sources No external funding was received for the research, the statement "No external funding was received for
this study" Data Availability Statement The data supporting the findings of this study are available from the corresponding author, Hamse Khalif
Hassan, upon reasonable request. Due to privacy and confidentiality concerns, the raw data cannot be
publicly shared. However, summarized data and relevant statistical analyses will be provided to
interested parties upon request and with appropriate permissions from the relevant institutional review
boards. PHCs: Primary Health Centers, SPSS: Statistical package for Social Sciences, OR: Odss Ratio, AOR:
Adjusted Odss Ratio PHCs: Primary Health Centers, SPSS: Statistical package for Social Sciences, OR: Odss Ratio, AOR:
Adjusted Odss Ratio Ethical Approval Ethical approval was obtained from the Ethics Committee of the AAMUC Institutional Review Board
Informed consent was obtained from the mothers with an assurance of confidentiality. Declarations The authors, Hamse Kh. Hassan., Abdeta M. Ahmed, and Mohamed A. Ahmed, declare no conflicts of
interest regarding the publication of this research article. No financial or non-financial interests have
influenced the study design, data collection, analysis, interpretation, or reporting of the findings
presented in this manuscript. Abbreviations WHO: World Health Organization, IYCF: Infant and Young Child Feeding Practice FSNAU: Food Security
and Nutrition Assessment Unit ICFI: Infant and Child Feeding Index, MDD: Minimum Dietary Diversity,
MMF: Minimum Meal Frequency, MAD: Minimum Adequate Diet, LGA: Local Government Administration, Page 17/20 Page 17/20 Author Contributions: Conceptualization: Hamse Kh. Hassan Data curation: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. Formal analysis: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed.. Investigation: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. Methodology: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. Project administration: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. Resources: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. Software: Hamse Kh. Hassan Data curation: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. Page 18/20 Page 18/20 Writing – original draft: Hamse Kh. Hassan Writing – review & editing: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. Writing – review & editing: Hamse Kh. Hassan, Abdeta M. Ahmed, Mohamed A. Ahmed. References 1. Abeshu, M. A., A. Lelisa and B. Geleta (2016). "Complementary feeding: review of recommendations,
feeding practices, and adequacy of homemade complementary food preparations in developing
countries–lessons from Ethiopia." Frontiers in nutrition 3: 41. 1. Abeshu, M. A., A. Lelisa and B. Geleta (2016). "Complementary feeding: review of recommendations,
feeding practices, and adequacy of homemade complementary food preparations in developing
countries–lessons from Ethiopia." Frontiers in nutrition 3: 41. 2. Alemu, Z. A., A. A. Ahmed, A. W. Yalew, B. S. Birhanu and B. F. Zaitchik (2017). "Individual and
community level factors with a significant role in determining child height-for-age Z score in East
Gojjam Zone, Amhara Regional State, Ethiopia: a multilevel analysis." Archives of Public Health
75(1): 1-13. 3. Appleton, J., R. Laws, C. G. Russell, C. Fowler, K. J. Campbell and E. Denney-Wilson (2018). "Infant
formula feeding practices and the role of advice and support: An exploratory qualitative study." BMC
pediatrics 18: 1-11. 3. Appleton, J., R. Laws, C. G. Russell, C. Fowler, K. J. Campbell and E. Denney-Wilson (2018). "Infant
formula feeding practices and the role of advice and support: An exploratory qualitative study." BMC
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Commission, ICF International. 4. Commission, N. P. (2013). Nigeria demographic and health survey 2013, National Population
Commission, ICF International. 5. Davis, E. M., J. M. Clark, J. A. Carrese, T. L. Gary and L. A. Cooper (2005). "Racial and socioeconomic
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95(9): 1539-1543. 6. Demographic, N. (2019). Health Survey 2013. National Population Commission (NPC)[Nigeria] and
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ability and willingness to participate in a web-based intervention to improve hypertension control."
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Ministry of Health. 8. Health, N. F. M. o. (2010). National Strategic Health Development Plan (NSHDP) 2010-2015, Federal
Ministry of Health. 9. Jones, L. R., C. D. Steer, I. S. Rogers and P. M. Emmett (2010). "Influences on child fruit and
vegetable intake: sociodemographic, parental and child factors in a longitudinal cohort study." Public
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inappropriate complementary feeding practices in infant and young children in Bangladesh:
secondary data analysis of Demographic Health Survey 2007." Maternal & child nutrition 8: 11-27. Page 19/20 Page 19/20 11. Kassa, T., B. Meshesha, Y. Haji and J. Ebrahim (2016). "Appropriate complementary feeding
practices and associated factors among mothers of children age 6–23 months in Southern Ethiopia,
2015." BMC pediatrics 16(1): 1-10. 12. Kassa, T., B. Meshesha, Y. Haji and J. Ebrahim (2016). "Appropriate complementary feeding
practices and associated factors among mothers of children age 6–23 months in Southern Ethiopia,
2015." BMC pediatrics 16: 1-10. 13. Kinyoki, D. K., J. A. Berkley, G. M. Moloney, E. O. Odundo, N.-B. Kandala and A. M. Noor (2016). "Environmental predictors of stunting among children under-five in Somalia: cross-sectional studies
from 2007 to 2010." BMC public health 16(1): 1-9. 14. Mitchodigni, I. M., W. Amoussa Hounkpatin, G. Ntandou-Bouzitou, H. Avohou, C. Termote, G. Kennedy and D. J. Hounhouigan (2017). "Complementary feeding practices: determinants of dietary
diversity and meal frequency among children aged 6–23 months in Southern Benin." Food Security
9: 1117-1130. 15. Ndiokwelu, C., A. Maduforo, C. Amadi and C. Okwy-Nweke (2014). "Breastfeeding and
complementary feeding practices of mothers of children (0-24 months) attending infant welfare
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Ituku-Ozalla Enugu." 16. Nutrition, I. (2013). "The achievable imperative for global progress New York." NY United Nations
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knowledge, practices, and dietary diversity among mothers of under-five children in an urban
community in Lagos State, Nigeria." International Journal of MCH and AIDS 6(1): 46. 18. Sharma, S. V., A. D. Gernand and R. S. Day (2008). "Nutrition knowledge predicts eating behavior of
all food groups except fruits and vegetables among adults in the Paso del Norte region: Qué
Sabrosa Vida." Journal of nutrition education and behavior 40(6): 361-368. 18. Sharma, S. V., A. D. Gernand and R. S. Day (2008). "Nutrition knowledge predicts eating behavior of
all food groups except fruits and vegetables among adults in the Paso del Norte region: Qué
Sabrosa Vida." Journal of nutrition education and behavior 40(6): 361-368. 19. White, J. M., F. Bégin, R. Kumapley, C. Murray and J. References Krasevec (2017). "Complementary feeding
practices: Current global and regional estimates." Maternal & child nutrition 13: e12505. 19. White, J. M., F. Bégin, R. Kumapley, C. Murray and J. Krasevec (2017). "Complementary feeding
practices: Current global and regional estimates." Maternal & child nutrition 13: e12505. 20. WHO, U. (2008). USAID. Indicators for assessing infant and young child feeding practices, Geneva,
World Health Organization. 20. WHO, U. (2008). USAID. Indicators for assessing infant and young child feeding practices, Geneva,
World Health Organization. Page 20/20
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https://www.emerald.com/insight/content/doi/10.1108/IMDS-05-2022-0293/full/pdf?title=coping-with-covid-19-using-contact-tracing-mobile-apps
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Coping with COVID-19 using contact tracing mobile apps
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Industrial management + data systems/Industrial management & data systems
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Abstract Purpose – To cope with the COVID-19 pandemic, contact tracing mobile apps (CTMAs) have been developed
to trace contact among infected individuals and alert people at risk of infection. To disrupt virus transmission
until the majority of the population has been vaccinated, achieving the herd immunity threshold, CTMA
continuance usage is essential in managing the COVID-19 pandemic. This study seeks to examine what
motivates individuals to continue using CTMAs. Design/methodology/approach – Following the coping theory, this study proposes a research model to
examine CTMA continuance usage, conceptualizing opportunity appraisals (perceived usefulness and
perceived distress relief), threat appraisals (privacy concerns) and secondary appraisals (perceived response
efficacy) as the predictors of individuals’ CTMA continuance usage during the pandemic. In the United States,
an online survey was administered to 551 respondents. Findings – The results revealed that perceived usefulness and response efficacy motivate CTMA continua
usage, while privacy concerns do not. Originality/value – This study enriches the understanding of CTMA continuance usage during a pub
health crisis, and it offers practical recommendations for authorities. Originality/value – This study enriches the understanding of CTMA
health crisis, and it offers practical recommendations for authorities. Keywords COVID-19, Contact tracing, Mobile application, Continuance usage, Coping theory
t
R
h Keywords COVID-19, Contact tracing, Mobile application, Continuance usage, Coping theory
Paper type Research paper 1440 Shaoxiong Fu
College of Information Management, Nanjing Agricultural University,
Nanjing, China Received 10 May 2022
Revised 15 October 2022
19 December 2022
Accepted 2 February 2023 The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/0263-5577.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/0263-5577.htm Industrial Management & Data
Systems
Vol. 123 No. 5, 2023
pp. 1440-1464
Emerald Publishing Limited
0263-5577
DOI 10.1108/IMDS-05-2022-0293 © Chenglong Li, Hongxiu Li and Shaoxiong Fu. Published by Emerald Publishing Limited. This article
is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce,
distribute, translate and create derivative works of this article (for both commercial and non-commercial
purposes), subject to full attribution to the original publication and authors. The full terms of this licence
may be seen at http://creativecommons.org/licences/by/4.0/legalcode y
p
g
y
g
This research received no specific grant from any funding agency in the public, commercial, or not-
for-profit sectors. 1. Introduction
A
h
f h At the onset of the COVID-19 pandemic, many countries deployed contact tracing mobile
apps (CTMAs) to prevent the spread of the coronavirus (Rowe et al., 2020; Trang et al., 2020). Installed on smartphones, these apps use Bluetooth or GPS signals to detect when two users
come into close contact with each other (such as within two meters for 15 min) (Ferretti et al.,
2020; Trang et al., 2020). Once an individual has tested positive for infection, these apps can
report their case anonymously and alert all other users who had been in close contact with the
infected person, allowing exposed users to quarantine or arrange a test and, thus, preventing
further transmission (Ferretti et al., 2020). However, such apps’ success in controlling the
spread of COVID-19 depends on their use by individuals. To sufficiently disrupt coronavirus
infection, at least 56% of a country’s population, as Hinch et al. (2020) estimated, should use
such apps. Many European countries (e.g. Germany, Finland and France) and the United
States have deployed CTMAs on a voluntary basis (Rowe et al., 2020; Trang et al., 2020). It is © Chenglong Li, Hongxiu Li and Shaoxiong Fu. Published by Emerald Publishing Limited. This article
is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce,
distribute, translate and create derivative works of this article (for both commercial and non-commercial
purposes), subject to full attribution to the original publication and authors. The full terms of this licence
may be seen at http://creativecommons.org/licences/by/4.0/legalcode © Chenglong Li, Hongxiu Li and Shaoxiong Fu. Published by Emerald Publishing Limited. This article
is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce,
distribute, translate and create derivative works of this article (for both commercial and non-commercial
purposes), subject to full attribution to the original publication and authors. The full terms of this licence
may be seen at http://creativecommons.org/licences/by/4.0/legalcode y
p
g
y
g
This research received no specific grant from any funding agency in the public, commercial, or not-
for-profit sectors. necessary for authorities to understand how to motivate individuals to keep using CTMAs
during the pandemic, which is vital for achieving the goal of a high rate of CTMA use in a
country’s population to disrupt coronavirus infection, especially if the CTMA use is on a
voluntary basis. Therefore, understanding the factors that may facilitate or obstruct citizens’
CTMA continuance usage is crucial. 1. Introduction
A
h
f h Continuance
usage of
contact tracing
apps Recent studies have examined individuals’ CTMA usage from different theoretical
perspectives, such as privacy concerns and diffusion of innovation theory (Lin et al., 2021),
unified theory of acceptance and use of technology (UTAUT) (Ojo and Rizun, 2021; Walter Thiee
etal.,2021),privacycalculusmodelandherdingheuristics(Wagneretal.,2021),appspecifications
(Trang et al., 2020) and critical social theory (Rowe et al., 2020). However, these studies focused
only on adoption and largely ignored continuance usage, which – unlike adoption’s focus on the
first-time use of apps – emphasizes the long-term use (Bhattacherjee, 2001). The initial adoption
does not always guarantee continuance usage (Bhattacherjee, 2001; Yan et al., 2021). Individual
users may stop using CTMAs after using it for some time. For instance, a Corsair study on
National Health Service (NHS) COVID-19 App in the United Kingdom has reported that 2% of
users deleted the app and 72% of users switched off the contact tracing function in the first two
months since the app was launched in September 2020 (Potts, 2020). The reasons for CTMA
continuance usage could be different from the ones explaining initial adoption. Users’
continuance usage could be influenced by their prior information systems (IS) use experience
such that their use experience could lead to their perception change on IS use, such as post-
expectation regarding usefulness, confirmation of pre-expectations following actual use and
satisfaction with prior IS use (Bhattacherjee, 2001). Thus, recent research findings on the
antecedents of CTMA adoption cannot fully explain the continuance usage of CTMA. To disrupt
virus transmission until the majority of the population has been vaccinated, achieving the herd
immunity threshold, people’s continuance usage of CTMA is essential in managing the COVID-
19pandemic (Wymantetal., 2021). Moreover, sinceglobal COVID-19infectionskeep spreadingin
the world (World Health Organization, 2021a), understanding how to promote CTMA
continuance usage is paramount to managing the COVID-19 pandemic. 1441 2. Literature review
2 1 R
h
th 2.1 Research on the use of contact tracing mobile apps (CTMA) during the pandemic 2.1 Research on the use of contact tracing mobile apps (CTMA) during the pandemic
Recently, CTMA use has attracted considerable research attention in many domains, with
researchers focusing on factors that compel individuals to install and use such apps from
different theoretical lenses. The determinants of this adoption can be grouped into two main
types: (1) usage benefits and (2) threats to usage. Regarding usage benefits, recent CTMA
studies have employed traditional IS theories to examine what determines user adoption. For
instance, centering the diffusion of innovations theory, Lin et al. (2021) found that relative
advantage positively influences users’ CTMA adoption. Also, the initial research on these apps
has highlightedtheir dual benefit appeal for not onlyindividuals(such as informing them about
high-risk contacts) but also society (informing other people about close contact with an infected
person), which are important motivations for the adoption of these apps (Trang et al., 2020). The most significant threats to CTMA adoption—since it involves a compromise between
privacy and health—are privacy concerns and risks (Chan and Saqib, 2021; Lin et al., 2021). People are afraid to disclose their private information, such as COVID-19 test results,
exposure to infection, location and their communities’ or social groups’ COVID-19 status;
moreover, they worry about potential hacking and data misuse by app service providers or
governments (Riemer et al., 2020). Such privacy risks, as Wagner et al. (2021) found, . esea ch on the use of contact t acing mobile apps (C
) du ing the pandemic
Recently, CTMA use has attracted considerable research attention in many domains, with
researchers focusing on factors that compel individuals to install and use such apps from
different theoretical lenses. The determinants of this adoption can be grouped into two main
types: (1) usage benefits and (2) threats to usage. Regarding usage benefits, recent CTMA
studies have employed traditional IS theories to examine what determines user adoption. For
instance, centering the diffusion of innovations theory, Lin et al. (2021) found that relative
advantage positively influences users’ CTMA adoption. Also, the initial research on these apps
has highlightedtheir dual benefit appeal for not onlyindividuals(such as informing them about
high-risk contacts) but also society (informing other people about close contact with an infected
person), which are important motivations for the adoption of these apps (Trang et al., 2020). 1441 1441 g
p
g g
p
Additionally, prior studies have examined CTMA adoption from the view of benefits and
threats and highlighted privacy concerns as a threat inhibiting individuals’ adoption of
CTMA (Chan and Saqib, 2021; Lin et al., 2021) but largely ignored the role of users’
assessment of coping options available to respond to privacy issues related to CTMA in
explaining their CTMA continuance usage during the public health crisis. The significance of
privacy protection regarding CTMA has been highlighted in the literature (Riemer et al.,
2020). Privacy-preserving techniques have also been applied in CTMA development to
protect CTMA users’ privacy, such as anonymity and decentralization (Apple and Google,
2021). However, it is not clear how CTMA users’ assessments regarding the coping resources
for privacy protection in their CTMA use will affect their continuance usage of CTMA in the
public health crisis. Hence, it is necessary to investigate whether users’ assessments of coping
resources related to privacy protection can predict their continued usage of CTMA, in
addition to users’ perceived benefits and threats of using CTMAs. Furthermore, IS researchers have developed an extensive body of knowledge on IS
continuance (Bhattacherjee, 2001). Their research contexts mainly focus on normal daily life
or work environments. Mobile app continuance usage during a public health crisis might
have different causes than IS continuance usage in daily life or work-environment contexts. CTMA continuance usage during the pandemic may be influenced by CTMA as a public good
(Rehse and Trem€ohlen, 2022; Riemer et al., 2020). A public good is non-rivalrous and non-
exclusive; everyone can benefit from it, and one person’s use does not obstruct another’s
(Riemer et al., 2020). Therefore, some individuals may consider more about the social impact
of CTMA use rather than their personal costs, thus continuing to use CTMAs during a public
health crisis to contribute to the public goods (Campos-Mercade et al., 2021; Riemer et al., 2020). For instance, some people may be willing to sacrifice their private information to help
slow the spread of COVID-19 and protect others’ health during the unprecedented and
emergent public health crisis. The knowledge on IS continuance in daily life or work
environment might not explain individuals’ CTMA continuance usage during the COVID-19
pandemic. However, research on citizens’ CTMA continuance usage during a public health
crisis is limited in the literature. 1441 Therefore, it is needed to investigate CTMA continuance
usage to explain IS continuance usage during unprecedented situations. 1442 g
p
g
g
p
To close this research gap, this study examines the factors that determine CTMA
continuance usage during the COVID-19 pandemic by developing a research model based on
coping theory. According to coping theory, how people cope with a stressful situation
depends on a combination of opportunity appraisals (i.e. perceived usefulness and perceived
distress relief), threat appraisals (i.e. privacy concerns) and secondary appraisals (i.e. perceived response efficacy) (Beaudry and Pinsonneault, 2005; Lazarus and Folkman, 1984). CTMAs can be an effective coping tool for the pandemic, but it does entail uncertainties; to
guide their app use during the pandemic, users cannot draw from or build on their prior
knowledge of technology use in daily life and work environments. Thus, CTMA users may
appraise the benefits and threats of CTMA use as well as the coping resources for privacy
protection over their usage based on first-time use experience to decide their continuance
usage of CTMAs. Therefore, to investigate CTMA continuance usage, this study deemed a
research model based on coping theory to be appropriate. The proposed research model was tested using the survey data collected in the United
States (n 5 551). The findings demonstrate that CTMA continuance usage is determined by
users’ perceived usefulness (opportunity appraisals) and perceived response efficacy
(secondary appraisals). Thus, this study contributes to the extant literature on IS continuance
usage by using the coping theory to explain CTMA continuance usage and distinguishing
differences between opportunity appraisals (i.e. perceived usefulness and perceived distress
relief), threat appraisals (i.e. privacy concerns) and secondary appraisals (i.e. perceived
response efficacy) as antecedents that determine individuals’ CTMA continuance usage
during the COVID-19 pandemic. In doing so, this study offers an in-depth appreciation of how
individuals’ different appraisals regarding CTMAs determine their continuance usage of
CTMAs to cope with the pandemic. 2. Literature review
2 1 R
h
th Th
i
ifi
h
CTMA d
i
i
i i
l
i
b The most significant threats to CTMA adoption—since it involves a compromise between
privacy and health—are privacy concerns and risks (Chan and Saqib, 2021; Lin et al., 2021). People are afraid to disclose their private information, such as COVID-19 test results,
exposure to infection, location and their communities’ or social groups’ COVID-19 status;
moreover, they worry about potential hacking and data misuse by app service providers or
governments (Riemer et al., 2020). Such privacy risks, as Wagner et al. (2021) found, negatively affect individuals’ willingness to adopt these apps. To reduce this reluctance, apps
that offer a high level of privacy protection and user data confidentiality are crucial
(Bhattacharya and Ramos, 2021; Lin et al., 2021; Trang et al., 2020). Continuance
usage of
contact tracing
apps Prior CTMA studies in IS fields have principally focused on these apps’ adoption (see
Table 1), largely ignoring continuance usage despite recent research stating that users’
continued app use is important for controlling the pandemic before vaccines achieve
sufficient population-level protection (Wymant et al., 2021). Additionally, although recent
research has examined the benefits and threats of using such apps, few studies have explored
how users’ assessment of available coping resources for privacy issues over CTMA use
affects their continuance usage of CTMA. It is still unclear what is the role of users’
assessments of available coping resources for privacy issues over their CTMA use in
explaining their continuance usage of CTMA in the public health crisis. As per previous
literature, coping theory may help explain individuals’ coping responses to stressful events,
based on a combination of opportunity, threat and secondary appraisals (Beaudry and
Pinsonneault, 2005; Lazarus and Folkman, 1984). This draws attention to the necessity of
exploring what motivates CTMA continuance usage from the coping theory perspective. 1443 2.2 Coping theory
d i
h 2.2 Coping theory
d i
h Rooted in psychology, coping theory aims to explain how people cope with stressful
situations and perform effective and appropriate coping behaviors. Lazarus and Folkman
(1984, p. 141) defined coping as “constantly changing cognitive and behavioral efforts to
manage specific external and internal demands that are appraised as taxing or exceeding the
resources of the person.” Individuals’ coping responses, according to coping theory, are based
on their cognitive appraisals—that is, their evaluations of whether a stressful event is
relevant to them and, if so, how (Beaudry and Pinsonneault, 2005; Lazarus and Folkman,
1984). The cognitive appraisal process comprises two steps: primary appraisals and
secondary appraisals. Primary appraisals refer to an individual’s evaluation of whether an
encounter causes potential benefits or harms. It, in turn, comprises opportunity appraisals and
threat appraisals. Opportunity appraisals refer to an individual’s assessment of whether
potential consequences are positive (Lazarus and Folkman, 1984). If they believe an event
allows them to achieve beneficial outcomes, they tend to perform such behavioral responses
(Bala and Venkatesh, 2016; Gong et al., 2020). Meanwhile, threat appraisals refer to an
individual’s evaluation of whether an event’s anticipated consequences are negative (Lazarus
and Folkman, 1984). If they believe an event will bring unfavorable outcomes, they will likely
perform avoidant behavioral responses (Bala and Venkatesh, 2016; Lazarus and Folkman,
1984). Encounters are complicated and frequently perceived as encompassing both
opportunities and threats. Secondary appraisals refer to an individual’s cognitive
assessment of whether coping resources are available to respond to an encounter (Beaudry
and Pinsonneault, 2005; Lazarus and Folkman, 1984). Such assessment determines the coping
options available to individuals and their sense of control over the encounter (Fadel and
Brown, 2010; Lazarus and Folkman, 1984). Coping theory’s three types of appraisals have been used to examine IS adaptation in
numerous contexts, such as the use of new IT in work environments (Beaudry and
Pinsonneault, 2005), wearable healthcare devices (Marakhimov and Joo, 2017), mandatory
IT use (Bhattacherjee et al., 2018) and mobile payments (Gong et al., 2020). For instance, in the
context of mobile payment services, Gong et al., (2020) found that opportunity appraisals
(measured by perceived value) and secondary appraisals (measured by perceived
controllability) positively influence individuals’ intentions to use mobile payment services,
whereas threat appraisals (measured by perceived threat) negatively influence use intentions. Table 2 summarizes previous studies on the three types of cognitive appraisals in the IS field. 2.2 Coping theory
d i
h Reference
Focus
Theory
Method
Findings
Bhattacharya
and Ramos
(2021)
Privacy and
confidentiality
related to
CTMA
/
Qualitative
study
CTMA should emphasize privacy protection and data confidentiality
Bitzer et al. (2021)
The
algorithmic
transparency of
CTMA
Algorithmic
transparency
Experiment
Algorithmic transparency disclosure is positively and significantly related to app selection, user comprehension and
trust
Chan and
Saqib (2021)
Unwillingness
to download
and use CTMA
/
Experiment
-COVID-19 concerns decrease intentions to download and use CTMA
-Privacy concerns and social conservatism mediate the impact of COVID-19 concerns on intentions to
download and use CTMA
Lin et al. (2021) CTMA use
intentions
-Diffusion
of innovation theory
-Privacy
concerns
Survey
-Trust
beliefs, compatibility and relative advantage positively affect citizens’ adoption intentions
-Privacy concerns negatively influence trusting beliefs and positively affect risk beliefs
Ojo and Rizun
(2021)
CTMA use
UTAUT
Computational
grounded
theory
The top-five most prevalent topics are the need for tracing apps and mandatory use, tracing apps’ launch supported
by private-sector organizations, anonymity in reporting positive test results, a new business continuity tracker app
for employee safety and apps for tracking symptoms
Riemer et al. (2020)
IT governance
-IT governance
Conceptual
paper
The selection of approaches to IT governance is influenced by factors regarding health risks to the population, prior
pandemic experience, societal culture and values, governments’ role in societies and trust in governments and
technologies
Rowe et al. (2020)
Failure in
CTMA design
and adoption
-Critical social theory
Critical
research
methodology
Alienation from the natural and social reality regarding the COVID-19 pandemic leads to inadequate app design and
adoption failure
Walter Thiee
et al. (2021)
CTMA use
UTAUT
Survey
-Thefactors influencing individuals’ intentions concerning CTMA include ethical considerations, social
influence, effort expectancy, popular opinions related to COVID-19 and CTMA
-Users’ behavioral intentions positively affect their actual app use
Trang et al. (2020)
CTMA
acceptance
-App
specifications Experiment
-Convenience and privacy design positively influence mass acceptance, whereas self-benefit appeal and
self-societal-benefit appeal decrease mass acceptance
-Mass
acceptance varies across groups
Urbaczewski
and Lee (2020)
CTMA use
/
Difference in
difference
-CTMA are highly and significantly associated with a decreased spread of COVID-19
Wagner et al. Table 1.
Summary of studies on
contact tracing mobile
apps (CTMA) from the
information systems
(IS) field 2.2 Coping theory
d i
h (2021)
Use intentions
and
continuance
intentions
-Privacy
calculus model
-Herding
heuristics
Longitudinal
online survey
-Privacy risks negatively affect use intentions, while perceived benefits and imitating others positively influence
use intentions
-Updated benefits and imitating others positively affect continuance intentions
Note(s): IT 5 information technology; CTMA 5 contact tracing mobile apps; UTAUT 5 unified theory of acceptance and use of technology;/5 no theory applied Reference
Focus Table 1. Summary of studies on
contact tracing mobile
apps (CTMA) from the
information systems
(IS) field Reference We selected coping theory as the theoretical framework for several reasons. First,
it highlights cognitive appraisal’s role in determining coping responses to stressful
situations (Beaudry and Pinsonneault, 2005; Lazarus and Folkman, 1984). This theory can We selected coping theory as the theoretical framework for several reasons. First,
it highlights cognitive appraisal’s role in determining coping responses to stressful
situations (Beaudry and Pinsonneault, 2005; Lazarus and Folkman, 1984). This theory can Continuance
usage of
contact tracing
apps Reference
Context
Method
Appraisals
Outcomes
Bala and
Venkatesh (2016)
IT adaptation
Field study
-Primary
appraisal
-Perceived
opportunity
-Perceived
threat
-Secondary
appraisal
-Perceived
controllability
-Avoidance
-Exploration-to-revert
-Exploration-to-
innovate
-Exploitation
Beaudry and
Pinsonneault
(2005)
Information
technology (IT)
use
Qualitative method
(interviews, annual
reports and job
descriptions)
-Primary
appraisal
-Opportunity
-Threat
-Secondary
appraisal
-IT control
-Minimization
of an IT event’s negative
consequences
-Individual
efficiency and
effectiveness
-Restoring
personal emotional
stability
Bhattacherjee et al. (2018)
Mandatory IT
use
Interviews
-Primary
appraisal
-Opportunity
-Threat
-Secondary
appraisal
-IT control
-Reluctant
response
-Engaged
response
-Deviant
response
-Compliant
response
Chen et al. (2019)
Mobile shopping
applications
Experiment
-Primary
appraisal
-Perceived
information overload
-Perceived
intrusiveness
-Perceived
reward
-Secondary
appraisal
-Perceived
information control
-Mobile computing
self-efficacy
-Discontinuance
behavior
D’Arcy et al. (2014) Security-related
stress (SRS)
Survey
-Primary
appraisal
-Secondary
appraisal
-Information
security policy violation
intentions
Christophe et al. (2011)
Disruptive IT
Experiment
-Primary
appraisal
-Opportunity
-Threat
-Secondary
appraisal
-IT control
-Benefitsatisficing
-Benefitmaximizing
-Self-preservation
-Disturbance
handling
(continued)
Table 2. Summary of studies
that used coping theory
in the information
systems (IS) field
apps
1445 Bhattacherjee et al. (2018)
Mandatory IT
use
Interviews Chen et al. (2019)
Mobile shopping
applications
Experiment Chen et al. (2019)
Mobile shopping
applications
Experiment D’Arcy et al. (2014) Security-related
stress (SRS)
Survey Christophe et al. (2011)
Disruptive IT
Experiment Reference
Context
Method
Appraisals
Outcomes
Gong et al. 2.2 Coping theory
d i
h (2020)
Mobile payments Survey
-Primary
appraisal
-Perceived
value
-Perceived
threat
-Secondary
appraisal
-Perceived
controllability
-Intentions
to use mobile payment
services
Liang et al. (2019)
IT security
Experiment
-Perceived
threat
-Perceived
avoidability
-Inward
emotion-focused coping
-Problem-focused
coping behavior
-Outward emotion-
focusedcoping
Marakhimov and
Joo (2017)
Wearable
healthcare
devices
Survey
-Primary
appraisal
-Challenge
-Threat
Extended use
Nach and Lejeune
(2010)
IT challenges to
users’ identity
Qualitative method
(narrative synthesis)
-Primary
appraisal
-Positive
emotional response
-Negative
emotional response
-Secondary
appraisal
-Controllability
-Anti-identity
-Reinforced
identity
-Ambivalent
identity
-Redefined
identity
Wu et al. (2017)
New IT systems
Survey and panel data
-Giving-network
closure
-Seeking-network
closure
-Post-adoption
IT use
-Cognitive
adaptation
-Behavioral
adaptation
-Affective
adaptation
Table 2. IMDS
123,5
1446 Table 2. help explain how individuals appraise stressful situations and how their appraisals form
their behavioral responses (Folkman and Moskowitz, 2004; Salo et al., 2020). Second, this
theory offers a theoretical framework with which to understand individual behaviors in
psychology and post-adoption use in IS (Beaudry and Pinsonneault, 2005; Fadel and Brown,
2010; Salo et al., 2020). This complements this study’s aims. Therefore, following the previous
literature, coping theory was used in this study to investigate continuous usage of CTMA. Construct
Definition
Perceived usefulness
Users’ expected benefits from using Contact Tracing Mobile Apps (CTMA) in
protecting their health during the COVID-19 pandemic (Bhattacherjee, 2001; Davis,
1989)
Perceived distress relief
Users’ perception of relieving their COVID-19-related distress through CTMA
(Dholakia, 2006)
Privacy concerns
Users’ concerns about opportunistic behavior related to private information
collected by CTMA (Dinev and Hart, 2006)
Perceived response
efficacy
Users’ perception of the efficacy of protection offered by the CTMA providers in
successfully attenuating privacy threats in CTMA use (Posey et al., 2015)
Continuance usage
Users’ continued use of CTMA during the COVID-19 pandemic (Bhattacherjee,
2001)
Table 3.
Definitions of the five
constructs 3. Research model and hypothesis development
3.1 Research model development p
Users’ cognitive appraisals help explain their behavioral responses to stressful events
(Beaudry and Pinsonneault, 2005; Lazarus and Folkman, 1984; Salo et al., 2020). The research
model was built based on coping theory to study how the three types of cognitive appraisals
affect users’ behavioral responses to CTMA. Specifically, perceived usefulness and perceived
distress relief were conceptualized as opportunity appraisal, privacy concerns as threat (
y
,
;
,
;
,
)
model was built based on coping theory to study how the three types of cognitive appraisals
affect users’ behavioral responses to CTMA. Specifically, perceived usefulness and perceived
distress relief were conceptualized as opportunity appraisal, privacy concerns as threat appraisal, perceived response efficacy as secondary appraisal and continuance usage as a
behavioral response. The definitions of the five constructs are defined in Table 3. Continuance
usage of
contact tracing
apps p
In this study, we adopted variables (e.g. perceived usefulness, perceived distress relief,
privacy concerns and perceived response efficacy)from prior studies because thecoping theory
does not provide specific constructs to measure appraisals. As suggested by prior literature
(Bhattacherjee et al., 2018), we accommodated existing relevant variables in the literature to
reflect different appraisals and to fit to the CTMA context. Some studies have accommodated
theIScontinuance enablers(e.g.perceived usefulness) (Bhattacherjee,2001; Yan et al., 2021) and
inhibitors (e.g. privacy concerns) (Zhou, 2016; Zhou and Li, 2014) to reflect users’ primary
appraisals of opportunity and threat, respectively (Bhattacherjee et al., 2018). Prior studies have
also adopted different variables regarding the external resources to deal with IS usage from
other theories, such as facilitating conditions, to uncover users’ assessments of available
resources to respond to IS usage (Bhattacherjee et al., 2018; Venkatesh et al., 2003). Even though
these variables (e.g. perceived usefulness, perceived distress relief, privacy concerns and
perceived response efficacy) are developed as part of different theoretical paradigms, the prior
research applying coping theory shows that they could also be applied in our study to reflect
users’ different cognitive appraisals under the theoretical framework of the coping theory. 1447 g
pp
p g
y
Specifically,inthis study, opportunity appraisals reflectthe degreetowhich auserperceives
using CTMA to help achieve beneficial outcomes (Bala and Venkatesh, 2016; Beaudry and
Pinsonneault, 2005). Past IS literature has used perceived usefulness (Bhattacherjee et al., 2018)
and stress reduction (Cheikh-Ammar,2020; Eisel etal., 2014) to reflect the beneficial outcomes of
using an IS. Since CTMA can offer timely alerts and instructions to users who have been near
an infected person, CTMA use will instrumentally enhance users’ performance in protecting
their health. Also, these apps’ timely alerts and guidance may relieve the distress caused by
infection-related uncertainties and worries (Lin et al., 2020). Thus, perceived usefulness and
perceived distress relief are assumed to constitute CTMA-related opportunity appraisal. Threat appraisals represent the degree to which a user feels that CTMA use disrupts their
personal benefits and well-being (Bala and Venkatesh, 2016; Beaudry and Pinsonneault,
2005). Previous IS research has highlighted that privacy concerns are a critical threat to IS
users if the IS collects, stores and uses users’ private information (Dinev and Hart, 2006;
Zhang et al., 2018). This also applies to CTMA since apps collect users’ private information—
such as their close contacts and COVID-19 testing results—and use it to detect potential
exposure. Therefore, privacy concerns can present a key threat for individual users, and app
use compromises between health and privacy. Indeed, recent studies have highlighted
individuals’ privacy concerns as the major threat to CTMA adoption (Lin et al., 2021; Riemer
et al., 2020; Trang et al., 2020). In light of this, privacy concerns are assumed to represent
threat appraisals regarding CTMA. Construct
Definition
Perceived usefulness
Users’ expected benefits from using Contact Tracing Mobile Apps (CTMA) in
protecting their health during the COVID-19 pandemic (Bhattacherjee, 2001; Davis,
1989)
Perceived distress relief
Users’ perception of relieving their COVID-19-related distress through CTMA
(Dholakia, 2006)
Privacy concerns
Users’ concerns about opportunistic behavior related to private information
collected by CTMA (Dinev and Hart, 2006)
Perceived response
efficacy
Users’ perception of the efficacy of protection offered by the CTMA providers in
successfully attenuating privacy threats in CTMA use (Posey et al., 2015)
Continuance usage
Users’ continued use of CTMA during the COVID-19 pandemic (Bhattacherjee,
2001)
Table 3. Definitions of the five
constructs Secondary appraisals refer to an individual’s cognitive assessment of whether coping
resources are available to respond to an encounter (Bala and Venkatesh, 2016; Beaudry and
Pinsonneault, 2005; Lazarus and Folkman, 1984). 1447 Coping theory has indicated that if users
believe there exist adequate and sufficient coping resources, the threat is apt to be minimal or
absent (Bhattacherjee et al., 2018; Lazarus and Folkman, 1984). Since privacy issues are one
major threat to CTMA use, users’ evaluations of privacy protection are particularly critical in
this study’s context. Indeed, many preventive measures and techniques have been used by
CTMA providers to protect private information, such as no location data collection,
anonymity and decentralization (Apple and Google, 2021; Sowmiya et al., 2021). These
privacy protections provided by CTMA providers offer the coping resource to CTMA users to
respond to privacy issues when using CTMA. CTMA users could assess the efficacy of these
coping resource for privacy protections in preventing privacy threats in their CTMA use. Thus, perceived response efficacy fits well with the definition of secondary appraisals in our
studied context and can be assumed to constitute secondary appraisals regarding CTMA. 1448 In this study, behavioral response was operationalized as continuance usage, which refers to
users’ continued CTMA use during the COVID-19 pandemic (Bhattacherjee, 2001). Prior
literature has adopted continuance usage to represent a behavioral response. For instance, Yang
et al. (2016) developed behavioral response as continuous use when employing coping theory to
study users’ reactions to mobile shopping channels. Therefore, in this study, continuance usage
of CTMA is assumed to reflect users’ behavioral responses to the COVID-19 pandemic. p
p
According to coping theory, individuals’ opportunity, threat and secondary appraisals
jointly determine their behavioral responses to stressful events (Beaudry and Pinsonneault,
2005; D’Arcy et al., 2014; Salo et al., 2020). Aligning with this theory, perceived usefulness,
distress relief, privacy concerns and perceived response efficacy are proposed as the essential
determinants of CTMA continuance usage. Meanwhile, age, gender, education and
coronavirus test status are moderators in the model (see Figure 1). Figure 1.
Research model 3.2 Hypothesis development Perceived usefulness has been widely considered an important driver of IS continuance usage
(Bhattacherjee, 2001). In this study’s context, by using CTMA, if users were near a person
reported to have been infected with COVID-19, they could use CTMA to be rapidly informed
about theirpotential exposureandreceive furtherinstructionson how toself-isolateand protect
themselves (Trang et al., 2020) or even quickly access testing and treatment if needed. Consequently, users will likely perceive such apps as useful, improving their ability to protect
their health during the COVID-19 pandemic. These usefulness perceptions can facilitate CTMA
continuance usage (Bhattacherjee, 2001). Therefore, the following hypothesis is suggested. Figure 1. Research model H1. Perceived usefulness is positively associated with CTMA continuance usage. The COVID-19 pandemic has created distress and fear (Li et al., 2021), and one leading cause
of this distress is informational uncertainty regarding the pandemic (Lin et al., 2020; Rettie
and Daniels, 2021). For instance, people do not know whether they have been exposed to
infection, and they are unsure of the correct response to virus exposure or diagnosed
infection. Such uncertainties have contributed to intense distress during the COVID-19
pandemic (Lin et al., 2020). Past research has suggested that IS can reduce the distress caused
by a specific event by providing information that supports decision-making (Eisel et al., 2014). Similarly, recent research has demonstrated that transparent, timely communication helps
reduce COVID-19-related distress (Lin et al., 2020). CTMA can convey such messages by
offering timely alerts and clear guidance, so users may perceive such apps as helpful in
alleviating distress. Therefore, they would likely continue using these apps. Hence, the
following hypothesis is proposed. Continuance
usage of
contact tracing
apps 1449 H2. Perceived distress relief is positively associated with CTMA continuance usage. For effective contact tracing, CTMA must collect and process individuals’ data (Lin et al.,
2021; Riemer et al., 2020; Trang et al., 2020). For instance, a GPS-based tracing app collects
users’ GPS information, disclosing their precise location. Although some apps rely on
Bluetooth techniques and do not collect precise location information, people remain
concerned about the use and storage of their sensitive data and possible surveillance (Riemer
et al., 2020; Trang et al., 2020). Such CTMA privacy concerns may decrease users’ CTMA
continuance usage. Privacy concerns, prior literature has indicated, are the primary inhibitor
of mobile app continuance intention (Zhou, 2016; Zhou and Li, 2014). 3.2 Hypothesis development For instance, according
to Zhou and Li (2014), in the context of mobile social network services, privacy concerns are
negatively linked to continuance intentions. Moreover, according to recent CTMA research,
privacy concerns are the main reason for unwillingness to download and use CTMA (Chan
and Saqib, 2021). Hence, it is reasonable to presume that privacy concerns decrease CTMA
continuance usage, yielding the following hypothesis. H3. Privacy concerns are negatively associated with CTMA continuance usage. Perceived response efficacy reveals the extent to which users perceive that preventative
measures effectively protect their privacy (Boss et al., 2015). To protect users’ privacy, many
efforts have sought to limit data collection and control data transmission and storage
(Sowmiya et al., 2021). Typically, a smartphone app generates and transmits a Bluetooth
signal (a series of unique strings of random numbers and letters) that does not contain any
personal information (such as names and identities), and when a user is infected, they can
report their infection via the app, which will send a unique Bluetooth signal to a central
database. The app will automatically check the report for other users and alert them if they
have been in close contact with an infected user. Neither app users, the infected person,
smartphone operating system developers, nor the government can infer any private
information from the data collected by these apps (Apple and Google, 2021). These preventive
measures may decrease users’ worries about the collection and use of their private
information. While users who perceive high response efficacy are more likely to feel secure
about their privacy and continue using such apps, users who perceived low response efficacy
may continue to feel uncertain and threatened regarding privacy security, avoiding the
continued use of such apps. Thus, the following hypothesis is proposed. H4. Perceived response efficacy is positively associated with CTMA continuance usage H4. Perceived response efficacy is positively associated with CTMA continuance usage. In this study, age, gender, education and coronavirus test status are considered as
moderators to explore the possible effects of user-related characteristics on these proposed In this study, age, gender, education and coronavirus test status are considered a
moderators to explore the possible effects of user-related characteristics on these propose IMDS
123,5 relationships. 3.2 Hypothesis development Such as senior CTMA users might be more vulnerable to COVID-19 than other
age groups (World Health Organization, 2021b), they might be more eager to seek
interventions to help them to cope with this stressful situation, thus, their cognitive
appraisals of using CTMA, such as opportunity appraisals, could exert a stronger influence
on their continuance usage than other age groups. Regarding gender, women have been
found to be more sensitive to stressful situations than men (Matud, 2004), therefore, women’s
opportunity, threats and secondary appraisals of using CTMA to cope with COVID-19
pandemic could have different impacts on their continuance usage than men. Considering
education background, prior studies found that people with higher education tend to use new
technologies more than those with less educational background (Owusu Kwateng et al., 2019). Furthermore, regarding COVID-19 testing status, users who have been tested are obligated to
report their test results via the app, particularly when they are diagnosed with an infection,
thereby, they might be more concerned about the benefits and threats of using CTMA as well
as the privacy protection related to CTMA compared with those who have not been tested. Hence, the effects of cognitive appraisals on continuance usage may be different among users
with different characteristics, and it is meaningful to explore the moderating effects of age,
gender, education and coronavirus test status in this study. 1450 4. Research method
4.1 Instrument development p
We adapted well-established scales from prior research to measure all constructs using a
seven-point Likert scale. Specifically, perceived usefulness and continuance usage were
measured with items derived from the work of Bhattacherjee (2001), perceived distress relief
using items derived from the study of Dholakia (2006), privacy concerns using items derived
from the research of Dinev and Hart (2006) and perceived response efficacy using items derived
from the work of Posey et al. (2015). More details of these measures can be found in Table 5. 4.2 Participants
Current CTMA users in the USA comprised the target population of this study for the
following reasons. First, CTMA use in USA was depended on a voluntary basis among
individuals. Second, many individuals had used CTMA for a while before our study. According to a survey on public’s attitude toward CTMA use in USA conducted in June, 2020,
42% of respondents supported CTMA use during the pandemic (Zhang et al., 2020). The
Amazon Mechanical Turk (MTurk) was used to recruit participants since obtaining a large
sample of respondents from various backgrounds who respond fast is easy in USA. A concise
explanation of the study’s objectives and a link to its online questionnaire were presented
with the invitation messages. To investigate CTMA continuance usage, it is necessary that
the required respondents have had certain usage experiences. Thus, the two following
screening questions were implemented to record respondents’ prior CTMA use, including
“Have you installed and used a mobile tracing app on your smartphone before?” and if so,
“What is the mobile tracing app you are using?”. Only those respondents who answer yes to
the first question and write the name of a specific tracing app they have used to the second
question can proceed with the rest parts of the questionnaire. In addition, two attention-
checking questions were incorporated into the questionnaire to ensure sufficient attention by
respondents. Each response was checked carefully to eliminate unreliable responses. The online survey was delivered to prospective respondents at the beginning of December
2020, when mass vaccination efforts had not yet publicly begun in the United States. In total,
783 responses were collected in one week. The 190 respondents who had not installed a
contact tracing app on their smartphone or failed to offer the specific names of the contact tracing app they had used were excluded in this study. 4. Research method
4.1 Instrument development Furthermore, we excluded 25
respondents who failed to answer two attention-checking questions correctly and 17
respondents who answered unreliably—such as by providing the same response option for
all measurement items. Finally, a valid sample of 551 responses was obtained. Considering
the sample size, prior literature suggests that a data sample size of about 10 times the number
of items used in a study can be appropriate (Hair et al., 2014). In this study, there are 20
measurement items in total. Thus, the sample size of about 200 will be appropriate for this
study. Our sample size (N 5 551) is greater than 200 and can be considered as adequate for
this study. Among the 551 final respondents, 60.4% were male and 39.4% were female. Most
respondents were 20–39 years old (64%), and most had been previously tested for
coronavirus (61.3%). Table 4 presents respondents’ demographic information. Continuance
usage of
contact tracing
apps 1451 4.3 Common method bias and collinearity 4.3 Common method bias and collinearity
To assess common method bias, this study employed Harman’s single factor test, as
recommended by Podsakoff et al. (2003). This test revealed that the highest variance explained
by a single factor was 39.2%, which was lower than the suggested threshold value of 50%
(Podsakoff et al., 2003). Therefore, common method bias was not a serious concern for this study. h
lli
i
f
h i d
d
i bl
d
d i
d b
k
d 4.3 Common method bias and collinearity
To assess common method bias, this study employed Harman’s single factor test, as
recommended by Podsakoff et al. (2003). This test revealed that the highest variance explained
by a single factor was 39.2%, which was lower than the suggested threshold value of 50%
(Podsakoff et al., 2003). Therefore, common method bias was not a serious concern for this study. The collinearity of each independent variable was measured, as advised by Kock and
Lynn (2012), by assessing the values of the variance inflation factor (VIF). The VIF values
ranged from 1.189 to 2.416, which was below the recommended threshold of 3.3 (Kock and
Lynn, 2012). Therefore, collinearity was not a critical issue for this study. y
To assess common method bias, this study employed Harman’s single factor test, as
recommended by Podsakoff et al. (2003). This test revealed that the highest variance explained
by a single factor was 39.2%, which was lower than the suggested threshold value of 50%
(Podsakoff et al., 2003). Therefore, common method bias was not a serious concern for this study. The collinearity of each independent variable was measured, as advised by Kock and
Lynn (2012), by assessing the values of the variance inflation factor (VIF). The VIF values (
,
)
,
y
The collinearity of each independent variable was measured, as advised by Kock and
Lynn (2012), by assessing the values of the variance inflation factor (VIF). The VIF values
ranged from 1.189 to 2.416, which was below the recommended threshold of 3.3 (Kock and
Lynn, 2012). Therefore, collinearity was not a critical issue for this study. 4.4 Data analysis This study utilized partial least squares (PLS) to analyze the data. Prior literature suggests
that compared with covariance-based structural equation modeling (CB-SEM), PLS-SEM is
more suitable for studies when the research objective is for prediction and when the data
distribution is lack of normality (Chin et al., 2003; Hair et al., 2019). This study aims to examine
what factors predict CTMA continuance usage. Additionally, prior literature suggests that
when testing the multivariate normality of the data, if the value of Skewness is between 2
and þ2, and the value of Kurtosis is between 7 and þ7, data distribution can be considered
as normal (Curran et al., 1996; West et al., 1995). Our test results showed that the values of both Variable
Items
Count
Percentage (%)
Gender
Male
333
60.4
Female
217
39.4
Unwilling to disclose
1
0.2
Age
20–29
172
31.2
30–39
181
32.8
40–49
97
17.6
50–59
70
12.7
60–69
31
5.6
Education
Basic education
8
1.5
Upper secondary education (or vocational school)
33
6.0
Bachelor’s or equivalent level
334
60.6
Master’s or equivalent level
170
30.9
Doctoral or equivalent level
6
1.1
Coronavirus test status
Tested
338
61.3
Not tested
207
37.6
Unwilling to disclose
6
1.1
Table 4. 4.4 Data analysis Respondents’
demographic profile Constructs
Reference
Item
Factor
loading
CA
CR
AVE
Continuance usage (CU)
Bhattacherjee
(2001)
CU1: I continue using CTMA, rather than discontinuing their use in recent days
0.849
0.866 0.903 0.650
CU2: I continue using CTMA, rather than using any alternative means of tracing in
recent days
0.788
CU3: I will continue my CTMA use in the following days during the COVID-19
pandemic
0.825
Perceived distress relief
(PDR)
Dholakia (2006)
PDR1: Using CTMA makes me feel self-assured
0.823
0.823 0.883 0.653
PDR2: Using CTMA makes me feel relieved
0.820
PDR3: Using CTMA makes me feel contented
0.820
PDR4: Using CTMA makes me feel calm
0.794
PDR5: Using CTMA makes me feel tranquil
0.773
Perceived usefulness (PU) Bhattacherjee
(2001)
PU1: Using CTMA improves my performance in protecting myself during the COVID-
19 pandemic
0.825
0.758 0.861 0.674
PU2: Using CTMA increases my productivity in protecting myself during the COVID-
19 pandemic
0.797
PU3: Using CTMA enhances my effectiveness in protecting myself during the
COVID-19 pandemic
0.825
PU4: Overall, using CTMA is useful in protecting myself during the COVID-19
pandemic
0.785
Privacy concern (PC)
Dinev and Hart
(2006)
PC1: I am concerned that my information collected by CTMA could be misused
0.824
0.871 0.911 0.719
PC2: I am concerned that someone else can access my information through CTMA. 0.855
PC3: I am concerned about information collected by CTMA because of what others
might do with it
0.871
PC4: I am concerned about information collected by CTMA because it could be used in
a way I did not foresee
0.843
Perceived response
efficacy (PRE)
Posey et al. (2015)
PRE1: The efforts of CTMA providers to keep my privacy safe from information
security threats are effective
0.783
0.788 0.863 0.611
PRE2: The available measures taken by CTMA providers to protect my privacy from
security violations are effective
0.773
PRE3: The preventive measures available to stop people from accessing my privacy
taken by CTMA providers are adequate
0.783
PRE4: The preventive measures available to keep people from misusing my privacy
taken by CTMA providers are adequate
0.788
Note(s): CR 5 composite reliability; CA 5 Cronbach’s alpha; AVE 5 average variance extracted Skewness and Kurtosis did not meet the recommended thresholds, indicating that our data is
not normal. Thus, PLS-SEM technique is a proper method for this study. The data analysis
includes the test of the measurement model and structural model. 4.4 Data analysis Continuance
usage of
contact tracing
apps 4.4.1 Measurement model. Convergent validity and discriminant validity were tested to
assess the measurement model. To test convergent validity, this study evaluated the factor
loading of each item, Composite Reliability (CR), Cronbach’s alpha (CA) and Average Variance
Extracted (AVE). As Table 5 shows, all factor loading, CA and CR values met the recommended
threshold of 0.70, 0.70 and 0.70, respectively, and AVE values all exceeded 0.5, indicating
satisfactory convergent validity for this study (Fornell and Larcker, 1981). 1453 In contrast, perceived distress relief and privacy concerns
do not significantly influence continuance usage, thus supporting H1 and H4 while not
supporting H2 and H3. The Stone–Geisser Q2 was further measured to estimate the predictive relevance of the
proposed research model (Geisser, 1974; Hair et al., 2017; Stone, 1974). The results of this test
showed that the Q2 value for continuance usage was 0.363, greater than zero, indicating good
predictive relevance (Hair et al., 2017). The standardized root mean square residual (SRMR) was applied to assess the goodness
of fit for the proposed research model (Hair et al., 2017; Henseler et al., 2014). This test’s result
was 0.061, which is less than the recommended threshold of 0.08, indicating a good model fit
(Hair et al., 2017; Henseler et al., 2014; Hu and Bentler, 1998). 4.4.3 Moderation analysis. The moderation analysis was conducted to explore the
moderating effects of age, gender, education and coronavirus test status on the proposed
relationships. Gender and coronavirus test status was operationalized as a dummy variable. Age and education were operated as a dummy variable that took ordinal values to represent
increasing levels of age and education background, respectively. Specifically, respondents
were divided into three sub-groups by age: (1) young, aged 20–29 years; (2) middle-aged, 30 to
49; and (3) senior, 50 to 69. Prior research has focused on the young, middle-aged and senior
groups to explore how age moderates certain IS relationships (e.g. Czaja et al., 2006; Tams
et al., 2014; Zhao et al., 2018). Recent literature also noted there are differences between young,
middle and senior people in terms of their vulnerability to COVID-19 (World Health
Organization, 2021b; Bhopal et al., 2021). Thus, this study also explores whether there are
Construct
PDR
PU
CU
PC
PRE
Perceived distress relief (PDR)
Perceived usefulness (PU)
0.792
Continuance usage (CU)
0.774
0.822
Privacy concerns (PC)
0.449
0.358
0.365
Perceived response efficacy (PRE)
0.890
0.804
0.877
0.369
Figure 2. Model-testing results
Table 8. Correlations’
heterotrait-monotrait
ratio (HTMT)
123,5
1454 g
The model explains 57.6% of continuance usage variance. Perceived usefulness
(β 5 0.284, p < 0.001) and response efficacy (β 5 0.448, p < 0.001) significantly positively
influence continuance usage. In contrast, perceived distress relief and privacy concerns
do not significantly influence continuance usage, thus supporting H1 and H4 while not
supporting H2 and H3. 1453 y
g
y
y (
,
)
Discriminant validity was evaluated using two means: (1) the criteria proposed by Fornell
and Larcker (1981) and (2) the Heterotrait–Monotrait (HTMT) ratio developed by Henseler
et al. (2015). First, according to Fornell and Larcker (1981), discriminant validity is established
when (1) the square root of each construct’s AVE exceeds the correlations between the
construct and others and (2) the item loadings are higher on their intended construct than on
other constructs. Tables 6 and 7 show that this study’s results met these criteria. Second,
according to Henseler et al. (2015), an HTMT of 0.90 is an acceptable threshold for
discriminant validity. Table 8 shows that this study’s discriminant validity was confirmed. 4.4.2 Structural model. The structural model was tested by measuring not only the
significance and size of the path coefficients and the coefficient of determination (R2 value) Construct
Construct item
CU
DR
PU
PC
RE
Continuance usage (CU)
CU1
0.849
0.553
0.551
0.259
0.627
CU2
0.788
0.521
0.513
0.261
0.563
CU3
0.825
0.482
0.538
0.221
0.567
Perceived distress relief (PDR)
PDR1
0.576
0.823
0.630
0.355
0.628
PDR2
0.541
0.820
0.562
0.313
0.604
PDR3
0.501
0.820
0.554
0.323
0.610
PDR4
0.468
0.794
0.495
0.303
0.576
PDR5
0.445
0.773
0.456
0.292
0.550
Perceived usefulness (PU)
PU1
0.550
0.555
0.825
0.249
0.561
PU2
0.519
0.558
0.797
0.247
0.533
PU3
0.539
0.539
0.825
0.248
0.507
PU4
0.491
0.530
0.785
0.244
0.493
Privacy concern (PC)
PC1
0.222
0.275
0.224
0.824
0.225
PC2
0.277
0.350
0.260
0.855
0.290
PC3
0.290
0.367
0.294
0.871
0.288
PC4
0.218
0.337
0.249
0.843
0.234
Perceived response efficacy (PRE)
PRE1
0.544
0.602
0.492
0.246
0.783
PRE2
0.547
0.542
0.512
0.204
0.773
PRE3
0.580
0.588
0.531
0.251
0.783
PRE4
0.561
0.574
0.492
0.266
0.788
Construct
PDR
PU
CU
PC
PRE
Perceived distress relief (PDR)
0.806
Perceived usefulness (PU)
0.675
0.808
Continuance usage (CU)
0.632
0.650
0.821
Privacy concern (PC)
0.395
0.305
0.301
0.848
Perceived response efficacy (PRE)
0.738
0.649
0.714
0.310
0.782
Table 7. Loadings and cross-
loadings
Table 6. Correlation matrix and
discriminant validity but also the model’s predictive relevance (Q2 value) and goodness of fit. Figure 2 presents the
results using PLS. results using PLS. The model explains 57.6% of continuance usage variance. Perceived usefulness
(β 5 0.284, p < 0.001) and response efficacy (β 5 0.448, p < 0.001) significantly positively
influence continuance usage. 1453 1454 The Stone–Geisser Q2 was further measured to estimate the predictive relevance of the
proposed research model (Geisser, 1974; Hair et al., 2017; Stone, 1974). The results of this test
showed that the Q2 value for continuance usage was 0.363, greater than zero, indicating good
predictive relevance (Hair et al., 2017). The standardized root mean square residual (SRMR) was applied to assess the goodness
of fit for the proposed research model (Hair et al., 2017; Henseler et al., 2014). This test’s result
was 0.061, which is less than the recommended threshold of 0.08, indicating a good model fit
(Hair et al., 2017; Henseler et al., 2014; Hu and Bentler, 1998). 4.4.3 Moderation analysis. The moderation analysis was conducted to explore the
moderating effects of age, gender, education and coronavirus test status on the proposed
relationships. Gender and coronavirus test status was operationalized as a dummy variable. Age and education were operated as a dummy variable that took ordinal values to represent
increasing levels of age and education background, respectively. Specifically, respondents
were divided into three sub-groups by age: (1) young, aged 20–29 years; (2) middle-aged, 30 to
49; and (3) senior, 50 to 69. Prior research has focused on the young, middle-aged and senior
groups to explore how age moderates certain IS relationships (e.g. Czaja et al., 2006; Tams
et al., 2014; Zhao et al., 2018). Recent literature also noted there are differences between young,
middle and senior people in terms of their vulnerability to COVID-19 (World Health
Organization, 2021b; Bhopal et al., 2021). Thus, this study also explores whether there are Construct
PDR
PU
CU
PC
PRE
Perceived distress relief (PDR)
Perceived usefulness (PU)
0.792
Continuance usage (CU)
0.774
0.822
Privacy concerns (PC)
0.449
0.358
0.365
Perceived response efficacy (PRE)
0.890
0.804
0.877
0.369
Figure 2. Model-testing results
Table 8. Correlations’
heterotrait-monotrait
ratio (HTMT) Construct
PDR
PU
CU
PC
PRE
Perceived distress relief (PDR)
Perceived usefulness (PU)
0.792
Continuance usage (CU)
0.774
0.822
Privacy concerns (PC)
0.449
0.358
0.365
Perceived response efficacy (PRE)
0.890
0.804
0.877
0.369
Figure 2. Model-testing results
Table 8. Correlations’
heterotrait-monotrait
ratio (HTMT) significant differences among these three user groups. The respondents were also divided
into three sub-groups by education following the prior literature (i.e. low, middle and high)
(e.g. 1455 )
As Table 9 shows, a significant difference was observed between the young and senior age
sub-groups concerning the relationship between perceived usefulness and continuance
usage. Specifically, senior group exhibited the most significant effect of perceived usefulness
on continuance usage (β 5 0.586, p < 0.001), followed by the middle-aged sub-group
(β 5 0.267, p < 0.001) and young sub-group (β 5 0.198, p < 0.05). As Table 10 shows, a significant difference was observed between the tested and not-
tested groups concerning the relationship between perceived response efficacy and
continuance usage. Specifically, perceived response efficacy more strongly and
significantly influenced continuance usage for the tested group (β 5 0.548, p < 0.001)
compared to the not-tested group (β 5 0.276, p < 0.01). Additionally, distress relief was
found to significantly affect continuance usage only in the not-tested group
(β 5 0.283, p < 0.05). Comparison by coronavirus test status
Path coefficients of separate structural models
Tested vs not tested
Not tested (n 5 207)
Tested (n 5 338)
H1
n.s. 0.325***
0.280***
H2
n.s. 0.283*
n.s. H3
n.s. n.s. n.s. H4
p < 0.05
0.276**
0.548***
Note(s): *** 5 p < 0.001; ** 5 p < 0.01; * 5 p < 0.05; n.s. 5 not significant
Comparison by age
Separate structural models’ path coefficients
Young vs
senior
Young vs middle-
aged
Young vs
senior
Young
(n 5 172)
Middle
(n 5 278)
Senior
(n 5 101)
H1
p < 0.01
n.s
n.s
0.198*
0.267***
0.586***
H2
n.s
n.s
n.s
n.s
n.s
n.s
H3
n.s
n.s
n.s
n.s
n.s
n.s
H4
n.s
n.s
n.s
0.464***
0.432***
0.496**
Note(s): *** 5 p < 0.001; ** 5 p < 0.01; * 5 p < 0.05; n.s. 5 not significant
Table 10. Coronavirus test status
as a moderator
Table 9. Testing for age as a
moderator Comparison by age
Separate structural models’ path coefficients
Young vs
senior
Young vs middle-
aged
Young vs
senior
Young
(n 5 172)
Middle
(n 5 278)
Senior
(n 5 101)
H1
p < 0.01
n.s
n.s
0.198*
0.267***
0.586***
H2
n.s
n.s
n.s
n.s
n.s
n.s
H3
n.s
n.s
n.s
n.s
n.s
n.s
H4
n.s
n.s
n.s
0.464***
0.432***
0.496**
Note(s): *** 5 p < 0.001; ** 5 p < 0.01; * 5 p < 0.05; n.s. 5 not significant
Table 9. 1453 Cruz-Jesus et al., 2016): (1) low, having only completed basic education or upper secondary
education (or vocational school); (2) middle, having completed a bachelor’s degree; and (3)
high, having completed a master’s or doctoral degrees. significant differences among these three user groups. The respondents were also divided
into three sub-groups by education following the prior literature (i.e. low, middle and high)
(e.g. Cruz-Jesus et al., 2016): (1) low, having only completed basic education or upper secondary
education (or vocational school); (2) middle, having completed a bachelor’s degree; and (3)
high, having completed a master’s or doctoral degrees. Continuance
usage of
contact tracing
apps Multi-group analysis (MGA) was applied in this study to assess the moderating
effect (Hair et al., 2017). Prior to MGA, as recommended by Henseler et al. (2016), the
Measurement Invariance of Composite Models (MICOM) procedure was used to
ascertain whether measurement invariance existed among the different groups. The
results showed no measurement invariance among the sub-groups regarding gender
and education, so the MGA was not used to analyze differences among user groups in
these categories. For age and coronavirus test status, partial measurement was
established, so MGA was used to analyze differences among these sub-groups (Henseler
et al., 2016). 1455 Testing for age as a
moderator Comparison by coronavirus test status
Path coefficients of separate structural models
Tested vs not tested
Not tested (n 5 207)
Tested (n 5 338)
H1
n.s. 0.325***
0.280***
H2
n.s. 0.283*
n.s. H3
n.s. n.s. n.s. H4
p < 0.05
0.276**
0.548***
Note(s): *** 5 p < 0.001; ** 5 p < 0.01; * 5 p < 0.05; n.s. 5 not significant
Table 10. Coronavirus test status
as a moderator Our findings raised several points of interest. First, our results reveal that perceived
usefulness (β 5 0.284, p < 0.001) and perceived response efficacy (β 5 0.448, p < 0.001)
determine CTMA continuance usage. This finding aligns with coping theory, according to
which primary appraisals and secondary appraisals predict users’ behavioral responses
related to new IS (Bala and Venkatesh, 2016; Gong et al., 2020). Generally, when deciding to
continue CTMA use, users assess the consequences of their use (primary appraisal) and
evaluate the coping resources over this use (secondary appraisal). They tend to continue
using these apps during a pandemic when they perceive them as highly useful in protecting
their health and highly efficacious in protecting their privacy. Specifically, perceived response efficacy was found to be a stronger driver for CTMA
continuance usage than perceived usefulness. Users’ privacy concerns considerably influence
their CTMA use during a public health crisis (Chan and Saqib, 2021; Riemer et al., 2020). Using
such apps involves a compromise between users’ privacy and health. Thus, continuance
usage is not only influenced by such apps’ perceived usefulness in protecting users’ health
but also—more importantly—determined by users’ evaluation of whether an app’s available
measures can effectively protect their privacy. If users perceive CTMA’s privacy protections
as effective and adequate, this study’s findings suggest, they will likely continue using these
apps—even if they do not fully recognize CTMA’s usefulness in protecting their health
during a pandemic. This finding highlights the importance of perceived response efficacy in
explaining CTMA continuance usage during a public health crisis. Contrary to our expectations, privacy concerns do not significantly influence CTMA
continuance usage. This finding contradicts some prior research findings in normal life
contexts. For instance, users’ privacy concerns have been discovered to negatively affect
their continuance usage regarding social network sites (SNS) (Zhou and Li, 2014) and
location-based services (Zhou, 2016). 1455 A possible explanation for this research finding is that
CTMA users might be willing to sacrifice their privacy for personal and public health benefits
during a public health crisis. Privacy calculus theory (Culnan and Armstrong, 1999; Dinev
and Hart, 2006) suggests that people may intend to compromise privacy for certain benefits,
such as health and security. Prior research has found that, when people perceive a pressing
security threat, they will more likely accept a compromise between privacy and personal
safety (Davis and Silver, 2004). The COVID-19 pandemic has become a serious public health
crisis, threatening individuals’ life and health, so users may accept CTMA as necessary for
the public benefits. Despite CTMA-related privacy concerns, they may continue using CTMA
to help manage the pandemic. Thus, privacy concerns could lose their importance and even
have no impact on individuals’ CTMA continuance usage during this pandemic. The finding on the insignificant impact of privacy concerns on CTMA continuance usage
is also not consistent with recent research findings on CTMA adoption. For instance, Chan
and Saqib (2021) found that privacy concerns can reduce individuals’ willingness to
download CTMA. A possible explanation is that, if users perceive that preventative measures
or designs (such as anonymity and decentralization) of CTMA can protect their privacy
effectively based on their prior CTMA use experience, privacy concerns will not obstruct
their continuance usage of CTMA (Trang et al., 2020). In addition, Chan and Saqib (2021)
focused on initial CTMA acceptance, while this study has focused on continuous usage. When individuals lack experience using CTMA during the COVID-19 pandemic, they are less
likely to recognize the extent to which CTMA will protect their privacy and their possible
benefits than individuals who have already used CTMA. Therefore, privacy concerns are a
critical factor in reducing individuals’ initial acceptance of CTMA. After individuals have
already used CTMA voluntarily for some time during the COVID-19 pandemic, they can
better understand the measures CTMA use to protect their privacy based on their usage
experiences, which could enhance their perceptions concerning privacy protection. Thus, their privacy concerns related to CTMA use become less critical and might not reduce their
CTMA continuance usage during the COVID-19 pandemic. Continuance
usage of
contact tracing
apps g
g
p
This study also found perceived distress relief has no significant influence on CTMA
continuance usage in general. 1457 Finally, this study found some differences among the factors determining individuals’
continuous CTMA use across different user groups. The results show that age and COVID-19
testing status moderate cognitive appraisal’s impact on continuance usage. Specifically,
regarding age, perceived usefulness was found to influence continuance usage the most among
senior users (β 5 0.586, p < 0.001) and the least among young users (β 5 0.198, p < 0.05), and
significant differences are visible between these two user groups. This significant difference
might be due to senior users’ vulnerability to COVID-19 than other age groups (World Health
Organization, 2021b). Therefore, senior people may eagerly seek effective interventions to
prevent infections. In contrast, the risk of severe coronavirus disease and mortality is low
among young people in general (Bhopal et al., 2021), so they might worry less about infection
than other age groups. Thus, the perception of the usefulness of CTMA will have the strongest
impact on their continuance usage of CTMA in the senior group than other age groups. In examining coronavirus test status, this study found that perceived response efficacy
has a stronger impact on continuance usage for users who have been tested for COVID-19
(β 5 0.548, p < 0.001) than users who have not (β 5 0.276, p < 0.01), and there a significant
difference between these two groups. One possible reason for this finding is that users who
have been tested are obligated to report their results to the apps if they are diagnosed with
infection; therefore, they may be more concerned about disclosing their information and
require more privacy protection. Accordingly, when they perceive that these apps can
effectively protect their privacy, they may intend more strongly to continue using them. 6. Conclusion 6.1 Theoretical contributions
Thi
d
ib
h 1455 This is not consistent with prior findings in normal life or work
contexts. For instance, the findings of Cheikh-Ammar (2020) indicated that distress relief
offered by a SNS can motivate users to continue their usage of SNS. A possible reason is that
not all CTMA users emphasize the value of distress relief when using CTMA, as we found
that perceived distress relief has a significant influence on continuance usage particularly for
users who had not been tested for COVID-19 (β 5 0.283, p < 0.05). These users worry more
about their health than those who have been tested since they do not know whether they have
been infected with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, the virus
that causes COVID-19). These uncertainties can cause distress (Peters et al., 2017), leaving
users eager for timely, relevant information from CTMA to help them to release the stress. CTMA can convey such information via timely alerts and instructions. Thus, users who have
not been tested might tend to consider CTMA helpful for reducing distress related to COVID-
19. Accordingly, when they perceive that these apps can relieve their distress, they will
continue using them during the pandemic. 6.1 Theoretical contributions
Thi
d
ib
h This study contributes to the research related to CTMA on several fronts. First, it makes
contextual contributions to the IS continuance literature by extending the context of IS use
from the normal daily life and work contexts to a larger societal public health crisis context
and by clarifying cognitive appraisals’ role in explaining individuals’ CTMA continuance
usage in the specific abnormal context of a public health crisis, grounded on coping theory. Investigating cognitive appraisals’ role in explaining CTMA continuance usage enriches IS
continuance research using coping theory, and it explains how technology can help people
cope with public health crises. p
p
Second, this study offers new insights explaining CTMA-related privacy issues. Unlike
previous research, which indicated that privacy concerns are one of the key obstacles to IS continuance in normal life contexts (Zhou, 2016; Zhou and Li, 2014), as well as CTMA
adoption (Chan and Saqib, 2021; Lin et al., 2021), this study found that privacy concerns do
not inhibit continuance usage of CTMA though privacy concerns have been highlighted to be
critical in understanding CTMA adoption. Individuals’ appraisal of privacy issues regarding
an IS can be different due to context differences (e.g. normal work and life situation and
abnormal public health crisis). Specifically, in a public health crisis context, users are willing
to compromise their privacy to protect their personal and public health, and their privacy
concerns will not hinder them to continue using CTMA during the pandemic. Thus, threat
appraisal (such as privacy concerns) is not a hinder for users’ CTMA continuance usage, and
the second appraisal (such as perceived response efficacy) and opportunity appraisal (such as
perceived usefulness) are important appraisals for users’ CTMA continuance usage in a
public health crisis context. 1458 p
Third, the theoretical lens of coping theory yields new insights into the mechanisms
underlying CTMA continuance usage from a multi-dimension cognitive appraisal view, that
is, individuals appraise not only the benefits and threats of CTMA usage but also the coping
resources to privacy issues in their continuance usage of CTMA. Unlike recent research on
CTMA largely focusing on the initial adoption and ignoring the impact of users’ assessment
of coping options available to respond to privacy issues on their continuance usage of CTMA
(e.g. 6.1 Theoretical contributions
Thi
d
ib
h Chan and Saqib, 2021; Lin et al., 2021), this study’s findings show that opportunity
appraisals (perceived usefulness) and secondary appraisals (perceived response efficacy)
motivate users’ continuance usage, indicating that – even during unprecedented situations –
coping theory is a powerful tool with which to explain IS continuance usage in a public health
crisis. Fourth, this study has provided new insights specific to emotion-related benefits of
CTMAs. The results of this study show that perceived distress relief has a significant
influence on CTMA continuance usage only for users who have not been tested for COVID-19. This finding offers further evidence that CTMAs can not only help users protect their health
but also reduce emotional distress for specific user groups. This finding indicates that the role
of perceived distress relief in predicting continuance usage can change due to the different
contexts and user characteristics. Finally, the moderated-related findings (including age and coronavirus test status)
provide a finer-grained understanding of the role of user features in explaining how cognitive
appraisals affect CTMA continuance usage during the COVID-19 pandemic. Specifically, the
findings highlight the crucial role of user age in explaining how perceived usefulness
influences continuance usage, as well as coronavirus test status’s role in explaining how
perceived response efficacy affects continuance usage. These findings suggest that CTMA
continuance usage is a complex and nuanced phenomenon where user-specific factors do
matter. 6.2 Practical implications for public policy 1459 g
p
Third, motivated by the finding that perceived response efficacy positively influences
continuance usage that authorities should adopt strategies to enhance users’ perceptions
of CTMA’s response efficacy during the COVID-19 pandemic. On the one hand, authorities
should provide individual users with clear, detailed information about CTMA’s privacy
protection to demonstrate that sensitive user information is not captured and that
collected user data will only be used strictly following data and privacy protection rules. For instance, to protect individuals’ privacy, GPS techniques should not be used. On the
other hand, authorities should clearly explain how these apps protect user privacy to
reduce the negative impacts of rumors or misconceptions related to these apps, such as
explaining in detail what data will be captured, stored and used, who owns the data, who
can access the data and how user privacy will be protected, following data protection and
privacy rules. These strategies will increase individuals’ perceptions concerning CTMA’s
response efficacy, which could lead to continuous CTMA use during the COVID-19
pandemic. The moderator-related findings inform the fourth recommendation: authorities should
consider user-specific factors (including age and coronavirus test status) when promoting
CTMA use. Specifically, they could customize promotions or educational programs to
specific user groups. For instance, since senior users are more vulnerable to COVID-19
and their usefulness perceptions most strongly influence continuance usage, explaining
CTMA’s usefulness in protecting their health will more likely motivate continuance usage
for this group. For users who have been tested for COVID-19, perceived response efficacy
more strongly affects continuance usage, so clearly explaining these apps’ privacy
protection will likely encourage them to keep using these apps. 6.2 Practical implications for public policy This study also provides practical implications for authorities about how to use mobile
technologies to manage the ongoing COVID-19 pandemic, as well as future public health
crises. First, the findings on CTMAs during the COVID-19 pandemic suggest that authorities
should use mobile technologies to manage public health crises. Although individuals will
concern about privacy when using mobile technologies to manage public health crises, these
concerns will not hinder their continuous use of such technologies if appropriate privacy
protection measures are implemented in the design and development of the technologies and
if they also help individuals navigate such crises. Second, in light of the finding that perceived usefulness motivates CTMA continuance
usage, authorities are recommended to promote CTMA continuance usage by improving users’ perceptions of these apps’ usefulness. For instance, authorities can provide authorized
information about how CTMAs have been used to disrupt virus transmission and protect
public health in managing the COVID-19 pandemic. Such disclosures can help individuals
recognize the benefits of CTMA use. Additionally, authorities can implement promotional or
educational programs that clarify these apps’ benefits in controlling the COVID-19 pandemic,
enhancing individuals’ awareness of their utility, which could trigger continuous CTMA use
during the COVID-19 pandemic. Continuance
usage of
contact tracing
apps 6.3 Limitations and future research directions This study faced some limitations. First, it used an online survey of US users to test its
research model. The country’s national culture and political atmosphere may affect CTMA
continuance usage. Therefore, more empirical evidence from respondents with different
cultural and political backgrounds is needed to generalize the findings across countries or
regions. Second, due to the survey method’s inherent limitations, the collected data may not
fully capture the complex nature of how people perceive CTMA. Accordingly, future research
should apply qualitative or mixed research methods to explore and comprehensively explain
continuous CTMA use. Third, the data collection took place in December 2020, when the
United States saw a record number of deaths from COVID-19 (Maxouri, 2020). Individuals’
opinions about these apps may have been influenced by this dire situation. Since the
pandemic situation keeps changing, future research could investigate CTMA continuance
usage via longitudinal research, examining whether changes to the pandemic and coping
appraisals related to COVID-19 outbreaks affect continuance usage. Finally, while this study
examined how cognitive appraisals directly affect continuance usage, it did not test coping
strategies. A user may engage in different coping strategies (such as benefit maximizing and
self-preservation) when using apps during the pandemic (Beaudry and Pinsonneault, 2005). Therefore, future research could also examine coping strategies’ role in determining IS
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pp. 408-412. Corresponding author
Ch
l
Li
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EDITORIAL
40 AÑ0S UNIDOS A TRAVÉS DEL CONOCIMIENTO
La Facultad de Estomatología Roberto Beltrán está cumpliendo su 40 Aniversario y profesores, alumnos
y personal administrativo que la integran, así como sus egresados deben sentirse orgullosos de su
existencia por la labor desarrollada en la formación de cientos de jóvenes odontólogos, especialistas en
las diferentes ramas de la odontología, magísteres, doctores y también de técnicos y asistentes dentales.
Todos ellos, con seguridad, juegan un papel positivo e importante en el cuidado de la salud bucal y en
el ejercicio de una profesión cada vez más orientada a la prevención, a la utilización de tecnología
avanzada y con un gran sentido de responsabilidad para asumir el papel que les toca jugar en los
ámbitos de la vida económica, social y cultural de nuestro país.
Podemos afirmar que se ha cumplido con los propósitos y la filosofía que los fundadores de esta gran
institución establecieron en el año 1969; principios que destacan, entre otros, el impartir educación
odontológica del más alto nivel, formar profesionales que entienden y desean contribuir con la solución
de los problemas de salud bucal de las poblaciones más necesitadas, dispuestos a seguir estudiando
para obtener los conocimientos y las habilidades para brindar a cada uno de sus pacientes el mejor
tratamiento.
Debemos reconocer que durante estos 40 años la tarea no ha sido fácil y ha demandado de parte de
sus miembros mucho trabajo, esfuerzo, dedicación y compromiso personal para remontar obstáculos y
llegar al escenario en que hoy nos encontramos, el de una Institución cada día más fuerte, más visible,
más propositiva y con mayor reconocimiento a nivel nacional e internacional.
En este proceso de formación de profesionales verdaderamente universitarios indudablemente no
estamos satisfechos y debemos seguir aportando, no podemos dudar, no podemos bajar la guardia y
debemos seguir reconociendo que no hay progreso sin conocimiento y no hay conocimiento sin educación
y que esta simbiosis educación y conocimiento no es únicamente responsabilidad de las autoridades
sino de todos los miembros que integran la Facultad.
A lo largo de sus 40 años de vida académica la Facultad de Estomatología Roberto Beltrán, ha realizado
innumerables aportes a nuestro país en el campo de su competencia dentro de los cuales podemos
señalar:
- Formación de Odontólogos de alto nivel científico, técnico y de gran sensibilidad social.
- Formación de Asistentes Dentales a nivel universitario.
- Formación de Técnicos Dentales a nivel universitario.
- Creación del primer Programa de Internado Odontológico Hospitalario.
- Creación del primer Programa de Internado Odontológico Rural.
- Creación de las primeras Clínicas Odontológicas Periféricas y Escolares.
- Creación del Sistema de Servicio Estomatológico Docente Asistencial.
- Creación del Programa de Diplomado en Estomatología.
- Creación del primer Programa de Especialización en Estomatología.
- Creación de los programas de Maestría en Estomatología, Maestría en Estomatología con Mención
en Especialidad y Doctorado en Estomatología.
La Facultad de Estomatología Roberto Beltrán cuenta con una excelente plana de profesores, la
mayoría con títulos de Especialista y grados de Magíster y de Doctor formados en la propia Facultad
y en prestigiosas universidades de Estados Unidos y Europa. Para la formación clínica de pre y
73
postgrado dispone de dos modernas clínicas dentales, así como laboratorios y otras facilidades
que garantizan una enseñanza de primer nivel.
Los avances logrados por la Facultad de Estomatología son el resultado del trabajo del selecto
grupo de docentes que la conforman, de la calidad de sus estudiantes y del dedicado trabajo de
su personal administrativo.
Estamos seguros que sus miembros seguirán trabajando y aunando voluntades en pro del progreso
de su Facultad. Sus esfuerzos y dedicación lograran mantener permanentemente el reconocimiento
de Institución con Acreditación Internacional en su carrera de Estomatología que es el
reconocimiento a la alta calidad de este programa.
No dudamos que el espíritu de ciencia que reina en las aulas heredianas, servirán también para
profundizar en estudiantes y profesores el sentido de responsabilidad social que seguirá poniéndose
de manifiesto en cada una de las acciones de servicio que se brinda a las comunidades de
nuestro país. Se continuará cultivando en los estudiantes el pensamiento crítico e independiente
y la capacidad de aprender durante toda la vida, fomentando la innovación y la diversidad.
En el cuadragésimo aniversario de la Facultad de Estomatología Roberto Beltrán, la Revista
Estomatológica Herediana saluda y felicita a todos sus miembros y se compromete a seguir
siendo una publicación seria con miras a alcanzar el reconocimiento que le permita figurar en
todas las bases de datos de revistas científicamente reconocidas.
74
Rev Estomatol Herediana. 2009; 19(2)
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Correction: Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli
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CORRECTION
Correction: Novel Functions and Regulation
of Cryptic Cellobiose Operons in Escherichia
coli CORRECTION Vinuselvi Parisutham, Sung Kuk Lee In the Funding section, a grant number from the funder Next-Generation Bio Green 21 Pro-
gram is listed incorrectly. The correct grant numbers are: SSAC, PJ011181. The complete, cor-
rect funding statement is: This work was supported by the National Research Foundation of Korea (NRF) through
grants funded by the Ministry of Science, ICT & Future Planning (MSIP) (NRF- This work was supported by the National Research Foundation of Korea (NRF) through
grants funded by the Ministry of Science, ICT & Future Planning (MSIP) (NRF-
2009-C1AAA001-2009-0093491), by the Intelligent Synthetic Biology Center of Global Fron-
tier Project funded by the MSIP (2011–0031948), and by a grant from the Next-Generation Bio
Green 21 Program (SSAC, PJ011181), Rural Development Administration, Republic of Korea. g
y
y
g
2009-C1AAA001-2009-0093491), by the Intelligent Synthetic Biology Center of Global Fron-
tier Project funded by the MSIP (2011–0031948), and by a grant from the Next-Generation Bio
Green 21 Program (SSAC, PJ011181), Rural Development Administration, Republic of Korea. Reference 1. Parisutham V, Lee SK (2015) Novel Functions and Regulation of Cryptic Cellobiose Operons in Escher-
ichia coli. PLoS ONE 10(6): e0131928. doi: 10.1371/journal.pone.0131928 PMID: 26121029 1.
Parisutham V, Lee SK (2015) Novel Functions and Regulation of Cryptic Cellobiose Operons in Escher-
ichia coli. PLoS ONE 10(6): e0131928. doi: 10.1371/journal.pone.0131928 PMID: 26121029 OPEN ACCESS OPEN ACCESS
Citation: Parisutham V, Lee SK (2015) Correction:
Novel Functions and Regulation of Cryptic Cellobiose
Operons in Escherichia coli. PLoS ONE 10(11):
e0143463. doi:10.1371/journal.pone.0143463
Published: November 16, 2015
Copyright: © 2015 Parisutham, Lee. 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: Parisutham V, Lee SK (2015) Correction:
Novel Functions and Regulation of Cryptic Cellobiose
Operons in Escherichia coli. PLoS ONE 10(11):
e0143463. doi:10.1371/journal.pone.0143463 Published: November 16, 2015 PLOS ONE | DOI:10.1371/journal.pone.0143463
November 16, 2015 Published: November 16, 2015 Copyright: © 2015 Parisutham, Lee. 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. 1 / 1 PLOS ONE | DOI:10.1371/journal.pone.0143463
November 16, 2015 PLOS ONE | DOI:10.1371/journal.pone.0143463
November 16, 2015
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https://openalex.org/W4247827057
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https://www.qeios.com/read/WCX4ZB/pdf
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English
| null |
Sternal Manubrium
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Definitions
| 2,020
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cc-by
| 68
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Sternal Manubrium National Cancer Institute National Cancer Institute Qeios ID: WCX4ZB · https://doi.org/10.32388/WCX4ZB Source National Cancer Institute. Sternal Manubrium. NCI Thesaurus. Code C52730. The upper segment of the sternum, quadrangular in shape, as well as wider superiorly
and narrower inferiorly. The sternal manubrium articulates with the clavicle and first two
ribs. Qeios ID: WCX4ZB · https://doi.org/10.32388/WCX4ZB 1/1
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